Presentation of insulin and insulin a chain peptides to mouse T cells: Involvement of cysteine residues

016175890/91 $3.00+0.00 Pergamon Pressplc

MolecularImmunoiogy,Vol.28,No. 4/5,pp. 479487, 1991 Printed in Great Britain.

PRESENTATION OF INSULIN AND INSULIN A CHAIN PEPTIDES TO MOUSE T CELLS: INVOLVEMENT OF CYSTEINE RESIDUES* JOHANNE~HAMPL,? GERNOTGR~E~A~~T,~ DIMITRI~PLACHOV,?HANS-GREGORGATTNER,$ HUBERTKALBACXER,$ WOLFGANGVOEL~R,$ MARGOTME~R-DELIUS~~and ERWIN RijDntT ibtstitut fur Immunologic der Joh. Gutenberg Universit& Mainz, Germany; $The Deutsches Wollforschungsinstitut, Aachen, Germany; $Abteilung fur Physikalische Biochemie der Universitlt Tubingen, Tubingen, Germany; j/Max-Planck-Institut fur Immunbiologie, Freiburg i. Brsg., Germany (First receioed 31 May 1990; accepted in revised form 12 September 1990)

Abstract-The requirements for insulin presentation and recognition by AtA!+ and AiA$restricted mouse T c&s were studied using a variety of derivatives of the insulin A chain. It was found that A chain peptides with irreversibly blocked Cys residues are non-stimulator for the T cells. This suggests that at least one of the Cys residues is essentiai for recognition. On the other hand, all A chain peptides containing Cys residues modified in a way reversible by reaction with thiols are stimulatory yet differ in antigenic potency. All these A chain derivatives including a 14 amino acid fragment require uptake by antigen presenting cells (APC) for eflicient presentation. Differences in stimulatory potency between the A chain peptides derived from the same insulin appear to be mainly due to the efficiency of uptake and/or processing by APC. Based on these findings we propose that processing in the case of insulin and its A chain derivatives

involves the reductive deblocking of Cys residues or the rearrangement of disulfide bonds apart from a possible proteolytic cleavage. The same may apply to other proteins if Cys residues in the presented peptides are important for the interaction with Ia or the T ceil receptor.

INTRODUCTION The presentation of most protein antigens to MHC cIass II-restricted T cells requires internalization and processing by antigen presenting cells (APC). Since certain peptide fragments of these proteins in the size range of 7 to 25 amino acids can usually be presented without internalization and intracellular processing it is generahy accepted that enzymatic degradation of proteins in acidic intracellular compartments constitutes a major step in antigen processing (Kourilsky and Claverie, 1989). A requirement for processing has also been demonstrated for the presentation of insulin to T cell clones of corresponding specificity (Falo et al., 1986; Naquet er al., 1987; Gradehandt et ai., 1988). Therefore, this small protein antigen comprising 51 amino acid residues seems to behave like most other protein antigens and one could expect that proteolytic cleavage is important for its presentation. In previous studies employing T cell clones specific for pork (PI) or beef (BI) insulin in combination with A,bAk molecules we found that the isolated insulin A chains were stimulator for the T cells (Reske-Kunz and Rude, 1984, 1985; Spaeth and Rude, 1985; *This work was supported by the Deutsche Forschungegemeinschaft, SFB 3 11, Mainz, Germany. ‘;jAuthor to whom correspondence should be addressed: Erwin Rude, Institut fiir Immunologic, Obere Zahlbacher Str. 67, D-6500 Mainz. Germanv. ~b~reu~at~o~~:Acm, acetamidomethyl; SI, beef insulin; Cm, carboxymethyl; PI, pork insulin; SI, sheep insulin.

Plachov et al., 1988). In some of these A chain preparations the disulfide loop had been opened by S-sulfonation resulting in derivatives which differ in structure considerably from intact insulin. On the other hand, it was reported by Naquet et al. (1987) that I-A’- and f-Ad-restricted mouse T cell hybridomas recognize a conformational determinant of insulin comprising part of both the A and B chains (Al-14, B7-15) and including the A6-All disulfide loop as well as the A7-B7 interchain disulfide bridge. Such a fragment of insulin could, for instance, be produced by intracellular proteolysis of insulin (Semple et al., 1989). In order to obtain more info~ation about the structural requirements for insulin presentation and recognition, mainly with regard to the role of the Cys residues within the A chain, a variety of new derivatives or fragments were tested for their processing requirements and antigenicity for Ai Ai-and Ai Airestricted T cells. It is shown that a 14 amino acid fragment of the A chain is sufficient for recognition but still requires processing for efficient presentation. Furthe~ore, it is suggested that certain Cys residues are critical for recognition and become available by processing only in those derivatives that are modified at these Cys residues in a reversible way. MATERIALSAND METHODS Mice of strains C57BL/10 (BlO) and BlO.BR were originally obtained from the Jackson Laboratory,

479

J.

