Immunology Today, vol. 8, No. 9, 1987

Mutations of class II MHC molecules It remains undear how the tertiary interaction of T-cell receptor, la molecule and foreign antigen results in the extensive divemty of the helper T cell repertoire. Here Laurie Glimcher and inMn Griffith focus on what has been learned about the relationship beoNeen structure and function of the la molecule from the use of mouse strains with mutations in the genes coding for these glycoproteins. Halper T (TH) cells recognize antigen in the context of class II major histocompatibility complex (MHC) (la) molecules on the surfac~ of antigen-presenting cells (APC)~.z. Most evidence suggests that T cells have a single receptor (TCR) whose ligand is composed of a bimolecular complex of a class II molecule and a foreign antigen3-s. If la molecules can associate with only a limited number of foreign antigenic determinants, and if the TCR can recognize only a small number of la epitopes, the n~cessityfor this ternary interaction n';~ht impose constraints on the diversity of TH cell responses.

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274

Department of CancerBiology, HarvardSchool of Public Health, and Departmentof Medicine, HarvardMedicalSchool, Boston, MA 02175, USA

Laude H. r.,.'..h---J ~mmmmnuuul~ll ( l l l l l Irwin J. Grim Structural considerations The class II MHC or la antigens are heterodimeric cell-surface glycoproteins consisting of one heavy (33-34 kDa) chain ((~)and one light (28-29 kDa) chain (13)which are noncovalently associated and span the plasma membrane= Two isotypic forms of la exist in the mouse: the I-A melecule, formed by the pairing of the Ap and A~ chains, and the I-E molecule formed by the pairing of Ep and E, chains. Each chain consists of two extracellular domains of approximately 96 amino acids, a membraneproximal domain that is strongly homologous to immunoglobulin constant region domains and a polymorphic amino-terminal domain. These are followed by a hydrophobic transmembrane segment and an intracytoplasmic tail of 12-15 am;no acids. Information about the structural organization of the class II genes at the molecular level is accumulating rap;dly. The organization of the class II genes (Fig. 1) reveals a leader exon encoding a signal peptide that also includes the first 3-6 amino acids of the first extracellular domain. The second and third exons encode the 131/¢x1 and ~32/o=2domains, respectively. There are three additional exons for 13-chaingenes which encode the transmembrane, intracytoplasmic and 3' untranslated region, while exon 4 of the oL-chain genes encodes the transmembrane, intracytoplasmic and the beginning of the 3' untranslated region with a fifth exon encoding the remainder of the 3' untranslated region. This article will focus primarily on the structural features of the polymorphic amino-terminal (131,oL1)domains of the la molecule. Identification of functionally important sites by allelic comparisons One approach to ~u¢,,u,ymg "'J^-+'~' " T-cell recognition sites on the la molecule has been to compare the amino acid sequences predicted from the cloned genes of three class II molecules (As, Ap, Ep) [rom different haplotypes (Fig. 2). Many allelic forms of the four class II genes have now been cloned and sequenced6-13. Examination of the nucleotide sequences and of the predicted amino acid sequences reveals a substantial degree of polymorphism which is almost entirely concentrated in the aminoterminal (c~1, 131) domains for each of these genes. Further comparison reveals that there are clusters of highly p01ymorphic residues within this variable region that are marked by substitutions of amino acids with different charges. These amino acid alterations are likely to cause significant structural differences between thc allelic la molecules. Indeed exon shuffling experiments which exchange 13-domainsof one haplotype for another have shown that monoclonal antibody (mAb)-binding sites and T-cell recognition sites are localized to this first external domain 14 although more recent studies, discussed below, indicate that other parameters can influence the formation of such sites. la moleculeswith limited mutations Although they are useful in predicting important structural regions on the la molecule, allelic comparisons 1987. Elsevier Publications, Cambridge

