Cell, Vol. 54, 47-56, July 1, 1988, Copyright © 1988 by Cell Press

Evidence That Multiple Residues on Both the R-Helices of the Class I MHC Molecule Are Simultaneously Recognized by the T Cell Receptor

R Ajitkumar,* S. S. Geier,*t K. V. Kesari,* F. Borriello,* M. Nakagawa,* J. A. Bluestone,$ M. A. Saper,§ D. C. Wiley,§ and S. G. Nathenson* * Department of Cell Biology Albert Einstein College of Medicine Bronx, New York 10461 University of Chicago

Ben May Institute/Department of Pathology Chicago, Illinois 60637 §Department of Biochemistry and Molecular Biology Howard Hughes Medical Institute Harvard University Cambridge, Massachusetts 02138

Summary

Single amino acid substitutions at nine different posi- tions on the H-2K b molecules from in vitro-mutage- nized, immunologically altered, somatic cell variants were correlated with their patterns of recognition by monoclonal antibodies (MAbs) and allogeneic cyto- toxic T lymphocyte (CTL) clones. While MAbs were found to detect spatially discrete, domain-specific sites, CTLs interacted simultaneously with multiple residues on the ~1 and a2 domains of the K b mol- ecule. The computer graphic three-dimensional K b model structure showed that, of the seven CTL-spe- cific residues analyzed, six residues were located on the R-helical regions of the two domains. Every CTL clone was found to interact with a distinct pattern of residues composed of a specific subset of the CTL- specific residues.

Introduction

The murine (H-2) major histocompatibility complex (MHC) class I molecules, K, D and L, are the classical transplan- tation antigens that mediate allogeneic tissue graft re- jection (Snell et al., 1976). They play a crucial role in the cellular immune response by functioning as the antigen- presenting molecules that enable cytotoxic T lymphocytes (CTLs) to distinguish foreign (non-self) antigens from self antigens. A foreign antigen, either viral, tumor, or chemi- cally modified, must be associated with the self MHC mol- ecule, K, D, or L, on the cell surface in order to be recog- nized by CTL as a non-self antigen, a phenomenon known as MHC-restricted recognition (Zinkernagel and Doherty, 1979). This recognition event is thought to be brought about by the binding of the T cell receptor to a bimolecular complex formed by the MHC molecule and foreign anti- gen. Current evidence suggests that foreign antigens can be presented in the form of peptides by the class I mole- cules for recognition by CTL (Townsend et al., 1986). Bio-

t Present address: Transplantation Laboratory, VA Medical Center, 508 Fulton Street, Durham, North Carolina 27705.

chemical studies have demonstrated that the class I mole- cules are expressed on the surface of almost all somatic cells as heterodimers composed of a 45,000 dalton poly- morphic glycoprotein (heavy chain) noncovalently associ- ated with a 11,600 dalton invariant polypeptide, 1~2-micro- globulin (light chain) (Nathenson, et al., 1981; Kimball and Coligan 1983). The heavy chain, which anchors the com- plex in the cell membrane, consists of three amino-termi- nal extracellular domains (designated (~1, (~2, and (~3), a hydrophobic transmembrane region, and a carboxy-termi- nal cytoplasmic portion (Nathenson et al., 1981).

A major question in understanding the cellular immune response concerns the identification of the structural fea- tures of the MHC molecule that are crucial for its recogni- tion by the T cell receptor. Functional studies on a series of mouse bm mutants (bm refers to the mutant of the H-2 b haplotype mouse) expressing K b molecules with multiple changes (K b molecule is the product of the K gene of the H-2 b haplotype mouse) have demonstrated that the (~1 and (~2 domains are crucial for recognition by CTL (Melief et al., 1980; Whitmore and Gooding, 1981; Sherman, 1982a; De Waal et al., 1983; Clark and Forman, 1983; Hur- witz et al., 1983). Since the structural changes that affect recognition in these mutants are confined mostly to the polypeptide stretches of amino acids 70 to 90 in the (~1 do- main and 150 to 180 in the c(2 domain, we have proposed that these two stretches of primary sequence form the "recognition regions" for CTL (Nathenson et al., 1986).

The usefulness of the in vivo mouse K b mutants for analyzing the role of specific amino acid residues involved in recognition by MAbs and CTL is limited due to the pres- ence of multiple substitutions in these two regions of the K b molecule. Therefore, we have developed an ex- perimental strategy to isolate a series of in vitro somatic cell variants expressing functionally altered K b molecules with single amino acid changes. The mutations were in- duced by chemical mutagenesis, and the mutant pheno- type was selected for loss of reactivity with Kb-specific MAbs (Geier et al., 1986) that had earlier been mapped approximately to the postulated CTL recognition regions on the K b molecule (Hammerling et al., 1982). The use of such a panel of MAbs provided us with K b mutants that were altered in MAb recognition as well as CTL recogni- tion, thereby permitting a comparison of features of MAb- and CTL-specific epitopes. Functional analysis of the K b mutants was carried out using a panel of allogeneic anti- K b CTL clones raised in bm mutant mice (Bluestone et al., 1986). In this study, we characterize the mutant K b molecules at the nucleic acid level, and correlate the structural alterations with their MAb and CTL reactivity patterns. The mutant K b molecules were found to have single amino acid substitutions in either the (~1 or the (~2 domain, except for one mutant that had a change in the (~3 domain. The importance of these amino acid residues for the recognition of the K b molecule by MAbs and CTL was evaluated with respect to their location on a three- dimensional K b model structure constructed from the

Cell 48

Table 1. Sequence Changes in the In Vitro K b Mutants

Position of Domain of Nucleotide Amino Acid Mutants Mutat ion Mutation Change Change

R8.24/246 80 (zl ACC to ATC Thr to Ite R8.208 82 ml CTC to CCC Leu to Pro R8.313 82 (zl CTC to TTC Leu to Phe R8.125/187 90 (zl GGC to GAC Gly to Asp R8.127 138 m2 ATG to AAG Met to Lys R8.14 141 m2 CTG to CGG Leu to Arg R8.33t 150 (z2 GCT to CCT Ala to Pro R8.341 162 m2 GGC to GAC Gly to Asp R8.332/347/ 166 m2 GAG to AAG Glu to Lys

354•360 R8.353 174 (z2 AAC to AAA Asn to Lys R8.205 192 ct3 CAC to CAG His to Gin

known coordinates of the HLA-A2 crystal structure (Bjork- man et al., 1987a) using computer graphic techniques.

The MAbs were found to be sensitive to changes of residues at discrete sites localized either to the a l or to the a2 domain. On the other hand, recognit ion by the anti- K b CTL clones was sensitive to substitutions in both do- mains. Further, each CTL clone was found to recognize a specif ic pattern of residues on the MHC molecule, as re- vealed by the point mutations analyzed.

