8
Immunogenetics 34: 149-156, 1991 11 1B1111o- genetics © Springer-Verlag 1991 Limited regions of the ¢ 2-domain a-helix control anti-A2 allorecognition: an analysis using a panel of A2 mutants Giovanna Lombardi 1, Masanori Matsui 2, Robert Moots 3, Gerald Aichinger 1, Sid Sidhu 1, Richard Batchelor 1, Jeff Frelinger 3, and Robert Lechler 1 t Department of Immunology, Royal Postgraduate Medical School, London, England 2 Department of Microbiology and Immunology, University of North Carolina, USA 3 Institute of Molecular Medicine, Oxford University, England Received March 19, 1991; revised version received May 10, 1991 Abstract. The regions of the HLA-A2 molecule controll- ing anti-A2 alloreactivity were explored using naturally occurring allelic variants of HLA-A, and a panel of transfectants expressing the products of A2.1 genes that had been mutated at multiple positions encoding residues in the a 2 domain a-helix. As a means of detecting distant conformational effects, these altered A2.1 molecules were also examined serologically. Amino acid substitutions at the carboxy-terminal end of the a 2 domain a-helix led to diminished staining with the monoclonal antibody (mAb) MA2.1. The epitope for this antibody has previously been mapped to the a t domain a-helix (residues 62-65). This suggests that interdomain contacts may cause conforma- tional alteration, and that mutants can have distant, as well as local effects. Of the 24 positions where substitutions were made, only six led to loss of the anti-A2 alloresponse by the three clones and three lines that were tested. In addition, the mutations that altered the MA2.1 epitope, located on the c~ 1 domain c~-helix, did not inhibit allorecognition. This suggests that a limited number of regions on the A2.1 molecule are responsible for allodeterminant expression. The most influential substitu- tions were those at positions 152, 154, 162, and 166. It is notable that three of these are predicted to be T-cell receptor (Tcr)-contacting residues, and one (152) to con- tribute to peptide binding. These results suggest that the specificity of alloreactive T cells is determined by exposed polymorphisms, directly contacted by the Tcr, and by concealed polymorphisms which influence pepfide binding. Address correspondence and offprint requests to: R. I. Lechler, Depart- ment of Immunology, Royal Postgraduate Medical School, Du Cane Road, London, W12 ONN, England. Introduction Class I major histocompatibility complex (MHC) mole- cules function as recognition structures for MHC- restricted and alloreactive T lymphocytes. However, the relationship between MHC-restricted and allospecific T- cell recognition has been an enigma for many years. It has become increasingly clear that the T-cell repertoire is positively selected, in the thymus, for self MHC-restric- tion (Zinkernagel 1978; Kisielow et al. 1988). It is also generally agreed that the alloreactive and MHC-restricted T-cell repertoires are entirely overlapping (Matis et al. 1987; Lombardi et al. 1989a; Lechler et al. 1990). Insights about the molecular architecture of MHC molecules, and about how the structure of these molecules subserves their functions in T-cell recognition have ac- cumulated in recent years. This has furthered our understanding of how MHC-restricted T cells can simultaneously display alloresponses against foreign MHC products. These insights have arisen from the com- bined application of structural and functional approaches. Using serological and cellular read-outs, the effects of spontaneous and artificially induced variants have been investigated. Since the three-dimensional structure of HLA-A2 was solved, it has been possible to interpret the results of such studies (Bjorkman et al. 1987). Early models of allorecognition suggested that alloreactive T cells, like alloantibodies, recognize directly polymorphic residues on the exposed surface of foreign MHC molecules. More recent experimental evidence has emphasized the key contribution of MHC-bound peptides to allorecognition (Mentzer et al. 1986; Bernhard et al. 1987; Lombardi et al. 1989b; Heath et al. 1989). The hypothesis that alloreactive T cells are specific for binary complexes composed of allogeneic MHC

Limited regions of the α2-domain α-helix control anti-A2 allorecognition: an analysis using a panel of A2 mutants

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Immunogenetics 34: 149-156, 1991 11 1B1111o- genetics

© Springer-Verlag 1991

Limited regions of the ¢ 2-domain a-helix control anti-A2 allorecognition: an analysis using a panel of A2 mutants

Giovanna Lombardi 1, Masanori Matsui 2, Robert Moots 3, Gerald Aichinger 1, Sid Sidhu 1, Richard Batchelor 1, Jeff Frelinger 3, and Robert Lechler 1

t Department of Immunology, Royal Postgraduate Medical School, London, England 2 Department of Microbiology and Immunology, University of North Carolina, USA 3 Institute of Molecular Medicine, Oxford University, England