480

HAMPL et

Bar Harbor, ME, U.S.A. (BlO x BIO.BR)Fl hybrids and the parental strains were then bred in our own animal facility and were used for experiments at the age of 24 months.

Antigens

and reagents

BI and PI were a kind gift from Dr G. Seipke, Hoechst AC, Frankfurt, Germany. BI-A(SO<), was purchased from Sigma, Deisenhofen, Germany. The A chain peptides A(SSO;),, A(SSb as well as the oligomeric A chain [A(SS),], derived from either BI or PI were prepared according to published procedures (Naithani and Gattner, 1981; Gattner et al., 1981). The fragment AI-14(SSO;)j was obtained by chymotryptic cleavage of A(SSO,), in analogy to the procedure described by Busse et al. (1973). The fragment Al-14/Bl-16 was prepared by chymotryptic cleavage of intact BI (Busse et al., 1973). These A chain derivatives were purified by anion exchange chromatography and desalted by gel filtration (Naithani and Gattner, 1981; Gattner et al., 1981; Busse et a/., 1973). Purity was ascertained by HPLC on a reverse phase column (Lichrospher C-18, 0.5 pm, Merck, Darmstadt, Germany) equilibrated with 30 mM ammonium acetate, pH 4 (solvent A). For elution a linear gradient from 30 to 75% solvent B (60% acetonitril in solvent A) was applied. [Al14(SS)S], was derived from Al-14(SSO~)3 by refollowed by with mercaptoethanol duction air-oxidation and by gel filtration on Sephadex G25sf in analogy to A(SS)* (Naithani and Gattner, 1981). The preparation of PI-[Al-14(SS)S], used here was not homogeneous when tested by reverse phase HPLC. Probably it contained isomers differing in the arrangement of disulfide bonds. BI-A(SCm), was prepared by reduction of BIA(SS0; )4 with mercaptoethanol (Gattner et al., 1981), followed by blocking of the free Cys residues by reaction with an excess of iodoacetate at pH 8.6 according to standard procedures. Purification was carried out as for BI-A(SS0, )4. BI-A l- 14(Met), and BI-AI-14(Acm), were prepared by continuous flow solid phase synthesis, based on the Fmoc-polyamide strategy using a MilliGen 9050 peptide synthesizer (Dryland and Sheppard, 1986). The peptides were purified chromatographically and characterized by analytical HPLC and amino acid analysis. In BI-AI14(Met), the Cys residues are replaced by Met; in BI-Al-14(Acm), the Cys residues are blocked by acetamidomethyl (Acm) groups. OVA was obtained from Serva, Heidelberg, Germany. The peptide corresponding to the sequence 323-339 of OVA with additional C-terminal tyrosine was purchased from UCB Bioproducts S.A., BraineL’Alleud, Belgium. Monoclonal anti-I-A; antibody 10-2.16 (Oi et al., 1978) was obtained from the American Type Culture Collection. Rockville, MD, U.S.A., and was used as ascites fluid.

al.