Results and Discussion

Sequence Changes in the K b Mutants Sequence analysis was carried out on the K b mRNA from 13 of the 16 mutants chosen in this study (Table 1), as descr ibed by the strategy outl ined in Experimental Pro- cedures. Of the remaining three variants, the mutant K b gene from R8.313 has been cloned and sequenced (Nakagawa et al., 1986). The changes in the K b genes of R8.354 and R8.360 were determined by the hybridization of total mRNA with radiolabeled ol igonucleot ides specific for the mutant or native K b sequences (data not shown). A single nucleotide change, resulting in the substitution of a single amino acid, was found in each mutant K b mol- ecule, and these changes are listed in Table 1. A few sets of mutants had identical changes, although each one of them was independent ly isolated from a separately muta- genized pool of R8 cells (see Table 1).

Location of the Mutated Residues on the Three-Dimensional K b Model In order to locate the altered residues in a three- dimensional K b structure, a model structure of the ~1 and m2 domain port ion (residues 1-182) of the molecule was constructed by introducing those amino acid residues of the K b molecule which differed from the HLA-A2 se- quence into the crystal lographic structure of the HLA-A2 molecule (Bjorkman et al., 1987a) using the computer graphics program FRODO (Jones, 1982). The basic prem- ise for the construction of the model was that the three- dimensional structure of the or-carbon backbone and the side chain directions of the residues of the K b molecule would be the same as that of the HLA- A2 molecule. This contention is supported by the following facts: the K b

.-'" • '",.

, ............. i ° " //k.;:;'/ :" ........................ .U..... X Jll K '4//~ / 141 i /

~J/L%o I "-~ ....................

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Figure 1. Location of the Mutated Amino Acid Residues on a Schematic Representation of the H-2K b Model Structure

The K b model depicts the ml and a2 domain portion of the K b mole- cule. The amino-terminal is indicated by N, the I~-strands by flat arrow ribbons, and the m-helices by coiled ribbons. Residues 1-90 form the (zl domain, which is composed of the first four p-strands from the amino-terminal, and the (zl-helix (residues 58-84). The (z2 domain con- sists of residues 91-182, and is formed by the four 13-strands (immedi- ately following the (zl-helix), and the m2-helix (residues 138-180). The two (z-helices are separated by a groove that is the putative antigen binding site. Eight I~-strands, four from each domain, form the floor of the groove. Single amino acid substitutions are identified by one-letter codes, and their positions are indicated by numbers. The side chains shown are of the native K b residues that underwent substitution. (A) Groups of residues contributing to the epitopes recognized by the MAbs, EH-144 (Thr 80, Leu 82, Gly 90), Y-3 (Met 138, Leu 141, Ala 150), and 5F1-2-14 (Gly 162, Glu 166, Asn 174), are identified by the respec- tive circles. (B) An area of 30.~ by 20,~ (600.~2), encompassing the residues Leu 82 and Glu 166, which are recognized by nearly all of the CTL clones examined in this study, is identified by the rectangle superimposed on the m-helices (inclusive of the antigen binding site in between).

and the HLA-A2 molecules share the same number of residues and can be opt imal ly al igned without any gaps or insertions; the al igned sequences of the ~1 and ~2 domains of the two molecules show 73% identity, with an addit ional 7% conservative substitutions (Klein and Figueroa, 1986); and the patterns of polymorphic and con- served residues present in the R-helices of the ~1 and ~2 domains of the HLA-A2 molecule (Bjorkman et al., 1987b) are similar to those in the K b molecule, giving confi-

Immunodominant Sites on the H-2K b Molecule 49

Table 2. Monoclonal Antibody Binding Pattern on In Vitro-Selected K b Mutants

Monoclonal Antibody Reactivity Position of

Mutant Mutation EH-144 B8-24-3 K9-136 K9-178 28-13-3 5F1-2-14 Y-3

R8 - + + + + + + + R8.24/246 80 - + + + + + + R8.208 82 - - + + + + + R8.313 82 - - - + + + + R8.125/187 90 - + - + + + + R8.127 138 + + + + / - + + / - - R8.14 14t + + + + + + - R8.331 150 + + + + + + - R8.341 162 + + + + + + R8.332/347/ 166 + + + + + +

3541360 R8.353 174 + + + + + + R8.205 192 + + - + + + +

Serology notation: less than 10% of R8 (- ) ; 10% to 25% of R8 (+ / - ) ; more than 25% of R8 (+).

dence that the a-hel ices are or iented simi lar ly in both the molecules. This model makes no attempt to predict con- format ional changes of ne ighbor ing residues caused by the subst i tut ions of the side chains of the K b residues for those in the H LA-A2 structure. A schemat ic representat ion of the three-dimensional K b model structure is g iven in Figure 1. The sal ient features of the model include two a-hel ical regions, one formed by the a l domain residues 58 to 84 (a l -hel ix) and the other by the a2 domain residues 138 to 180 (a2-helix), and a groove (the putat ive ant igen binding site) between the a-helices. The residues in the a-helices, with side chains or iented towards the groove, form the sides of the ant igen binding site. The f loor of the site is formed by the side chains that project from the resi- dues on the central 13-strands of the antiparalle113-pleated sheet that run below the helices.

Residues that underwent subst i tut ion can be classif ied into the fol lowing groups based on their location and side chain or ientat ions: those on the exposed surface of the a-hel ices (Leu 82, Met 138, Ala 150, Gly 162, Glu 166, Asn 174), and hence presumed to interact direct ly with the T cell receptor (Bjorkman et al., 1987b); those with side chains project ing into the ant igen binding site, but located on the exposed surface of the a-hel ix (Thr 80); those with side chains point ing downward (toward the cell mem- brane) and away from the ant igen binding site and the a-hel ices (Leu 141); and those which are neither on nor near the a-hel ices (Gly 90, His 192).

Monoclonal Antibody Binding Patterns of the In Vitro K b Mutants A correlat ion of the posit ion and nature of the amino acid subst i tut ions in the R8 variants with their MAb binding profi les al lowed us to determine some of the residues that contr ibute to the ant igenic sites on the K b molecule for the MAbs EH-144, B8-24- 3, K9-136, 5F1-2-14, and Y-3. The results are summar ized in Table 2, and d iagrammed in Figure 1A. Binding of the MAb EH-144 to the K b mole- cule was affected by changes at Thr 80, Leu 82, and Gly 90. Al though the MAb B 8 - 2 4 - 3 was not sensit ive to the changes at Thr 80 or Gly 90, its binding was affected by

the subst i tut ions at Leu 82. Binding studies on cells from bm mutant mice (Hammer l ing et al., 1982) and formalde- hyde-f ixed H-2 b spleen (Hua et al., 1985) suggest that Lys 89 is also involved in the epi topes recognized by EH-144 and B8- 24-3. The f inding that nei ther of the two MAbs were sensit ive to subst i tut ions at posit ions 138, 141, 150, 162, 166, or 174 in the a2 domain, or at 192 in the a3 domain, is consistent with the conclusion that the resi- dues which contr ibute to the epi topes recognized by EH- 144 and B 8 - 2 4 - 3 are conf ined to the a l domain of the K b molecule.

The K9-136 MAb displayed a di f ference in its sensit iv i ty to the two subst i tut ions at Leu 82. Change of Leu to Phe, but not Leu to Pro, abol ished the binding of K9-136. Other residues whose subst i tut ions affected the binding of this MAb are Gly 90 and His 192. Since these two residues are separated by a distance of about 52, ~, in the K b model , it is diff icult to understand how the K b molecule is recog- nized by this antibody.