Received March 19, 1991; revised version received May 10, 1991

Abstract. The regions of the HLA-A2 molecule controll- ing anti-A2 alloreactivity were explored using naturally occurring allelic variants of HLA-A, and a panel of transfectants expressing the products of A2.1 genes that had been mutated at multiple positions encoding residues in the a 2 domain a-helix. As a means of detecting distant conformational effects, these altered A2.1 molecules were also examined serologically. Amino acid substitutions at the carboxy-terminal end of the a 2 domain a-helix led to diminished staining with the monoclonal antibody (mAb) MA2.1. The epitope for this antibody has previously been mapped to the a t domain a-helix (residues 62-65). This suggests that interdomain contacts may cause conforma- tional alteration, and that mutants can have distant, as well as local effects. Of the 24 positions where substitutions were made, only six led to loss of the anti-A2 alloresponse by the three clones and three lines that were tested. In addition, the mutations that altered the MA2.1 epitope, located on the c~ 1 domain c~-helix, did not inhibit allorecognition. This suggests that a limited number of regions on the A2.1 molecule are responsible for allodeterminant expression. The most influential substitu- tions were those at positions 152, 154, 162, and 166. It is notable that three of these are predicted to be T-cell receptor (Tcr)-contacting residues, and one (152) to con- tribute to peptide binding. These results suggest that the specificity of alloreactive T cells is determined by exposed polymorphisms, directly contacted by the Tcr, and by concealed polymorphisms which influence pepfide binding.

Address correspondence and offprint requests to: R. I. Lechler, Depart- ment of Immunology, Royal Postgraduate Medical School, Du Cane Road, London, W12 ONN, England.

Introduction

Class I major histocompatibility complex (MHC) mole- cules function as recognition structures for MHC- restricted and alloreactive T lymphocytes. However, the relationship between MHC-restricted and allospecific T- cell recognition has been an enigma for many years. It has become increasingly clear that the T-cell repertoire is positively selected, in the thymus, for self MHC-restric- tion (Zinkernagel 1978; Kisielow et al. 1988). It is also generally agreed that the alloreactive and MHC-restricted T-cell repertoires are entirely overlapping (Matis et al. 1987; Lombardi et al. 1989a; Lechler et al. 1990).

Insights about the molecular architecture of MHC molecules, and about how the structure of these molecules subserves their functions in T-cell recognition have ac- cumulated in recent years. This has furthered our understanding of how MHC-restricted T cells can simultaneously display alloresponses against foreign MHC products. These insights have arisen from the com- bined application of structural and functional approaches. Using serological and cellular read-outs, the effects of spontaneous and artificially induced variants have been investigated. Since the three-dimensional structure of HLA-A2 was solved, it has been possible to interpret the results of such studies (Bjorkman et al. 1987).

Early models of allorecognition suggested that alloreactive T cells, like alloantibodies, recognize directly polymorphic residues on the exposed surface of foreign MHC molecules. More recent experimental evidence has emphasized the key contribution of MHC-bound peptides to allorecognition (Mentzer et al. 1986; Bernhard et al. 1987; Lombardi et al. 1989b; Heath et al. 1989).

The hypothesis that alloreactive T cells are specific for binary complexes composed of allogeneic MHC

150 G. Lombardi et ai.: Mutational analysis of anti-A2 allorecognition

molecules and a derivative of a self protein was first pro- posed by Matzinger and Bevan (1977). The high level of occupancy of cell surface MHC class I molecules with bound peptides (Bjorkman et al. 1987), the influence of putative pepfide-binding residues on alloresponses (Hogan et al. 1989; Santos-Aguado et al. 1989), and the detection of species-specific and lineage-specific allo- recognition (Mentzer et al. 1986; Berhard et al. 1987; Lombardi et al. 1989b), all suggests that bound peptides contribute to the ligand recognized by many alloreacfive T cells. The influence of amino acid residues which are predicted to contact the T-cell receptor directly, and the recognition of "empty" class I molecules by anti-A2 and anti-H-2K b allospecific T cells has kept the debate about the specificity of alloreacfive T cells alive (Elliot and Eisen 1990; Ohlen et al. 1990).

There have been a number of previous studies of allorecognition of HLA-A2.1 using natural variants of A2 and in vitro-induced mutants (Santos-Aguado et al. 1989; Hogan et al. 1988a; Hogan et al. 1988b; Mattson et al. 1989). The conclusions from these earlier experiments were that positions 152 and 156, both of which are located in the a2-domain a-helix, and point into the peptide-bind- ing groove of the molecule, play a particularly important role in the responses of anti-A2.1 alloreactive T cells. In this study we present the results of testing a large panel of transfectants expressing A2.1 molecules that have been altered by mutation of the codons for conserved and polymorphic amino acid positions in the a-helix of the a2-domain. The separate effects of these mutations on the conformation of the molecule, detected by an anti-A2 monoclonal antibody (mAb), and on allospecific T cell responses are described.