Cell lines The T cell clones ST2jK9.6 (Spaeth and Rude, 1985) F2.1, F7.7, and F7.15A (Plachov er al., 1988) were derived from (BlO x BlO.BR)Fl mice immunized with PI (ST2/K9.6) or PI-A(SSO,), (F2.1, F7.7, and F7.15A), respectively. T cell clone F7.15A recognizes Ia molecules on APC without nominal antigen and should be regarded as autoreactive. The line BlO.BI was developed from lymph node T cells of BI-primed BlO mice; the line IIIBS was generated from lymph node T cells of OVA(323-339)Tyrprimed (BlO x BIO.BR)FI mice. For propagation these lines were periodically restimulated with their specific antigen and splenic APC followed by expansion with recombinant human IL 2. The mouse fibroblast line LBK (Landais et al., 1986) transfected with A,b and Ai genes, was kindly given to us by Dr Mathis, Strasbourg, France. The Ia-positive B-hybridoma line LB27.4 (H-2dxb) (Kappler et al., 1982) was a gift of Dr P. Marrack, Denver, CO, U.S.A. The IL 3-dependent cell line DA-I (Pierce er al., 1985) was donated by Dr J. Ihle, Frederick, MD, U.S.A. Cell culture Cells were cultivated in Iscove’s medium (IMDM, Gibco) supplemented with 2 mM L-glutamine (Gibco), 100 IU penicillin, 100 pg ml-’ streptomycin, 5 x 10m5 M mercaptoethanol (Sigma) and 5% heatinactivated FCS. For T cell proliferation assays each culture (200 ~1 final volume) contained 2 x IO“ T cells, 1 x 10’ gamma-irradiated (2000 rad) syngeneic spleen cells and antigen at varying concentrations. After 48 hr cultures were pulsed with 0.2 PCi (7.4 kBq) [3H]TdR (Amersham Buchler, Braunschweig, Germany) for 18 hr and were then harvested. Results are expressed as mean cpm of triplicate cultures. For inhibition of T cell activation serial dilutions of monoclonal anti-I-A; antibody (10-2.16) were added at the start of these cultures. For activation of T cells with antigen-pulsed and aldehyde-fixed APC the cell lines LBK (A,bAi) or LB27.4 (I-Ah, I-Ad) were used. These APC (lo6 ml-‘) were incubated with antigen at different concentrations for 3 hr and washed 3 times with PBS. The cells were then metabolically inactivated (fixed) by incubation with 0.05% glutardialdehyde for 40 sec. Fixation was stopped by adding L-lysine up to a final concentration of 0.1 M followed by extensive washing in culture medium. The T cell stimulation cultures contained 2 x IO4 T cells and I x IO5 antigen-pulsed, fixed APC in 200~1 medium. In tests with prefixed APC antigen was added to these stimulation cultures instead of pulsing the APC with antigen before fixation. After an incubation period of 48 hr at 37’C 50 ~1 of supernatants were sampled, frozen, thawed, and assayed for their IL 3 content. IL 3 assay The T cell supernatants were added at a 2-fold final dilution to lo4 of the IL 3-dependent DA-I cells in a

Presentation

total volume of 100~1 culture. After incubation for 18 hr, 0.1 /frCi (3.7 kBq) f3H]TdR was added for another 18 hr and cells were harvested as described above.

of insulin

I. Bl

II. Bl-Al-14/Bl-16 RESULTS

Stimufation of insulin -specific, I-A -restricted T cell lines by derivatives of the isolated A chain in BI Previous studies have shown that T cell clones or hybridomas derived from PI- or RI-immune (H-2b x H-2k)FI mice can be specifically restimulated by intact insulin or its isolated A chain when presented on accessory cells expressing the appropriate MHC class II molecules, mostly AkAi (Reske-Kunz and Rude, 1984; Reske-Kunz and Rude, 1985; Spaeth and Rude, 1985). The A chain preparations employed for these expe~ments were derivatized at their Cys residues by S-sulfonation or by intrachain disulflde formation (Fig. 1). For a more detailed study a series of additional A chain derivatives, depicted in Fig. 1, were tested for their ability to stimulate insulinspecific T cell clones or lines. Before presenting data on the stimulatory properties of these compounds it is important to describe some of their structural features. In addition to the S-sulfonated A chain (VI) and its Al-14 fragment (VII) “d~sulfide-1inke~’ A chain derivatives were used. These are obtained by air-oxidation of the A chain in its SH form [A(SH),]. This results in the formation of monomeric A chains, A(SS)2 (III), with two intrachain disulfide bridges as well as in the formation of oligomers (IV) with a mixed arrangement of intra- and interchain disulfide bonds. The monomer, A(SS),, represents a mixture of two isomers (Fig. 1, III) that can be separated by reversed phase HPLC (Gattner et al., 1981). For the experiments presented here the mixture of isomers was used. In an analogous way air oxidation of the A chain fragment, A1-14(SH)3, derived from Al14(SSO;),, results in formation of a disulfide-linked dimer [Al-14(SS)S], in addition to other isomers (V) which were partially separated by gel filtration (H. G. Gattner, unpublished). It is important to note that all of these derivatives including intact insulin (I) and a chymotryptic fragment of insulin (II) can be converted to A chain peptides with free Cys residues by reaction with thiols. In a second group of A chain derivatives the Cys residues were oxidized to cysteic acid, (A(SO;),) (VIII), blocked by Cm- [IX) or Acm-groups (X) or were replaced by methionine (M) residues (XI}. From the data presented in Table I it is apparent that none of these irreversibly modified A chain derivatives was stimulatory for the T cells. A comparison of the stimulatory capacity of some of the reversibly modified derivatives of BI for clone F2.1 T cells is presented in Fig. 2(A). Most notable is the SOO-fold higher efficiency of the oligometric A chain relative to intact insuiin. The monomeric BI-A chains including the S-sulfonated Al-14 fragment