Recogni t ion of K b molecules by the 5F1-2-14 MAb was abol ished by changes at Gly 162, Glu 166, and Asn 174 (Table 2), whi le the binding by other MAbs was unaffected. Previous serological studies (Sherman and Randolph, 1981; Hammert ing et al., 1982; Bluestone et al., 1984a; Hua et al., 1985) showed that 5F1-2-14 MAb did not bind to cells expressing K b molecu les with changes at 152, 155, and 156 in bml mice (Weiss et al., 1983; Schulze et al., 1983), or with changes at 163, 165, 167, 173, and 174 in bm4 or bm l0 mice (Gel iebter et al., submitted). Taken together, these data indicate that the epi tope recognized by 5F1-2-14 is contr ibuted by at least the residues at 162, 166, and 174, as well as one or more of the residues at 152, 155, 156, 163, 165, 167, and 173.

Mutants that showed lack of b inding by Y-3 had changes at Met 138, Leu 141, and Ala 150 (Table 2). All the other MAbs showed binding to these mutant K b molecules. Earl ier studies had shown that changes in the K bin1 or the K brn4 molecule did not affect the binding of Y-3 (Hua et al., 1985). Thus, the ep i tope recognized by Y-3 appears to be contr ibuted by at least the residues Met 138, Leu 141, and Ala 150. Consistent with the mapping of the epi topes

Cell 50

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tt 190

~ 15o

50 ,o ~ ~

.m,o. .c. ,c,o.E, .o,oo..c,,c~o"~,, ~c 'L,,'o.:c,,c,o.,' ' ~, ' " . t

B

230 220 21G 20C 19(

18C 17¢

16C 15(3

12c U IlQ

~ oc

7¢

5C

4C

2O 10

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,c o , , , o , , 0 , , , , , . , , . . . . c , , c , o . , , " " ' " . 1 . " ' " ' " " . l . ' c . . I , , I , , ,, bm I0 oK b CTLCLONES bm II ~KbCTLCLONES

Figure 2. Effect of the Single Amino Acid Substitutions in the K b Molecule on CTL Reactivity

Cytotoxicity expressed as percent specific 51Cr release, was calculated from triplicate cultures of R8 variants. Relative lysis of the mutants as com- pared with the parental R8 cells (wild-type) was calculated from the formula:

CTL reactivity on variant Percent cytotoxicity index (CI) . . . . . . . . x 100

CTL reactivity on R8

The CTL reactivity on R8 cells was determined separately for each experiment for the calculation of CI. Mean and standard error were calculated from at least three separate experiments and plotted as a function of the position of the point mutations. The X-axis shows the individual CTL clones (represented by the clone numbers, demarcated by short vertical lines), and polyclonal population (represented by PC) of bm8, bml0, and bm11 origin. Each symbol (described below) in the graph represents substitution at a specific amino acid position. Statistical evaluation of the deviations in the CI values of the mutants from the CI value for R8 (the horizontal broken line) was carried out using the Student's t-test (see text). These devia- tions, as graphically represented in the figure, demonstrate the effect of the substitutions on the reactivity of the polyclonal and monoclonal CTL. (A) Effect of the substitutions at positions 80 (O), 82 with the substitution Pro (A) or Phe ( [ ] ) , and 90 ( • ) on the reactivity of each of the bm mutant anti-K b CTL clones and polyclonal CTL population. Effect of the substitutions on the polyclonal CTL population from bm8 was not tested. (B) Effect of the substitutions at positions 138 (O), 141 (A), 162 ([~), 166 ( • ) , and 192 ( I ) , on individual CTL clones and polyclonal CTL population. Effect of the substitution at position Ala 150 was not tested.

Immunodominant Sites on the H-2K b Molecule 51

Table 3. CTL Recognition Pattern of In Vitro-Selected K b Mutants

Positions of Origin of Altered Amino Acid Residues Anti-K b CTL Affecting CTL Recognition CTL Clone Clones Number ~1 domain

Positions of Altered Amino Acid Residues NOT Affecting CTL Recognition

c~2 domain ~3 domain al domain ~2 domain a3 domain

bm8

4 80 82P 82F 138 162 166 192 90 141 6 82F 138 166 80 82P 90 141 162 9 80 NT 82F 138" 162 NT NT 90 NT

10 82P NT NT NT 166 192 80 NT 141 11 NT 82F 138 162 166" 192 80 90 141 12 80 82F 138 166" 82P 90 141 162 13 82P 82F 138 166 192 80 90 141 162 14 82P 82F 90* 138 162 166 192 80 141 15 80* 82P 82F NT NT 166 192 90 141

192

192

5 80 82P 82F* 162 166 90 138 141 192 6 80* 82P 82F NT NT 90 138 141 192

37 80 82P* 82F 162 166 NT 90 138 141 bml0 38 80 82P* NT NT NT NT NT 138 141 42 80 82P 82F 162 166 90 138 141 192 47 82P 82F NT NT NT 80 90 NT 192

28 82F* 166" 80 82P 90 138 141 162 192 37 82P 82F 166" 80 90 138 141 162 192 bin11 39 82P 82F 192 80 90 138 141 162 166 41 82P 82F 138 NT NT 80 90 NT 162

82P and 82F indicate the substitution of Pro and Phe, respectively, for Leu 82. An (*) sign on the numbers, which indicates the positions of muta- tions, shows partial reactivity of a particular CTL clone on the mutant having a substitution at that position. NT: not tested.

for Y-3 and 5F1-2-14 MAbs is the f inding that the changes at 80, 82, and 90 did not affect their binding.

Monoclonal Antibodies Recognize Spatially Discrete, Domain-Specific Sites The MAbs EH-144, 5F1-2-14 and Y-3, which were used for the isolat ion of the somatic variants, selected mutants with al terat ions of amino acid residues clustered at discrete sites on the K b molecule, and these regions are out l ined in Figure 1A. The residues, Thr 80, Leu 82, and Gly 90, which contr ibute to the binding site detected by EH-144, are situated near the junct ion of the end of the a l -he l ix and the beginning of the ~-sheet segment of the a2 domain. The residues Gly 162, Glu 166, and Asn 174, which partici- pate in the format ion of the ant igenic site for 5F1-2-14, are located toward the carboxy-terminal of the a2-hel ix.

The Y-3-specific site, contr ibuted by the residues Met 138, Leu 141, and Ala 150, is at the opposi te end of the ~2- helix. Thus, all of the residues that were found to contr ib- ute to epi topes recognized by the MAbs used for mu- tant select ion are located at spat ial ly discrete, domain- specific, and surface-exposed regions of the K b molecule. These results are consistent with the fact that surface ex- posed port ions of protein molecules have high ant igenic potential (Benjamin et al., 1984; Novotny et al., 1986).