Materials and methods

Mutagenesis of HLA-A2 and gene transfection. The mutant A2.1 genes were constructed by saturation mutagenesis (Matsui and Frelinger, manuscript in preparation). Briefly, four oligonucleotides of 45 bases each, which in tandem comprise the HLA-A2 at 2 exon, were synthesiz- ed on a DNA synthesizer (Applied Biosystems, Foster City, CA). Before synthesis, each nucleofide precursor reservoir was deliberately con- taminated with a 5.9 % equimolar solution of all four nucleotide precur- sors. Statistically this was calculated to give one coding change per oligo. The cx2-encoding exon of A2 (from the JY cell line) was subeloned into M13 and expanded in an u n g - d u t - strain of Escherichia Coli. The mutant oligos, were annealed to single stranded M13 template and se- cond strand synthesis achieved using T4 DNA polymerase. The derived mutant exons were sequenced, selected for transfection, and subcloned back into the intact A2 genomic clone in the pUC18 plasmid. Transfec- tion was achieved by electroporation. Mutant A2.1 genes in the pBS vec- tor were cotransfeeted with the pSV2-neo plasmid into an HLA-A and B deficient human B cell line C1R (Storkus et aL 1987). The cells were fed with fresh medium and Geneticin (G418, Sigma, Poole, UK) 200 ~tg/ml twice weekly. The list of transfectants with the amino-acid substitutions that were made, together with the orientation of the amino- acid side chains are shown in Table 1. Some mutations led to more than one substitution, as indicated in the Table. The surface expression was

Table 1. List of A2 mutants expressed on C1R cells.

Position Amino-acid change Orientation of side-chain

140 (A>D) ' , A > V ? 144 K > R D 145 H > L O 146 K > R U 148 (E>K) ' D 149 (A > G)- U 150 (A > D)- U 151 ( H > D ) ' U 152 V > E , (V>A)* I 154 E > K , ( E > A ) ~ O 155 Q > K , (Q> L)* U 156 L > F I 157 R > G D 160 L > M I 161 E > V D 162 (G > D) ̂ O i66 (E > G) ̂ O 170 R > G , R > S ?U 171 Y > F I 174 N > T , ( N > I ) " , (N>K)" ? 175 (G > V)" ? 176 K > M , ( K > R ) " ? 177 E > D , E > Q ? 181 (R> G)" ?

The amino acid substitutions resulting from A2.1 mutagenesis are shown. The positions in the c~2-domain c~-helix, the amino acid changes, and the orientation of their side chains are shown (D=down, O=out , U=up , I=in, and ?=indeterminate - with regard to the an- tigen-binding cleft). Some mutations led to more that one amino acid substitution; residues that were altered in combination are indicated by bracketing and use of a common symbol, e .g.*.

monitored before each experiment using the HLA-specific class I an- tibody W6/32.

Monoclonal antibodies. The mAbs used in this study were W6/32, a broadly reactive HLA-specific class I antibody, which reacts with all HLA class I polymorphisms (Ferrier et al. 1985) and MA2.1, an HLA- A2-specific antibody (McMichael et al. 1980). They were used as con- centrated cell-free supernatants for flow cytometric analysis.

Flow microfluorimetric analysis. For flow microfluorimetric analysis, 5 x 105 cells were incubated with 50 ~tl of the indicated mAb, or with medium alone for the negative control, at 4 °C for 30 min. After washing twice in phosphate-buffered saline (PBS) with 2.5% fetal calf serum (FCS), the cells were incubated for a further 30 rain at 4 °C with 100 Ixl of 1:50 dilution of fluoresceinated sheep anti-mouse Ig (Amersham, Amersham, UK). After two additional washes stained cells were analyz- ed using the EPICS Profile Flow Cytometer (Coulter Electronics, Luton, UK).