111.Bl-A(SS) 2 (two isomfir8)

IV. BI-[A(SS)2] n

V. Bi-[Al -14(SS)S] 2 (+ other isomers)

VI. Bi-A(SSO;),

VII. Bl-Al-14(SSO&

VIII. BI-A(SOj)

4

IX. BLA(SCm) 4

X. Bl-Al-14(Acm)

S

Xi. Bi-Al-14(Met) S

Fig. 1, Schematic representation of the structure of bovine insulin (BI) and of several derivatives of its A chain. Analogous derivatives were prepared from pork insulin (PI).

exhibited about an equal, but IO-fold lower potency than intact BI. As summarized in Table 1, a very similar pattern was observed for these BI derivatives with three other T cell clones or lines. It is important to note that these T cell lines are distinct with respect to their specificity patterns for species variants of insulin (PI, BI, SI). For instance, the AkAk,-restricted clones F2.1 and F7.7, originally developed from (BlO x BlO.BR)Fl mice immunized with PIA(SSO;),, show a heteroclitic response to BI (Plachov et af., 1988), whereas cione ST2jK.9 obtained upon immunization with PI is about equally reactive to PI/AkAi*, and BI/AiA$ (Spaeth and Rude, 1985). T cells of line BlO.BI are BI/AiA&specific and do not react to PI. It will also be noted from the data presented in Table 1 that these T cell lines differ considerably with respect to their antigen sensitivity. A chain derivatives of PI have stimulutory properties a~alo~a~s to those qf the corresponding BZ derivatives The results presented so far demonstrate that T cell clones or lines with distinct specificity both with respect to species variants of insulin and the Ia molecules are capable of recognizing the isolated A chain of BI or its Al-14 fragment. If the different antigenic potency of some of the A chain derivatives of BI including intact BI is primarily due to the efficiency of their uptake and/or processing by APC

482

J. HAMPL et ol. Table 1. Capacity of fragments and peptides derived from 81 or PI to stimulate insulin-reactive 7 cell lines* T cells Peptides

F2.1

F-T.7

BI

0.1 0.1

0.1 I.1 0.0002

27

JWWW,

1.4 0.0002

0.75 1.3 NS NS

BI-Al-14(Met),

0.8 1.1 NSt NS NS NS

90 87 NS NS NS NS

9 9 NS NS NS NS

IO 15 I.1 20 20

NS

BI-Al-14/B]-16 BI-A(%),

BI-~Al~l4(SS)S~~ BI-A(SSO,-), BI-AI-14(SS0,)3 BI-A(SO,), BI-A(SCm), ~I-Al-14(SAcm)~

;:- A(SS),

PI-[A(SS)zl, PI-[A~-lqSS)S]~ PI-A(SSOJ )4 PI-A I- 14(SSO; ),

ST2jK9

BIO.BI

27

0.1 3 0.01

60

3 12 0.08 4 20

20

*The data representthe ~on~ntration of antigen (pM) required to induce a half-maximal proliferation of the T cells in the presence of syngeneic, gamma-i~adiated spleen cells as APC. tNS = non-stimulatory.

one should expect to find a similar hierarchy for the corresponding derivatives of PI. The data presented in Fig. 2(B) and summarized in Table I indicate that this is indeed the case. It should be mentioned that in spite of chromatographic purification certain variations in the stimulatory potency of different batches of the same A chain derivative of both PI or BI were observed. This applies mainly to monomeric A(!Z& and the oligomeric form of the disulfide-linked A chain [A(SS),],. Since the latter is much more potent than the former, even a small contamination with ohgomers hardly detectable by HPLC will increase the apparent potency of a monomer preparation. Presentation of dzerent A chain derivatives of BI uppears to result in recognition of the same determinant by T cells Differences in the stimulatory

potency of antigens

for the same T cell clone can be either due to differences in the structure (amino acid sequence) of the peptide that specifically interacts with the MHC molecules and the T cell receptor after processing by APC or to differences in the efficiency of uptake and/or processing of the respective antigens by APC. If, as suspected, the latter effect is primarily responsible for the potency differences between BI and its A chain derivatives stimulation of a T cell clone by these antigens should be inhibitable by anti-la or anti-CD4 antibody to about the same degree. The results of such an experiment are presented in Fig. 3. Four different dilutions of anti-I-Ak antibody (I O-2.16) were used to inhibit the stimulation of clone F7.7 T cells by BI and two of its A chain derivatives. Each antigen was applied at three concentrations which in the absence of inhibitory antibody induced about lO-20%, 50%, or 80% of the maximal response. The inhibitory effect of antibody was very similar for the

- - - PI-[A(sqll,

Antigen concentr&ion

PM]

Fig. 2. Proliferative response of clone F2.1 T cells to BI (A) and PI (B) and their derivatives. Data represent mean values for (3H]TdR incorporation of triplicate cultures of 2 x 10’ T ceils and 1 x lo5 gammairradiated (BIO x BIO.BR)Fl spleen cells with various concentrations of antigen.