An assessment of the distance between the a-carbon atoms of the most distant residues that contr ibute to any part icular MAb-specif ic ep i tope can be used to est imate the min imum dimensions of the area of the discrete region recognized by that MAb. For example, in the case of the Y-3 epitope, the distance between the a-carbon atoms of Met 138 and Ala 150 is 21,~. This distance can be accom- modated by an an t igen-an t ibody interface area of approx-

imately 30~, by 20~, (600,~, 2) such as that observed in the an t igen-an t ibody complexes of nat ive proteins like lyso- zyme and influenza virus neuraminidase, which were stud- ied using X-ray crystal lographic methods (Amit et al., 1986; Sherif f et al., 1987; Co lman et al., 1987). Such esti- mates for the EH-144 and 5F1-2-14 MAbs were also found to be well within this specif ied area (data not shown).

AIIogeneic CTL Reactivity Patterns with the In Vitro K b Mutants The reactivity profi les of the anti-K b potyclonal and mono- clonal CTLs raised in the bm8, bin10 and bm11 mice on the MAb-selected R8 variants have been descr ibed previ- ously (Bluestone et al., 1986). In order to evaluate the quant i tat ive inf luence of single amino acid subst i tut ions in the K b molecules, we compi led these data and subjected them to statistical analysis. For this purpose, the cytotoxic- ity values of the CTLs on the var iants were measured rela- t ive to those on the parent R8 cells and expressed as per- cent cytotoxici ty index (CI; see legend to Figures 2A and 2B). The mean and standard error were calculated from at least three separate experiments. The CI of each CTL clone on each R8 var iant was plotted as a function of the dif ferent amino acid subst i tut ions (Figures 2A and 2B). The deviat ions in the CI values of the mutants from the CI value of parental R8 cells (the horizontal broken l ine in Figures 2A and 2B) were statist ically evaluated using the Student's t-test (Sokal and Rohlf, 1981). The CTL react ivi ty was considered to be severely affected when the CI values signif icant ly deviated from those of R8 (P < 0.05). However, in some cases, the CTLs were only part ia l ly af- fected (0.01 < P < 0.05). Table 3 presents a summary of these data showing the pattern of reactivi ty of the dif ferent

Cell 52

CTL clones as determined by the effect of the alteration of different residues in the K b molecule.

An examination of the pattern of reactivity of the anti-K b CTL clones on the R8 variants in the context of the se- quence information revealed several features of T cell rec- ognition of the MHC class I molecule. One of these was that substitutions of amino acid residues at certain posi- tions were found to affect the reactivity of almost all the CTL clones tested. For example, substitutions of Phe for Leu 82 and Lys for Glu 166 abolished the reactivity of nearly all the CTL clones of bin8, bml0, and bm11 origin (Figures 2A and 2B; Table 3). The only exception to this was bm11 CTL clone 39, which was not sensitive to the change at Glu 166. It is of interest to note that, unlike Phe 82, substitution of Pro for Leu 82 did not abrogate the re- activity of all the CTL clones. For instance, out of the 19 CTL clones tested, CTL clones 6 and 12 from bm8 and clone 28 from bin11 were not sensitive to the presence of Pro at position 82.

Only three CTL clones were tested for the change at po- sition 174. Two bml0 CTL clones, 37 and 47, and the bm11 CTL clone 28 showed loss of reactivity when Lys was sub- stituted for Asn 174 (data not shown). In our studies, whenever point mutations have affected CTL recognition, they have caused a decrease rather than an increase in CTL reactivity. However, two bin8 CTL clones, 13 and 14, reacted with the mutant having a change at Thr 80, with an affinity significantly greater than that for R8 cells (Fig- ure 2A). Residues, whose substitutions did not affect reac- tivity of nearly any of the CTL clones tested, were Gly 90 and Leu 141 (Figures 2A and 2B; Table 3). The only excep- tion to this was the bm8 CTL clone 14, which was partially sensitive to the change at Gly 90.

An interesting observation was that substitution of the ~3 domain residue His 192 affected CTL reactivity (Figure 2B; Table 3). Six out of eight bin8 CTL clones and one out of three bm11 CTL clones were sensitive to the change at His 192, although none of the bml0 CTL clones examined were affected by this substitution. From the analysis of the effect of substitutions of residues at nine positions on the recognition by 19 CTL clones, the residues Thr 80 and Leu 82 in the ~1 domain, Met 138, Gly 162, Glu 166, and Asn 174 in the e2 domain, and His 192 in the (~3 domain of the K b molecule were found to be important for CTL recogni- tion. The changes at Gly 90 (Gly to Asp) and Leu 141 (Leu to Arg) had no effect on T cell recognition. Reactivities of polyclonal CTL populations of bml0 and bm11 origin were not affected by any of the substitutions.

T Cell Receptor Interacts Simultaneously with Residues on Both c~-Helices The crucial information provided by the present study is that a number of residues from both the ~1 and ct2 do- mains are involved in the interaction with the T cell recep- tor. Of the seven CTL-specific residues analyzed (Thr 80, Leu 82, Met 138, Gly 162, Glu 166, Asn 174, and His 192), all except His 192 are located along the (~-helical regions of the two domains (Figure 1B). Every CTL clone tested (except bm11 CTL clone 39) was found to be sensitive to alterations of at least one residue each from the c~1- and

~2-helices (Table 3). For instance, recognition of the K b molecule by the bm8 CTL clone 4 was affected by changes at Thr 80 and Leu 82 in the ~l-helix, as well as those at Met 138, Gly 162, and Glu 166 in the c~2-helix. Similarly, the bin10 CTL clone 42 was sensitive to substitu- tions at Thr 80 and Leu 82 in the el-helix, and at Gly 162 and Glu 166 in the (z2-helix.

The finding that all of the CTL clones examined inter- acted with residues located on both ~1- and ~2-helices is consistent with the surface location of these contact residues. Further, the upward orientation of their side chains from the m-helices supports the idea that they inter- act directly with the T cell receptor (Figure 1B). In close concordance with this inference is the finding that neither Gly 90, which is not located on the c~-helix, nor Leu 141, with its side chain oriented away from the top surface of the ~2-helix and the antigen binding site, were important for recognition by the CTL clones examined. These obser- vations strongly suggest that in a single recognition event the T cell receptor interacts simultaneously with multiple amino acid residues on the al- and c~2-helical stretches of the class I molecule.

Although the examination of these MAb-selected mu- tants has confined our observations to residues at certain positions on the ~-helices, residues at other positions, such as the stretch of 58 to 79 in the cd-helix, could also be involved as contact sites for the T cell receptor. In fact, evidence for the involvement of the Arg 75 residue has al- ready been obtained with a mutant selection technique using CTL clones (P. Ajitkumar, S. G. Nathenson, and J. M. Shell, unpublished data). Since His 192 is located ap- proximately 50,~ below the a-helical surface, we conclude that this residue may not be directly involved in the interac- tion with the T cell receptor. The effect of the change at His 192 on T cell recognition might be indirect, and could be due to its influence on the binding of an accessory mol- ecule, such as Lyt-2. The finding that only certain CTL clones were sensitive to this change could be a reflection of the relative dependency of T cell-mediated cytolysis on accessory molecules, perhaps directly resulting from a lower avidity of these T cells for MHC molecules (Mac- Donald et al., 1982). This observation suggests that resi- dues that are not located on the ~-helices can also in- fluence the recognition process.