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Fig.2. Sequencesof ~1 and aT domainsof publishedallelicforms of Ap, A~ and Epgenes(seetext and Refs46-48). are not specific enough to identify precise relationships between la structure and function. To determine the role of individual amino acids in the formation of serologic determinants or T-cell restriction sites, several groups have begun to analyse la molecules with limited mutations. These efforts were prompted by the wealth of information provided by the study of one spontaneous I-region mutation (B6.C-H-2bm12)is. Molecular analysis revealed that this mutation involved a °gene conversion' event between E~ and A~ resulting 16.17in the alteration of three amino acids in the carboxy-terminal half of the A~I domain (lie --~ Phe 67; Arg --~ Gin 70; Thr --, Lys71)18. These alterations resulted in the loss of several serologic specificities including la.8 (Ref. i9), altered T-cell recognition specificities, and a change in immune respon=e (Ir) gene ph~notype2°-22. The valuable information provided by ~his single mutant class II molecule thus suggested that elucidation of la structure-function relationships could be aided by the development of a large number o| distinct mutants. This has been accomplished by a number of groups in two ways. Firstly, in vitro mutagenesis directed at the 1~ domain of the A~ (Ref. 23), Abin'2_ (F. Ronchese, R. Germai~, pers. commun.) E~I3(F. Ronchese, R. Schwartz, K Germain, pers. commun.) genes has been

used to construct several structurally distinct mutant A~ and EL genes. DNA-mediated gene transfer of these genes into a B-lymphoma cell 24--26 or into the L cell27 results in their successful synthesis and cell-surface expression. Secondly, panels of cloned populations of antigen-presenting cells with mutated i-Ak and I-Ek molecules have been produced by negative and positive immunoselection using monoclonal anti-la antibodies, and the mutant genes then isolated and sequenced to localize the mutation. Such mutant lines have been selected, therefore, for serologic alterations in surface lak molecules. Functional studies coupling both types of la muta3t cell lines with panels of antigen-specific, MHCrestricted T cells leads to the following three major conclusions, the evidence for which is presented below. Firstly, multiple determinants are present on a single I-A molecule; secondly, a single class II molecule and a simple globular protein can generate multiple antigenic determinants or TCR ligands. Thus the association of la molecules and foreign antigens can generate extraordinary diversity available for the TH cell immune response. Thirdly, and perhaps most important in the interpretation of studies which use such class II mutant cell lines, the tertiary conformation of the la molecule is critical in the formation of the majority of such sites.

275

Immunology Today, vol. 8, No. 9, ;987

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SilcHikectedmutantmolecules Specific alteration of DNA by in vitro mutagenesis has become a rapid and reliable means of correlating structure and function of chosen genes. Figure 3 illustrates a method pioneered by Zoller and Smith for producing such alterations simply, rapidly and with high efficiency. Three mutant A~ genes were constructed by Cohn et al. "~ using oligonucleotide site-directed mutagenesis; the choice of the mutation sites was based on the location of allelic polymorphisms in the b and k haplotypes (Fig. 4). Two of these mutants, 18.1 and 18.3, convert single amino acid residues at positions 9 and 13

to those in the k haplotype. A third mutant, 23.1, involves the replacement of three residues at positions 65, 66 and 67 (Pro-Glu-Ile) with a single Tyr residue. These mutant or wild-type A~ genes were co-transfected with the A~ gene into an la-negative B-lymphoma cell line, C3 (Ref. 36). The pattern of binding of a panel of od-Ab mAb by two of the mutant cells (18.1 and 18.3) was indistinguishable from that of the C3 cells transfected with wild-type molecules. This is interesting in view of the clearcut functional changes discussed below and highlights the need to screen potentially interesting mutant cell lines by both serology and antigenpresenting capability. The key result, however, is that the mutations resulted in the loss of sites on the I-Ab molecule important for T-cell recognition as determined by testing the ability of the A~ mutant cell lines to activate a panel of antigen-specific or alloreactive cloned T-cell hybridomas. Thus, the variable ability of both alloreactive and foreign antigen-specific T hybridomas to be activated by the three site-directed mutant cell lines and a bm12 mutant cell line25 subdivided these T cells into multiple groups. These mutant lines therefore appear to distinguish between multiple sites on the I-Ab molecule used for T-cell recognition. There appeared to be some correlation between the extent of the nucleotide substitution, loss of mAb binding and T-ceU activation since the more extensively altered 23.1 and bm12 mutants resulted in a more profound ablation of antigen presentation. Alternatively, the determinants formed by the 3' end of the 131 domain may be more critical for T-cell recognition than determinants formed by the 5' end. Most of these determinants depend on the homozygous pairing of the Ai3 and A~ molecule. Most importantly, the data obtained suggest that the tertiary conformation of the I-A molecule is critical in the formation of at least some of these restriction sites. This point can be best illustrated by a closer look at the results obtained with these four A~ mutant cell lines. One ovalbumin-specific T-cell hybridoma is not activated by the two mutants (18.3 and bm12) which lie 54 amino acids apart but is aGivated by the two other mutant cell lines (18.1 and 23.1) whose alterations are adjacent to those in the 18.3 and bm12 cells, respectively (Fig. 4). Failure of T-cell activation could reflect protein sequence alterations if either the 18.3 and bin12 regions are opF'osed in the tertiary structure of I-Ab in the process of T-cell recognition; or if the altered site disrupts interaction with the antigen and alters the interaction with the T-cell receptor. It is equally likely, however, that each of these mutations independently alters the T-cell recognition site by affecting tertiary conformation. The critical role of tertiary conformation of the la molecule in T-cell activation is also demonstrated by the pairs of mutations which have adjacent 23.1