483

Presentation of insulin

Bl-A(SSO-3)4

0.045

0.08

0.17

0.7 1.3 2.6 Antigen concentration (CIM]

Antigen concentrationWM]

m /_

t

+

Medium

Ea+-

OL

0.0017 0.0034 O.OOW AnUgenconcentr&n bM]

1112800 1151200 l&?OO Dllullonof anti-la aniibody

Medium

Fig. 3. Inhibition of the proliferative response of F7.7 T cells induced by BI, BI-A(SSO,),, and BI-[A(S),], presented on syngeneic spleen cells by anti-A! antibody. The cultures contain 2 x 10“T cells, 1 x 10’ gamma-irradiated (BIO x BlO.BR)FI spleen cells and three concentrations of antigen which in the absence of inhibitory antibody (*) induced 10 to 20%, 50% and 80% of the maxima1 response, respectively. Ascites fluid containing anti-A; antibody (10-2.16) was added at dilutions of l/3200 (O), l/12,800 (A), and l/51,200 (0). For control the same antibody was added to the T cells stimulated by 2 pg ml-’ ConA.

three antigens in spite of the fact that their antigenic potency varies widely. In a control experiment practically no inhibition of the ConA response by anti-IAk antibody was observed. The same result was obtained in a similar, previously published experiment in which the inhibitory effect of different concentrations of anti-CD4 antibody on the response of F2.1 T cells to BI, BI-A(SSOr ),, PI and PIA(SSOc), was compared (Gradehandt et al., 1988). Ejicient presentation of insulin and its A chain derivatives requires processing by APC The results of inhibition experiments with anti-la and anti-CD4 antibody indicate that the presentation of various A chain derivatives by APC results in recognition of a closely related or identical A chain peptide by the T cells. In view of the structural differences between some of these A chain derivatives including intact insulin the question arose whether these antigens require intracellular processing to

create a common A chain peptide. Figure 4(A-C) presents the results of an experiment in which the processing requirement for the presentation of intact BI and of its Al-14 fragment to three different T cell lines was compared. It can be seen that prefixed APC in contrast to antigen-pulsed and fixed APC can present the two antigens only with low efficiency to clone F2.1 T cells. In the case of ST2/K.9 T cells, which are less sensitive to antigen, only antigen taken up and presumably processed by the APC can stimulate the T cells, whereas soluble antigen in the presence of fixed APC is not stimulatory. The same is true for BlO.BI T cells which exhibit an even lower antigen sensitivity. Control experiments (Fig. 4D) showed that fixed APC can still stimulate the autoreactive T cell clone F7.15A and present the processing-independent peptide, (323-339)Tyr, corresponding to residues 323-339 of ovalbumin (Shimonkevitz et al., 1984) to the T cell line IIIBS. The stimulatory capacity of other A chain peptides

J. HAMPL et ul

484

1B.

, ,’ P” I’

ST2/K9

:

Antigen concentration WM]

C. 1.5

Antigen concentration WM]

D.

F7.15A

Bl O.BI

= E 3 c ’ .G tj i h q = 085 o _ o-

/*,

,’ ,i ,-,------‘e,L::_::::-;] ’ 087 a7

/’

Ill-B5

01

/”

4 Bi T-cells

Antigen concentration PM]

T-cells APC

+

T-Ceil9

T-cell9

T-cells

+ APC

OVA(323-339)Tyr

+ APC +

Fig. 4. Processing requirements for the presentation of BI (0) and BI-Al-14(SSOc), (0) to T cells. (A) T cell clone: F2.1, APC: L cell line LBK (A,hA;). (B) T cell clone: ST2/K9, APC: L cell line LBK (A,bA$). (C) T cell line: BIO.BI, APC: B hybridoma 27.4 (A,bAi). The APC were pulsed with various concentrations of antigen and were then fixed with glutardialdehyde (---). Alternatively the APC were fixed before antigen was added at different concentrations in soluble form to the test cultures (---). (D) Control experiments with the autoreactive T cell clone F7.15A, stimulated by fixed LBK cells as APC, and the T cell line IIIBS activated by fixed LBK cells in the presence (hatched bar) or absence (filled bar) of OVA(323-339)Tyr (10 ng ml-‘). T cell stimulation was estimated by measuring the IL 3 content of 48 hr supernatants using the IL 3-dependent cell line DA-l.