Similar to the assessment of the interface area for the antigen-antibody complex, we could estimate the poten- tial interface area of an MHC molecule-T cell receptor complex from the distance between two CTL-specific residues on the MHC molecule. An area of dimensions 30~, by 20,~ (600,&,2), which is equivalent to the interface area of an antigen-antibody complex (Amit et al., 1986), can accommodate the residues Leu 82 and Glu 166, which are recognized by nearly all of the CTL clones ex- amined (Figure 1B). However, our data suggest that the T cell receptor-MHC molecule interface could encompass a slightly larger area since the most distantly located CTL- specific residues, Leu 82 and Asn 174 reported in this study are separated by a distance of 44A which can be accommodated by an area of 39,~ by 20/~, (780,~2). The specific physical requirement that the T cell receptor

Immunodominant Sites on the H-2K b Molecule 53

should contact residues on both (~-helices necessitates such an extensive interface area. This feature of the inter- action may be correlated with the broader distribution of hypervariability in the variable regions of the T cell receptor (Patten et al., 1984; Davis, 1985; Kronenberg et al., 1986), in contrast to the three discrete areas of hyper- variability in the immunoglobulin molecule (Wu and Kabat, 1970).

An alternative explanation for the disruption of CTL rec- ognition of the mutant K b targets might be that the lack of recognition results from an alteration of peptide binding due to the substitution of residues on the (~-helices. How- ever, structural considerations based on the K b model make this explanation unlikely for the mutants examined in our study since the side chains of five of the six CTL- specific residues on the (~-helices are directed away from the antigen binding site. These residues should interact directly with the T cell receptor rather than cause an alter- ation in peptide binding. Thus, our studies were essen- tially focused on the structural features of the K b mole- cule involved in the direct interaction with the T cell receptor per se, rather than on those features, such as residues with side chains projecting into the antigen bind- ing site, that would influence peptide binding.

T Cell Receptor Recognizes a Distinct Pattern

of Residues on the MHC Molecule A major feature of T cell recognition revealed by these studies is that every CTL clone tested interacted with a specific subset of contact residues at Thr 80, Leu 82, Met 138, Gly 162, and Glu 166. For instance, recognition of the K b molecule by the bm8 anti-K b CTL clone 4 was affected by changes at Thr 80, Leu 82, Met 138, Gly 162, and Gtu 166 (Table 3). On the other hand, the CTL clone 13 of the same origin was sensitive to the same substitutions ex- cept for the changes at Thr 80 and Gly 162.

Furthermore, all of the CTL clones of a particular origin shared a common sensitivity or insensitivity to the change of a specific residue. For example, the change at Met 138 abolished the reactivity of all the CTL clones of bm8 origin, while none of the bml0 CTL clones were affected (Table 3). Similarly, the change at Thr 80 did not affect the reac- tivity of any of the bm11 CTL clones tested. Thus, with an analysis of the effect of ten substitutions on the reactivities of 19 CTL clones, we could differentiate between CTL clones of the same and different origins at the molecular level in terms of the distinct patterns of residues that they recognized on the K b molecule. However, irrespective of the genotypic differences between the MHC of the mouse in which CTL is raised and the alIo-MHC molecule, all of the allogeneic CTL clones appear to be programmed to in- teract with residues on the ~1- and ~2-helices of the alIo- MHC molecule.

Implicat ions Considerable structural homology between murine class I and class II MHC molecules has been documented (Hood et al., 1983; Germain and Malissen, 1986; Schwartz, 1986; Bjorkman et al., 1987b; Brown et al., 1988). In keeping with this structural similarity, both class

I and class II molecules have been found to present anti- gens in the form of peptides (Townsend et al., 1986; Bab- bitt et al., 1985; Buus et al., 1986; Guillet et al., 1987). Fur- thermore, there are several lines of evidence showing the expression of ~ and 13 polypeptide chains of the T cell receptor from the same pool of V(~ and VI3 gene segments in both cytotoxic and helper cells (Roehm et al., 1984; Acuto et al., 1985; Garman et al., 1986). In addition, the usage of identical T cell receptor VI3 genes by class I- and class II-restricted T cells also has been noted (Rupp et al., 1985). Considering these observations in the context of our studies, one can make a reasonable prediction that class II allorecognition would be governed by the interac- tion of the T cell receptor of the helper cell with a specific extensive pattern of residues on the class II MHC mole- cule, in a manner similar to that recognized on the class I MHC molecule.

Assuming that in a single recognition event one T cell receptor recognizes one MHC molecule, a simple view of the interaction implied by our studies is that as the T cell receptor simultaneously contacts residues on the two (~-helices, it must also interface with the foreign or self peptide in the putative antigen binding site between the helices. Although there is no direct evidence for the in- volvement of peptides in allogeneic recognition, the oc- currence of H-2K b mouse mutants, bm5, bm6, bm7, bin8, and bm9 identified by skin graft incompatibility (T cell- mediated), is consistent with such a possibility. The K b molecules from these mutants have changes exclusively at the residues, Tyr 22 (Tyr to Phe), Glu 24 (Glu to Ser), and Tyr 116 (Tyr to Phe) (Nathenson et al., 1986), whose side chains project from the 13-strands on the floor of the groove into the antigen binding site (Bjorkman et al., 1987b). Tak- ing the peptidic self model (Claverie and Kourilsky, 1986) into consideration, such substitutions in a K b molecule could potentially cause the binding of a set of self peptides different from that presented by the wild-type K b mole- cule and thereby evoke an allogeneic response. The residues on the (~-helices of these mutant K b molecules should not have evoked an allogeneic response since they are identical to those in the wild-type K b molecule. Consistent with this idea is the finding that K b molecules from all of the T cell-selected H-2K b mouse mutants have at least one altered residue with its side chain projecting into the antigen binding site (Bjorkman et al., 1987b). All of these observations taken together imply that in al- Iogeneic recognition the T cell recognizes a pattern formed by the residues on the (~-helices and on a peptide between the helices, in a manner which may be presumed to be the basis for antigen-specific recognition as well. In conformity with this idea is the demonstration (Bevan, 1977; Finberg et al., 1978; Pfizenmaier et al., 1980; Braciale et al., 1981; Ashwell et al., 1986; Shell et al., 1987) that a single T cell clone can have antigen-associated rec- ognition as well as allogeneic recognition properties.

Experimental Procedures

Isolation and Serological Characterization of the H-2K b In Vitro Mutants Isolation and serological characterization of the somatic cell variants

Cell 54

of the heterozygous (B6 x BALB/c)F1 (H-2 b x H-2 d) Abelson virus- transformed pre-B cell line R8 has been described in detail (Geier et al., 1986). Briefly, R8 cells were mutagenized with either Ethyl- methanesulphonate (EMS) or Ethylnitrosourea (ENU). Three classes of R8 cells were detected. These included wild-type cells with no struc- tural changes in K b, loss mutants with no detectable surface expressed product, and structural variants with an altered K b phenotype. Wild- type cells were eliminated by a negative selection with one of the three anti-H-2K b MAbs, EH-144, 5F1-2-14, or Y-3, in the presence of rabbit complement. The surviving K b mutants were subjected to a positive selection using a mixture of Kb-specific MAbs on a fluorescence- activated cell sorter (FACS). The H-2K b somatic cell variant population thus selected was cloned, and individual clones were serologicalty characterized using a panel of Kb-specific MAbs, EH-144, B8-24-3, K9-136, K9-178, 28-13-3, 5F1-2-14, and '(-3.