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Immunology Today, vol. 8, No. 9, 1987

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amino acid changes. The 23.1 and bm 12 mutation sites overlap at amino acid 67, yec this close proximity fails to predict how the mutant cell will function in T-cell activation. Thus, although it is clear that there are multiple functional sites on the I-Ab molecule, it is not .-,A~..... +L.~. . . . .~o,, . . be further -'-'-:--" IJl,y~,~.c~.y ~,~,, o, +k ,,,=y . localized . . .U. .l l L. I.I . .[.r l ~ crystallographic szructure of the la molecule is known. Recent experiments using site-directed mutagenesis to derive variant A~ genes encoding all possible mutations of the three amino acid difference between A~ and Al~m'2 may provide information on local versus distant conformational effects of amino acid replacements on la function. These experiments demonstrated significant differences in the importance of individual amino acids for antibody versus T-cell discrimination of A~ and -,13Ab~U since only residue 70 was critical for mAb binding while the residues 67, 70 and 74 were all important in T-cell recognition (F. Ronchese and R. Germain, pets. commun.). These data are consistent with the theory that the bm12 mutation may not significantly affect the overall configuration of the la molecule and that its profound effect on T-cell recognition reflects a dire:t disruption of a structure critical to TCR-la or la--antigen binding. Sero!ogicallyselectedla mutant antigen-presentingcell lines A large panel of functional lak mutant APC lines has been derived from a (k x d)F1 B-B hybridoma (TA3) by negative and positive immunoselection using various lak-specific mAb. These include cell lines wih mutations in the A~ (Refs 28-34), A~ (Refs 30, 35, 36) and EL (Ref. 37) molecules. The sequences of some of these mutant genes are available and the site of these mutations with the name of the mutant cell line is shown in Fig. 5. The serologic and functional characterization of these

mutant cell lines with panels of mAb and antigen-specific T cells has provided much useful information which can be summarized as follows: (1) Tertiary conformation is critical for the format!on of some serological epitopes but not others. 19~ F~rf~;n nnn_r~lunnnrnhlr ~,¢-I ~,...1~.! i.uww i | v v l i ~ l T i •,v,

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The EL mutant cell line 2B1 (Fig. 5) has a histidine substituted for an arginine at the non-polymorphic residue 49 (Ref. 38), and this cell line does not bind the EL-specific mAb 17.3.3 (Ref. 37). The antibody-binding site cannot be localized only to residue 49, however, since inbred mice which lack reactivity to the 17.3.3 mAb share the arginine at residue 49 but differ from antibodybinding strains at residue 48. It is therefore critical but not sufficient for the formation of the 17.3.3 determinant, and adjacent residues probably contribute to the tertiary conformation of the Ei~ molecule and formation of this antibody-binding site. Two A~ mutant cell lines, 3J9 (Refs 36, 39) and JE67 (Ref. 40), produced independently, have the same mutation at residue 75 (Fig 5). But here the serologic epitope recognized by the mAb 39J appears to be localized to one residue (Glu 75) since this residue is found only in the single other haplotype (H-29 which binds to the 39J mAb 41. While it is possible that the binding site of the antibody is formed by a distal effect of residue 75, it is more likely that the physical location of this epitope can be directly assigned to residue 75.