such as BI-A(SS), or BI-[A(SS)2], to fixed APC was essentially the same as shown in Fig. 4 for BI and BI-AI-14(SSO;), (Gradehandt et al., 1988). Furthermore, analogous results were obtained when APC with the lysosomotropic drug were treated chloroquine (up to 200 gg ml-‘) during pulsing with insulin and its derivatives (Fig. 5). Thus, BI and all of its antigenic A chain derivatives require uptake by APC for efficient presentation to T cells. DISCUSSION

The data presented in this study demonstrate that in agreement with previous observations (ReskeKunz and Riide, 1984; Reske-Kunz, 1985; Spaeth and Riide, 198.5; Plachov et al., 1988) insulin-reactive AiAi- and A,bAi-restricted mouse T cells recognize the isolated A chain of insulin. Within the A chain a peptide comprising the 14 N-terminal amino acid residues is still fully antigenic. It is not yet known whether an even smaller fragment would be stimulatory for the T cells.

From studies on the specificity of insulin-reactive T cell clones using species variants of insulin it is known that amino acid substitutions within the disulfide loop of the A chain (A8-AlO) influence the interaction with Ia and/or the TCR. Furthermore, residue A4 has been implicated as being essential for recognition because in the A chain of PI this is the only residue that differs from mouse insulin (Reske-Kunz and Riide, 1984; Reske-Kunz, 1985; Spaeth and Riide, 1985; Plachov et al., 1988). Within the same region of the A chain three Cys residues are located in positions A6, A7, and Al 1. Therefore, one could expect that some or all of these Cys residues may also influence the interaction with Ia or the TCR. The finding that the irreversible modification of these Cys residues or their replacement by methionine abrogates antigenicity supports this assumption since such modifications correspond to the replacement of Cys by a different amino acid. On the other hand, these Cys residues can be engaged in variably arranged intra- or interchain disulfide bonds or can be S-suldonated. Yet in spite of these structurally quite distinct

485

Presentation of insulin

no antigen

81

El-Al-14(ssoj,

81

Fig. 5. Inhibition of the presentation of BI and BI-Al-14(SSO; ), by the lysosomotropic drug chloroquine. LBK cells were pulsed with stimulatory concentrations of antigen (0.87rM Bi and 8.7pM BIAl-14(SSO; )If in the presence (hatched bar) or absence (filled bar) of chloroquine (200 pg ml-‘) before fixation with glutardialdehyde and addition of T ceils of clone F2.1. As control the autoreactive T cell clone F7.15A was used with the same APC. T cell stimulation was estimated by measuring the IL 3 content of 48 hr supernatants using the IL 3-dependent cell line DA-I.

modifications such derivatives, including intact insulin, retain full antigenicity. In this context it should be noted that all of these “disulfide linked” or S-sulfonated A chain derivatives including intact insulin, can be converted to A chains with free SH-groups by reaction with thiols. Thus, these modi~cations are principalIy reversible also under physiological conditions, for instance by giutathione. Since these data suggest that at least some of the Cys residues of the insulin A chain participate in specific recognition it appears likely that removal of S-sulfonate groups or opening of disulfide bonds may be important for the presentation of the various derivatives and occurs during intracellular processing. The following results support this assumption: (i) There is evidence that presentation of the various insulin derivatives results in recognition of the same or a very similar antigenic peptide to the T cells. This is indicated by the finding that the hierarchy of various A chain derivatives with respect to their stimulatory potency is essentially the same for T cell clones of distinct specificity. In addition, analogous derivatives of BI and PI show a very similar hierarchy pattern. Furthermore, in spite of the wide differences in antigenic potency the T ceil response to the various A chain derivatives of the same insulin is inhibited to about the same degree by anti-Ia or anti-CD4 antibodies. Thus, the affinity of interaction of these antigens with Ia and the TcR does not appear to differ significantlr, which would be in agreement with the conversion of the structurally distinct derivatives including intact insulin to a closely related or identical peptide. (ii) Insulin and all of its antigenic A chain derivatives, including the A 1- 14 fragment, require uptake by APC for efficient presentation. In contrast, peptides of similar size derived from other proteins can usually be presented by aldehyde-fixed APC without intracellular processing (reviewed in Kourilsky and Ciaverie, 1979).