T-cell lines and clones. The anti-A2.1 alloreactive T-cell lines and clones used for this study were obtained from five A2-negative donors with different HLA class I types. They were derived by culturing peripheral blood mononuclear cells (PBMC) from the responders in the presence of irradiated A2.1-expressing stimulator PBMC. After seven days, primed lymphoblasts were either maintained in culture by weekly stimulation with allogeneic PBMC and 10 units ml of rIL-2 or cloned

G. Lombardi et al.: Mutational analysis of anti-A2 allorecognition 151

by limiting dilution in the presence of 10 units ml of rIL-2 and a fresh alloantigenic challenge in Terasaki plates (Nunc), as described previous- ly (Lombardi et al. 1989b). The uncloned lines were depleted of CD4 + T cells using the OKT4 anti-CD4 mAb (Becton Dickinson, Mountain View, CA) and anti-mouse Ig-coated magnetic beads (Dynal, New Ferry Wirral, UK). T-cell lines and clones were used for functional assays one week after their last stimulation.

Proliferative T-cells assays. The T cells used in these experiments ex- hibited both cytotoxic and proliferative responses when cultured with A2-expressing cells, in the absence of added IL2. A proliferative assay was used throughout for the sake of convenience. T-cell clones (104 cells/well) were cultured in the presence of different numbers of stimulator cells in flat-bottomed microtiter plates in a total volume of 200 gl. The C1R transfectants were treated with mitomycin-C and B cell lines (B-LCL) with X-irradiation, before use as stimulator cells. After two days 1 ~tCi/well of 3H-thymidine (3H-TdR) was added and the cultures were harvested 18 h later onto glass fiber falters. Prolifera- tion was measured as 3H-TdR incorporation as determined by liquid scintillation spectroscopy. The results are expressed as cpm for triplicate cultures.

Results

Serological analysis of HLA-A2.1 mutants. Transfectants expressing wild type or altered forms of HLA-A2.1 were stained with the HLA-A2-specific antibody MA2.1,

which has been shown previously to bind to an epitope on the c~ 1 domain of the A2 molecule, and with the

broadly reactive mAb W6/32. For most of the A2.1 mutants, the ratio of fluorescence intensity achieved with

W6/32 and MA2.1 was the same as for wild-type A2.1. For three of the mutants, however, there was a marked reduction in staining with MA2.1. The patterns of staining of four different transfectants expressing mutated A2.1 genes and the wild type A2.1 gene with the W6/32 and MA2.1 mAb are shown in Figure 1. As can be seen, there is a marked reduction in the ratio of relative fluorescence intensity with W6/32 and MA2.1 for three of the mutants, compared to the ratio seen with wild type A2.1 and the 154 mutant. The staining was performed with a series of dilutions of both antibodies, and saturating conditions were achieved in all cases except for the three mutants stained with MA2.1 as shown in Figure 1. For all the other transfectants there was no reduction in mean fluorescence intensity (MFI) until the antibody was diluted beyond 1: 8. For the three mutants involving positions 170, 175, and 181, the MFI fell as soon as the antibody-containing supernatant was diluted two-fold. This clearly implies that these amino acid changes lead to alteration of the MA2.1 epitope. It is noticeable that the less radical change at posi- tion 170 (R > S v R > G) caused a less drastic reduction in MA2.1 staining.

The functional effects of amino acid substitutions on anti- A2 T-cell reponses. A series of T cell lines (TvG, PvG,

C1R A2.l E154K

E --a ¢--

<D O

! 1.4 A

R170S

=...:

R170G N174K, G175V, R181G

I

Relative fluorescence intensity Fig. 1. Flow cytometric profiles of transfectants expressing mutated and wild type HLA-A2. ] genes. Flow cytometric profiles of the parental C 1R cell iine and five transfectants, as indicated on each panel are displayed. Background staining is shown as a dotted line, staining with W6/32 as a thin line and staining with MA2.1 as a bold line.

152 G. Lombardi et al. : Mutational analysis of anti-A2 allorecognition

and RvR) and clones (K7, K20, and C8.1) were raised against HLA-A2.1-expressing stimulator cells from five different responder-stimulator combinations. The pro- liferative responses of the T-cell lines and clones were tested against the A2.1 and A2.3 subtypes of HLA-A2 and against the related HLA-A molecule Aw69 expressed on B-LCL. The results are shown in Table 2. The failure of all three T cell clones and lines to recognize the B-LCL expressing the Aw69 allele suggests that residues in the c~-helix of the c~ 1 domain, the only region that differs bet- ween A2 and Aw69, are crucial for their anti-A2 alloresponse.

All three T cell lines were able to recognize the two subtypes of A2, however the clones exhibited a different patterns of fine specificity, in that only C8.1 proliferated in response to A2.3-expressing stimulator cells.