Cytotoxic T Cell Assay Establishment of primary anti-K b cytotoxic T lymphocyte cultures from bm8, bml0, and bin11 mice, and the isolation, characterizat!on, main- tenance, and the assay of CTL clones derived from these cultures have been described in detail (Bluestone et al., 1986).

The rationale for raising CTLs in bm mutant mice (expressing K b molecules with multiple, but limited substitutions) was that since the M HC is known to influence the specificity of the alloreactive CTL reper- toire (Sherman, 1982b), the anti-K b CTL response of bm mutant mice could be expected to be less heterogeneous than that of mice from a different haplotype (Bluestone et al., 1984b). Cytolytic activity was measured using the 51Cr release assay. Percent specific 51Cr release was calculated in triplicate cultures from the formula:

CPMex p - CPMcontro I x 100 CPMtotal - CPMcontrol

It was established that all of the R8 variants used could be lysed by H-2Kd-specific CTL, and all CTL used in this study were H-2K b- spe- cific and did not react with H-2D b or H-2 d (for details see Bluestone et al., 1986). Lysis on the variants was expressed as percent of lysis on R8 by using a mean of at least three separate experiments (see legend to Figures 2A and 2B).

Preparation of Total Poly (A) + RNA from Mutant Cells The total RNA from about 10 l° cells was extracted at 60°C using phe- nol saturated with 100 mM sodium acetate buffer (pH 5.2) containing 1 mM EDTA (Soeiro and Darnell, 1969). Total poly (A) + RNA was pre- pared by oligo(dT)-cellulose column chromatography (Aviv and Leder, 1972), and washed once with 1 M lithium chloride solution.

Nucleic Acid Sequence Analysis of the K b Mutants In anticipation of the need to sequence a large number of mutant K b genes, we employed an RNA sequencing method previously estab- lished in our laboratory to analyze K b genes from mouse H-2K b mu- tants (Geliebter et al., 1987). In this method, the substitutions in the K b

molecules were determined directly at the total poly (A) + RNA level without the necessity of cloning the mutant K b genes. Sequencing was carried out, using end-labeled K b gene-specific oligonucleotides as primers, in the presence of deoxy and dideoxynucleotide tri- phosphates and reverse transcriptase. In order to confirm the muta- tions thus determined, we used an alternate sequencing approach. First, the cDNA for the K b mRNA was synthesized from the total poly (A) + RNA population using a Kb-specific, unlabeled oligonucleotide as primer. The uncloned cDNA for the K b mRNA was then sequenced by the dideoxy sequencing method (Sanger et aL, 1977). A number of refinements, however, were necessary in order to approach the analy- sis of the heterozygous tissue culture cell line R8 (H-2 b x H-2d), in part due to the very low K b mRNA content of the cell lines, as well as to the complications arising due to cross-hybridization of the H-2 d haplotype class I mRNAs.

in a typical sequencing reaction, 40 p,g of total poly (A) + RNA in 20 p.I of 10 mM Tris-HCI buffer (pH 8.3), containing 250 mM KCl, and 1 mM EDTA, was annealed at the appropriate hybridization temperature with 10 ng (in 1 pl) of 32p-labeled Kb-specific oligonucleotide for 1 hr. Four microliters each of the annealed RNA solution was added to five tubes, each containing 4 111 of reverse transcription buffer of the following composition: 100 mM Tris-HCl (pH 8.3), 20 mM each of MgCl2 and di-

thiothreitol, 1.25 mM each of dATP, dCTP, dGTP, and dTTP (PL Bio- chemicals), 33 ng of actinomycin D (Sigma Chemicals), and 14 U (21 U/p.I) of avian myeloblastosis virus reverse transcriptase (Life Sciences). In addition, each tube contained 2 #1 of one of the four ddNTPs (PL Biochemicals). The ddNTP concentrations were 1.66 mM ddATP, 0.4 mM ddCTP, 0.625 mM ddGTP, and 2 mM ddTTP. The reaction was allowed to proceed for 48 min at 50°C in order to avoid cross-hybridization of the K b mRNA with other class I mRNAs. One microliter of 10 mM EDTA solution in formamide, containing bromo- phenol blue and xylene cyanol dyes, was added to terminate the reac- tion. Half the sample from each tube was heated for 3 min in a boiling water bath, and subjected to electrophoresis on an 8% polyacrylamide gel (one meter in length) containing 7 M urea.

Owing to the considerable length of time and effort to sequence each K b mutant in all three external domains, a sequencing strategy was adopted as follows: chosen variants from each group of mutants, selected by the three selecting MAbs (EH-144, 5F1-2-14, and Y-3), were subjected to sequence analysis of the exons 2, 3, and 4 (corre- sponding to el, 0t2, and ~3 domains). These included the variants R8.24 (change at Thr 80), R8.127 (change at Met 1,38), R8.341 (change at Gly 162), R8. 353 (change at Asn 174), and R8.205 (change at His 192). The variant R8.313 gene (change at Leu 82) was cloned and se- quence determined for the exons 1 (leader sequence), 2, 3, 4, and 5 (transmembrane region) (Nakagawa et at., 1986). For none of these genes was an additional sequence change detected, except for the sin- gle base change noted in Results and Discussion. Nor was a second nucleotide sequence alteration ever noted in the sequence analysis of three additional mutants, R8 clone 15 and R8.10 (selected by MAbs), and R8.110.49 (selected by CTL; our unpublished results). Moreover, since the mutation rate for these structural variants was 1 in 106, one could anticipate that occurrence of a second mutation at the same lo- cus at a frequency of 1 in 1012 would be very unlikely. Therefore addi- tional K b mutants, R8.246, R8.208, R8.125, R8.187, R8.14, R8.331, R8.347, and R8.335, were sequenced for the exon containing the single nucleotide change only.

Acknowledgments

The authors gratefully acknowledge the outstanding technical as- sistance received from Joanne Trojnacki, Diane McGovern, and Ruchika naval. We thank Edith Palmieri-Lehner for efficient and prompt synthesis of a series of oligonucleotides used in this study, and Catherine Whetan for secretarial assistance. P. Ajitkumar was a recipi- ent of a postdoctoral fellowship from Cancer Research Institute, New York, during these studies. K. V. Kesari is supported by a postdoctoral fellowship from Cancer Research Institute, New York, and F. Borriello by an MSTP grant NIH T32 GM7288. R A. thanks Jan Geliebter for helpful suggestions, and Kim Hasenkrug for critical reading of the manuscript. The authors are grateful to James Shell and Margaret Kie- lian for thoughtful comments on the manuscript. The studies were sup- ported in part by Public Health Service grants, A1-10702, AI-07289, and NCI P30-CA13330 from the National Institutes of Health, and IM-236 from the American Cancer Society, to Stanley G. Nathenson who is a member of the Irvington House Institute for Medical Research, New York.