It is not known whether the presented peptide that is derived from insulin and its derivatives is an A chain fragment with free SH-groups or is a peptide with a defined arrangement of intrachain disulfide bonds, for instance with the native A6 to Al 1 loop. A peptide with free SH-groups could possibly be stabilized by its binding into the groove of the Ia moiecuIes. An indirect argument for the recognition of a peptide with free SH-groups may be the observation that the monomeric A chain preparation BI-A(%), (compound III in Fig. 1) which contains both possible intrachain disulfide isomers in about a 1:2 ratio still requires processing as do the other derivatives. If one of these isomers would represent the antigenic peptide with the correct disulfide configuration one could expect that this mixture will be presented more efficiently by aldehyde-fixed APC than the other derivatives. This was not the case (Gradehandt et al., 1988). High concentrations of insulin and its derivatives were found to be presented in an apparently processing independent way by aldehyde-fixed APC. The T cell response induced under these conditions is very limited and detectable only with T cell clones of relatively high antigen sensitivity such as F2.1 or F7.7. If the opening of disulfide bonds would indeed represent an essential step required for the presentation of these antigens it is conceivable that such a reaction could also occur, although with low efficiency, extracellularly, for instance by thiols released from the T cells. While Naquet et al. (1987) reported that a conformationai determinant comprising the A chain Ioop as well as B chain residues of insulin is recognized by I-Ad and I-Ab-restricted T cell hybridomas no indication for a participation of B chain residues was obtained in our studies which also included I-Ah-restricted T cells. As discussed above, it is possible that an intact A6 to All disulfide loop is important for recognition of the A chain pcptide by our T cells but

486

J. HAMPLet al.

such a loop structure is certainly not required for the antigen that is added to the APC. It could be created, however, by processing. In this context it should be mentioned that our T cell clones were obtained from mice immunized with either intact insufin (e.g. ST2iK.9 or BIO.BI) or with the S-sulfonated A chain, PI-A(SSO,)., (F2.1 and F7.7) (Plachov et af., 1988). Thus, an intact A chain loop is also not required for the in vitio stimulation of insulin-reactive T cells. In a study on the specificity of human BI-reactive T cell lines the insulin peptides used were also included in our experiments (Miller et al., 1988). The stimulatory pattern of these peptides was essentially the same as reported here. Furthermore, these insufin peptides with the exception of BI-[Al14(SS)S], also required processing for presentation. In general, the presentation and recognition requirements for insulin by human and mouse T cells appear to be similar. Insulin and its derivatives differ considerably in their stimulatory potency. Most remarkable is the high efficiency of the oligomeric (RI-[A(SS),J,) as compared to the monomeric (BI-A(SS),) A chain and the other derivatives including insulin. Since, as discussed before, there are good arguments to assume that the same antigenic peptide is recognized these potency differences cannot be attributed to differences in the affinity of interaction with la and the TCR but are most likely due to the efficiency of uptake and/or processing by APC. Differences in the stimulatory potency between antigens are mostly interpreted as being due to differences in the affinity of specific recognition. In most cases this is certainly correct if antigens of slightly different amino acid sequence, e.g. BI and PI, are compared. However, if the general structure differs one has to take into account that, as shown here, the efficiency of uptake or processing by APC can be of considerable importance for the antigenic potency. According to our knowledge only few antigenic peptides have been described in which Cys residues are located within the stretch of amino acids that presumably interact with Ia and/or the TCR. One example is apamin, an 18 amino acid peptide containing four Cys residues (Regnier-Vigouroux et al., 1988). This peptide, similar to insulin, requires processing for presentation to T cells. For some T cell clones this processing requirement is eliminated by the opening of intrachain disulfide bridges and blocking of the Cys residues by Acm groups, Other clones, however, are non-reactive to such blocked apamin derivatives and respond only to intact apamin in a processing-dependent way. For the latter, in analogy to insulin, Cys residues may be directly involved in recognition and processing may depend on the opening or rearrangement of disuifide bonds. Such a processing step in addition to proteolytic fragmentation may therefore be of general importance for cysteine-rich proteins.

REFERENCES

Busse W. D. and Gattner H. G. (1973) Selective cleavage of one disulfide bond in insulin: preparation and properties of insulin A7-B7-di-S-sulfonate. Hoppe Seyler’s Z. Physiol. Chem. 354, 147-155.

Dryland A. and Sheppard R. C. (1986) Peptide synthesis. Part 8. A system for solid phase synthesis under low pressure continuous flow conditions. J. Chum. Sot. Perkin Trans. 1, 125-157.