277

Immunology Today, vok 8, No. 9, 1987

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(2) N o n - p ~ ' ~ c rescues The 2B 1 E~ mutant cell line described above is the only mutant line with an alteration in d non-polymorphic residue. This mutation does not impair antigen presentation to many diverse T-cell hybrids and clones 37. This mutant, therefore, accords with the finding of McDevitt and his co-workers that non-polymorphic residues which do not drastically affect tertiary conformation may not be involved in T-cell recognition. (3) ~amutant linesand Y-cellreceptordiversity

The studies described above using the site-directed mutant cell lines show that there are several functionally important epitopes on a single I-A molecule. However, since T cells of many antigenic specificities were used, they do not address the issues of whether a single antigen can be presented to the T cell in the context of multiple la determinants and whether several peptides could b~ presented in the context oi a single la molecule. To add,'~:~ -~his issue Allen et al. 36 used hen egg white lysozyme (HEU as a model system. They used a panel of HEL-specific I-Ak-restricted T-cell hybridomas together with the panel of AI~ and A~ mutant cell lines28-36 shown in Fig. 5 and proteolytic fragments of HEL and related synthetic peptides to show that the HEL-specific T cell recognized I-Ak in several distinct ways and responded to at least three distinct determinants on the HEL molecule. Combining the response patterns to the panel of I-Ak mutant APC lines with the antigen specificity revealed that the 10 T-cell hybrids tested recognize at last 8 unique determinants formed by the A~ chains, A~ chains and HEL peptides36, This analysis provides direct evidence that a large number of different determinants or TCR ligands can be generated from a single la molecule and a simple globular protein.

278

Condudom Attempts to localize sites on the class II molecules •~.-.m., int~rdCt wlm IL.I~ proteins and with toreign antigen have yielded much information which must still be regarded with healthy skepticism. Comr)arison of class II allelic polymorphisms together with exonshuffling experiments have confirmed the critical role of the highly polymorphic e=l and 131 domains in T-cell recognition. The role of the non-polymorphic regions of the ia molecule cannot be ignored, since recent experiments by Germain and co-workers indicate that, for an Ap molecule, additional contributions from the 132 domain and/or the paired A~ chain were necessary to produce the native struGure42,43. The production of la mutant cell lines by site-directed mutagenesis and serologic immunoselection suggests that the e~and 13chains of an la molecule interact to produce multiple sites which can differentially stimulate individual T lymphocytes. What remains unclear, however, is the extent to which ihe tertiary conformation of the la heterodimer affects the formation of these sites. The behaviour of certain mutant cell lines demands a critical role for three dimensional structure rather than linear sequence. Indeed results obtained to date highlight three-dimensional configurations (chain pairing and folding) in the creation of B-cell (mAb-binding) and T-cell recognition sites. Thus it is not possible to state definitively that there are multiple physically distinct regions on a single la molecule used by T cells or B cells since the apparent complexity

may be due to quantitative variations in the avidity for a single site. Indeed, recent studies from Gefter's laboratory~ suggest that each la molecule possesses only one binding site for all peptides with which it normally associates. Thus peptides capable of being presented by a given class II molecule compete with each other for T-cell activation. Such peptides bind to class II molecules through a region that shows homology to the class II molecule itself. The polymorphic regions of the class II molecules discussed above must therefore be involved in the binding of peptide to la. A definitive elucidation of the precise role of different sites on the la molecule in interaction with TCR molecules and in binding to antigen should be forthcoming from studies analysing recombinant soluble TCR molecules, recombinant, secreted class II molecules and well-characterized synthetic peptides which bind to class II molecules44.4s. This functional analysis together with the crystallographic structure of the wild-type and mutant class II molecules may ultimately provide us with a clearer picture of structure-function relationships. We thank Ronald Germain for his comments on this review. We are grateful to David McKean and Ronald Germain for communication of unpublished data. References

1 Rosenthal,A.S. and Shevach,E.M. (1973)J. Exp. Med. !38, 1194 2 Schwartz,R.HI'(1984)in Fundamental Immunology 37S-438, RavenPress 3 Kappler,J.W.,'Skidmore, B., White, J. and Marrack, P. (1981) J. Exp. Med. 15.~, 1198 4 Heber-Katz,E., Schwartz, R.H., rv;.~tis,L.A. etal. (1982) J. Exp. Med. 155, 1086 S Hunig,T.R.and Bevan, M.J. (1982)J. Exp. Med. 155, 111 § Choi, E., Mclntyre, I