The costs of publication of this manuscript were defrayed in part by the payment of page charges. This manuscript must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. Section 1734 solely to indicate this fact.

Received March 11, 1988; revised April 5, 1988.

References

Acuto, O., Campen, T. J., Royer, H. D., Hussey, R. E., Poole, C. B., and Reinherz, E. L. (1985). Molecular analysis of T cell receptor (Ti) vari- able region (V) gene expression. Evidence that a single Ti 13 V gene family can be used in formation of V domains on phenotypically and functionally diverse T cell populations. J. Exp. Med. 161, 1326-1343.

Amit, A. G., Mariuzza, R. A., Phillips, S. E. V., and Poljak, R. J. (1986). Three-dimensional structure of an antigen-antibody complex at 2.8A resolution. Science 233, 747-753.

Ashwell, J, D., Chen, C., and Schwartz, R. H. (1986). High frequency

Immunodominant Sites on the H-2K b Molecule 55

and nonrandom distribution of alloreactivity in T cell clones selected for recognition of foreign antigen in association with self class II mole- cules. J. Immunol. 136, 389-395.

Aviv, H., and Leder, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69, 1408-1412.

Babbitt, B. R, Allen, R M., Matsueda, G., Haber, E., and Unanue, E. R. (1985). Binding of immunogenic peptides to la histocompatibility mole- cules. Nature 317, 359-361.

Benjamin, D. C., Berzofsky, J. A., East, I. J., Gurd, R R. N., Hannum, C., Leach, S.J., Margoliash, E., Michael, J.G., Miller, A., Prager, E. M., Reichlin, M., Sercarz, E. E., Smith-Gill, S. J., Todd, P. E., and Wilson, A. C. (1984). The antigenic structure of proteins: a reappraisal. Annu. Rev. Immunol. 2, 67-101.

Bevan, M. J. (1977). Killer cells reactive to altered-self antigens can also be alloreactive. Proc. Natl. Acad. Sci. USA 74, 2094-2098.

Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L., and Wiley, D. C. (1987a). Structure of the human class I histocom- patibility antigen, HLA-A2. Nature 329, 506-512.

Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J. L., and Wiley, D. C. (1987b). The foreign antigen binding site and T cell recognition regions of class I histecompatibility anti- gens. Nature 329, 512-518.

Bluestone, J A., McKenzie, I. R C., Melvold, R. W., Ozato, K., Sandrin, M. S., Sharrow, S. O., and Sachs, D. H. (1984a). Serological analysis of H-2 mutations using monoclonal antibodies. J. Immunogenet. 11, 197-207.

Bluestone, J. A., Palman, C., Foe, M., Geier, S. S., and Nathenson, S. G. (1984b). Analysis of major histocompatibility complex class I anti- gens using in vitro-derived somatic cell mutants. In Regulation of the Immune System, UCLA Symposium on Molecular and Cellular Biol- ogy New Series, Volume 18, R Sercarz, H. Cantor, and L. Chess, eds. (New York: Alan R. Liss, Inc.), pp. 89-97.

Bluestone, J. A., Langlet, C., Geier, S. S., Nathenson, S. G., Foo, M, and Schmitt-Verhulst, A.-M. (1986). Somatic cell variants express al- tered H-2K b allodeterminants recognized by cytolytic T cell clones. J. Immunol. 137, 1244-1250.

Braciale, 3-. J., Andrew, M. E, and Braciale, V. L. (1981). Simultaneous expression of H-2-restricted and alloreactive recognition by a cloned line of influenza virus-specific cytotoxic T lymphocytes. J. Exp. Med. 153, 1371-1376.

Brown, J. H., Jardetzky, T., Saper, M., Samroi, B., Bjorkman, R J., and Wiley, D. C. (1988). A hypothetical model of the foreign antigen binding site of class 11 histocompatibility molecules. Nature 332, 845-850.

Buus, S., Colon, S., Smith, C., Freed, J. H., Miles, C., and Grey, H. M. (1986). Interaction between a "processed" ovalbumin peptide and la molecules. Proc. Natl. Acad. Sci. USA 83, 3968-3971.

Clark, S. S., and Forman, J. (1983). AIIogeneic and associative recog- nition determinants of H-2 molecules. Transplant. Proc. 15, 2090-2092.

Claverie, J.-M., and Kourilsky, R (1986). The peptidic self model: a reassessment of the rote of the major histocompatibility complex mole- cules in the restriction of the T cell response. Annu. Inst. Pasteur/ Immunol. 137D, 425-442.

Colman, P. M., Laver, W. G, Varghese, J. N, Baker, A. T, Tultoch, R A., Air, G. M., and Webster, R. G. (1987). Three-dimensional struc- ture of a complex of antibody with influenza virus neuraminidase. Na- ture 326, 358-363.

Davis, M. M. (1985). Molecular genetics of the T cell receptor beta chain. Annu. Rev. Immunol. 3, 537-560.

De Waal, L. P., Nathenson, S. G., and Melief, C. J. M. (1983). Direct demonstration that cytotoxic T lymphocytes recognize conformational determinants and not primary amino acid sequences. J. Exp. Med. 158, 1720-1726.

Finberg, R., Burakoff, S. J., Cantor, H., and Benacerraf, B. (1978). Bio- logical significance of alloreactivity: T cells stimulated by Sendai virus- coated syngeneic cells specifically tyse allogeneic target cells. Proc. Natl. Acad. Sci. USA 75, 5145-5149.

Garman, R. D., Ko, J.-L., Vulpe, C. D., and Raulet, D. H. (1986). T cell

receptor variable region gene usage in T cell populations. Proc. Natl. Acad. Sci. USA 83, 3987-3991.

Geier, S. S., Zeff, R. A., McGovern, D. M., Rajan, T. V., and Nathenson, S. G. (1986). An approach to the study of structure-function relation- ships of MHC class I molecules: Isolation and serologic characteriza- tion of H-2K b somatic cell variants. J. Immunol. 137, 1239-1243. Geliebter, J. (1987). Dideoxynucleotide sequencing of RNA and un- cloned cDNA. Focus 9, 5-8.

Germain, R. N., and Malissen, B. (1986). Analysis of the expression and function of class II major histocompatibility complex-encoded mol- ecules by DNA-mediated gene transfer. Annu. Rev. Immunol. 4, 281-315.

Guillet, J.-G., Lai, M.-Z., Briner, T. J., Buus, S., Sette, A., Grey, H. M., Smith, J. A., and Getter, M. L. (1987). Immunological self, nonself dis- crimination. Science 235, 865-870.

Hammerling, G. J., Rusch, E., Tada, N., Kimura, S., and Hammerling, U. (1982). Localization of allodeterminants on H-2K b antigens deter- mined with monoclonal antibodies and H-2 mutant mice. Proc. Natl. Acad. Sci. USA 79, 4737-4741.

Hood, L, Steinmetz, M., and Malissen, B. (1983). Genes of the major histocompatibility complex of the mouse. Annu. Rev. Immunol. 1, 529-568.