Faio L. D. Jr., Benacerraf B. and Rock K. L. (1986) Phospholipase treatment of accessory cells that have been exposed to antigen selectively inhibits antigen-specific la-restricted, but not allospecific, stimulation of T lymphocytes. Proc. natn. Acai. Sci. U.S.,4. 83, 69946947. Gattner H. G.. Krail G.. Danho W.. Knorr R.. Wieneke H. J., Bfiflesbach E. E., Schaftmann B., Brandenburg D. and Zahn H. (1981) Eine verbesserte Methode der Kombination von Insulinketten zur Darstellung van Insulinanalogen. Hoppe SeyIer’5 Z. Physioi. Chew. 362, 1043-1~9. Gradehandt G., Hampl J., Plachov D., Reske K. and Rude E. (1988) Processing requirements for the recognition of insulin fragments by murine T cells. bnmun. Rev. 106, 59-75. Kappler J., White J., Wegmann D., Mustain E. and Marrack P. (1982) Antigen presentation by Ia + B cell hybridomas to H-2-restricted T cell hybridomas. Proc. nafn. Acad. Sci. U.S.A. 79, 36043607.

Kourilsky P. and Claverie J.-M. (1989) MHC-antigen interaction: what does the T cell receptor see? In Ado. Imrnu~u~. (Edited by F. Dixon). Vol. 45, pp. 107-IY3. Academic Press, San Diego. Landais D,, Beck B. N., Buerstedde J.-M.. Deraw S.. Klein D., Koch N., Murphy D., Pierres M., Tada T., Yamamoto K., Benoist C. and Mathis D. (1986) The assignment of chain specificities for anti-la monoclonal antibodies using L cell transfectants. J. fmmun. 137, 3002-3005. Miller G. G., Hoy J, F., Nell L. J. and Thomas J. W. (1988) Antigen processing and the human T cell receptor repertoire for insulin. J. Immun. 141, 3293-3298. Naithani V. K. and Gattner H. G. (1981) Semisynthesis of human proinsulin. I. Preparation of arginyl-A-chain cyclic bis-disulfide. Hoppe Seyler’s Z. Physioi. Chem. 362, 685695.

Naquet P., Ellis J., Singh B.. Hodges R. S. and Delovitch T. L. (1987) Processing and presentation of insulin. 1. Analysis of immunogenic peptides and processing requirements for insulin A loop-specific T cells. J. Immun. 139, 3955-3963. Oi V. T., Jones P. P., Goding J. W., Herzenberg L. A. and Herzenberg L. A. (1978) Properties of lnonoclonal antibodies to mouse IgG allotypes, H-2 and 1 antigens. Curr. Top. Microbial. Immun. 81, 115120.

Pierce J. H., DiFiore P. P., Aaranson S. A., Potter M., Pumphrey J., Scott A. and lhle J. N. (1985) Neoplastic transformation of mast cells by abelson-MuLV: abrogation of IL3-dependences by a nonautocrine mechanism. Cell 41, 685-693. Plachov D., Fischer H.-G., Reske-Kunz A. B. and Riide E. (1988) The specificity of the interaction between the agretope of an antigen and an Ia-molecule can depend on the T cell clonotype. Molec. Immun. 25, 61 l-620. Regnier-Vigouroux A., Ayeb M. E., Defendini M.-L. Granier C. and Pierres M. (1988) Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid ueutide. J. Immure. 140. . . 1069-1075.

Reske-Kunz A. B. and Rude E. (1984) Analysis of the (H-2b x H-2k)FI-restricted response to insulin. Scami. /. Immun. 20, 97-104. Reske-Kunz A. B. and Rude E. (1985) Insulin-sp~i~c f cell hybridomas derived from (H-2b x H-2”)Fl mice preferably employ F,-unique restriction element for antigen recognition. Eur. J. Immun. 15, 1048 1054.

Presentation of insulin Semple J. W., Ellis J. and Delovitch T. L. (1989) Processing and presentation of insulin. II. Evidence for intracellular, plasma membrane-associated and extracellular degradation of human insulin by antigen-presenting B cells. J. Immun. 142, 41844193. Shimonkevitz R., Colon S., Kappler J., Marrack P. and Grey H. M. (1984) Antigen recognition by H-2-

487

restricted T cells. II. A typic ovalbumin peptide that substitutes for processed antigen. J. Immun. 133, 2067-2914. Spaeth E. and Riide E. (1985) Development of T cell clones reactive to two defined restriction elements in conjunction with two defined epitopes of antigen. Eur. J. Zmmun. 15, 1177-1183.