Hua, C., Langlet, C., Buferne, M., and Schmitt-Verhulst, A. -M. (1986). Selective destruction by formaldehyde fixation of an H-2K b serological determinant involving lysine 89 without loss of T cell reactivity. Im- munogenet. 21, 227-234.

Hurwitz, J. L, Pan, S., Wettstein, P.J., and Doherty, P.C. (1983). Cross-reactivity patterns of vaccinia-specific cytotoxic T lymphocytes from H-2K b mutants. Immunogenet. 17, 79-87.

Jones, T. A. (1982). FRODO: a graphics fitting program for macro- molecules. In Computational Crystallography, D. Sayre, ed. (Oxford: Clarendon Press), pp. 303-317.

Kimball, E. S., and Coligan, J. E. (1983). Structure of class I major histocompatibility antigens. Contemp. Top. Mol. Immunol. 9, 1-63.

Klein, J., and Figueroa, R (1986). The evolution of class I MHC genes. Amino acid sequences of class I MHC molecules from mouse, rat, rab- bit and man. Immunol. Today 7, No. 2, the center page diagram.

Kronenberg, M., Siu, G., Hood, L. E., and Shastri, N. (1986). The mo- lecular genetics of the T cell antigen receptor and T cell antigen recog- nition. Annu. Rev. Immunol. 4, 529-591.

MacDonald, H. R., Gtasebrook, A. L, Bron, C., Kelso, A., and Cerot- tini, J.-C. (t982). Clonal heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T lymphocytes (CTL): possible implica- tions for the affinity of CTL antigen receptors. Immunol. Rev. 68, 89-115.

Metief, C. J. M., De Waal, L. P., Van Der Meuten, M. Y., Melvold, R. W., and Kohn, H. I. (1980). Fine specificity of alloimmune cytotoxic T lym- phocytes directed against H-2K. A study with K b mutants. J. Exp. Med. 151, 993-1013.

Nakagawa, M., Zeff, R. A., Geier, S. S., Bluestone, J. A., and Nathen- son, S. G. (1986). Somatic cell variants of H-2Kb: a point mutation in the first extracellular domain results in altered immune recognition. Im- munogenet. 24, 381-385.

Nathenson, S. G., Uehara, H., Ewenstein, B. M., Kindt, T. J., and Coli- gan, J. E. (t981). Primary structural analysis of the transplantation anti- gens of the murine H-2 major histocompatibility complex. Annu. Rev. Biochem. 50, 1025-1052.

Nathenson, S. G., Geliebter, J., Pfaffenbach, G. M., and Zeff, R. A. (1986). Murine major histocompatibility complex class I mutants: mo- lecular analysis and structure-function implications. Annu. Rev. Im- munol. 4, 471-502.

Novotny, J., Handschumacher, M., Haber, E., Bruccoleri, R. E., Carl- son, W. B., Fanning, D. W., Smith, J. A., and Rose, G. D. (1986). Anti- genic determinants in proteins coincide with surface regions accessi- ble to large probes (antibody domains). Proc. Natl. Acad. Sci. USA 83, 226-230.

Patten, P., Yokota, T., Rothbard, J., Chien, Y., Arai, K., and Davis, M. M. (1984). Structure, expression and divergence of T cell receptor 6-chain variable regions. Nature 312, 40-46.

Cell 56

Pfizenmaier, K., Stockinger, H., Rollinghoff, M., and Wagner, H. (1980). Herpes-simplex-virus-specific, H-2Dk-restricted T lymphocytes bear receptors for H-2D d alloantigen. Immunogenet. 11, 169-176.

Roehm, N., Herron, L., Cambier, J., DiGuisto, D., Haskins, K., Kappler, J., and Marrack, P. (1984). The major histocompatibility complex- restricted antigen receptor on T cells: distribution on thymus and pe- ripheral T cells. Cell 38, 577-584.

Rupp, E, Acha-Orbea, H., Hengartner, H., Zinkernagel, R., and Joho, R. (1985). Identical V~ T cell receptor genes used in alloreactive cyto- toxic and antigen plus I-A specific helper T cells. Nature 315, 425-427.

Sanger, E, Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463- 5467.

Schulze, D. H., Pease, L. R., Geier, S. S., Reyes, A. A., Sarmiento, L. A., Wallace, R. B., and Nathenson, S. G. (1983). Comparison of the cloned H-2K brnl variant gene with the H-2K b gene shows a cluster of seven nucleotide differences. Proc. Natl. Acad. Sci. USA 80, 2007- 2011.

Schwartz, R. H. (1986). Immune response (Ir) genes of the murine ma- jor histocompatibility complex. Adv. Immunol. 38, 31-201.

Sheil, J. M., Bevan, M. J., and Lefrancois, L. (1987). Characterization of dual-reactive H-2Kb-restricted anti-vesicular stomatitus virus and al- Ioreactive cytotoxic T cells. J. Immunol. 138, 3654-3660.

Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H:, Smith-Gill, S. J., Finzel, B. C., and Davies, D. R. (1987). Three-dimensional struc- ture of an antibody-antigen complex, Proc. Natl. Acad. Sci. USA 84, 8075-8079.

Sherman, L. A. (1982a). Recognition of conformational determinants on H-2 by cytotytic T lymphocytes. Nature 297, 511-513.

Sherman, L. A. (1982b). Influence of the major histocompatibility com- plex on the repertoire of atlospecific cytolytic T lymphocytes. J. Exp. Med. 155, 380-389.

Sherman, L. A., and Randolph, C. P. (1981). Monoclonal anti-H-2K b antibodies detect serological differences between H-2K b mutants. Im- munogenet. 12, 183-186.

Snell, G. D., Dausset, J., and Nathenson, S. G. (1976). Histocompatibil- ity (New York: Academic Press).

Soeiro, R., and Darnell, J. E. (1969). Competition hybridization by "pre- saturation" of HeLa cell DNA. J. Mol. Biol. 44, 551-562.

Sokal, R. R., and Rohlf, E J. (1981). Biometry. The principles and prac- tice of statistics in biological research (San Francisco: W. H. Freeman and Company).

Townsend, A. R. M., Rothbard, J., Gotch, E M., Bahadur, G, Wraith, D., and McMichael, A. J. (1986). The epitopes of influenza nucleopro- tein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 44, 959-968.

Weiss, E. H., Mellor, A., Golden, L., Fahrner, K., Simpson, E., Hurst, J., and Flavell, R. A. (1983). The structure of a mutant H-2 gene sug- gests that the generation of polymorphism in H-2 genes may occur by gene conversion-like events. Nature 301, 671-674. Whitmore, A. C., and Gooding, L. R. (1981). Specificities of killing by T lymphocytes generated against syngeneic SV40 transformants: Cross-reactivity of the H-2K bin1 through K bin4 H-2 mutant alleles in Kb-restricted SV40-specific killing. J. Immunol. 127, 1207-1211.

Wu, T. T., and Kabat, E. A. (1970). An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132, 211-250. Zinkernagel, R. M., and Doherty, IR C. (1979). MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplanta- tion antigens determining T cell restriction-specificity, function, and re- sponsiveness. Adv. Immunol. 27, 51-77.