In order to define the amino acids which determine the specificity of these populations of anti-A2.1 T cells with more precision, their responses to the panel of transfectants expressing wild type and mutated forms of HLA-A2.1 were tested. The results of four experiments for each mutant cell line are summarized in Table 3. The preservation or alteration of the MA2.1 epitope is also indicated. In all cases where a loss of response to mutated forms of HLA-A2.1 is reported another transfectant with the same, or a lower level of A2.1 expression induced a positive response in the same experiment. As can be seen, the large majority of amino acid substitutions resulting from these A2.1 mutations had no measurable effect on anti-A2 allorecognition by the T-cell lines and clones studied here. Furthermore, the patterns of responses of the bulk alloreactive T-cell lines was very similar to those shown by the T-cell clones.

Several of the transfectants were tested with the T-cell clones in a dose response titration, in order to detect a quantitative rather than a qualitative loss of recognition. Selected results are displayed in Figure 2. In the figure the responses of K20 and C8.1 to four mutants and wild

Table 2. Response of anti-HLA-A2 T cells to A2 subtypes and related HLA-A molecules.

T cells 3H TdR incorporation, A cpm

A2.1(BM16) A2.3(TUBO) Aw69(MAL-NF)

K7 54,624 9 -269 K20 142,830 136 330 C8.1 58,087 16,915 326 TvG 14,570 11,439 1,921 PvG 69,749 26,072 529 RvR 44,694 17,556 1,020

10 4 T cell clones (K7, K20, and C8.1) o r 10 4 T cell lines (TvG, PvG, and RvR) were co-cultured with different numbers of X-irradiated B- LCLs. After 48 h, cultures were pulse-labeled with 3H-Thymidine and harvested 18 h later. Proliferation is recorded as A cpm= (cpm ofT cells plus stimulator cells-cpm of stimulator cells alone).

type HLA-A2.1 are shown. The substitution of E for V at 152 led to a complete loss of response. The other three mutations had no effect on the reactivity of these clones. The proliferation of K20 and C8.1 to the three mutants is comparable to the wild type A2.1 response. The slight variation in the levels of response by the T-cell clones to the mutant molecules can be accounted for by differences in the levels of cell surface expression of A2, as detected by staining with W6/32. Interestingly, in all cases where a response was seen it appeared to be of comparable strength to that induced by wild type A2.1. In other words this series of mutations had an "all or nothing" effect on the alloresponse of these T-cell clones. It is of particular note that no loss of response was seen using the transfec- tants which expressed conformationally altered A2 molecules, as determined by alteration in the pattern of MA2.1 staining.

The results of this panel analysis can be summarized as follows: 1) amino acid substitution at only six out of 24 positions in the aa-domain a-helix led to any loss of allorecognition; 2) Alteration of the residue at position 152 (pointing in) from V to E lead to a complete loss of response by all the anti-A2 clones and lines; the more con- servative change from V to A, in conjunction with L for Q at position 155, had no effect; 3) Substitution of K or A for E at position 154 (pointing up) lead to loss of recognition by five of six of the T-cell populations tested; 4) The combined substitutions at positions 162 and 166 (pointing up) abolished the response of two of the three clones; 5)Alterations at positions 170, 175, and 181, lead to marked reduction in the expression of the MA2.1 epitope. Despite this alteration in the molecule these substitutions did not lead to loss of response by any of the T cells examined.

D i s c u s s i o n

This study was designed to explore the contribution of conserved and polymorphic amino acids in the a-helix of the a2 domain of the HLA-A2.1 molecule to allorecogni- tion. In addition, the substituted A2.1 molecules were analyzed serologically. The epitope recognized by MA2.1 has been mapped previously to a region on the al do- main a-helix between residues 62 and 65 as boxed in Figure 3 (McMichael et al. 1980; Santos-Aguado et al. 1988). The W6/32 epitope is formed by an exposed loop at the lateral margin of the floor of the a2 domain and the 132-microglobulin domain (Ferrier et al. 1985; Kahn- Perles et al. 1987). Given the robust nature of the W6/32 epitope, the level of fluorescence obtained with this mAb was used as the standard for each cell, and reduction in the ratio ofMA2.1 to W6/32 staining was taken to indicate a reduction in the expression of the MA2.1 epitope. Diminished expression of the MA2.1 epitope was seen as

G. Lombardi et al. : Mutational analysis of anti-A2 allorecognition

Table 3. Effects of mutations in HLA-A2 on serological and T cell allorecognition.

153

Position Substitution MA2.1 epitope T cell response

K7 K20 C8.1 TvG PvG RvR0

Wild type A2.1 ~ + + + + + + 140 A > V ---' + + + + + + 140 (A > D)' --, + + + + + +

144 K > R ~ + + + + + +

145 H > L ~ + + + + + + 146 K > R --' + + + + + + 148 (E > K)' --* + + + + + + 149 (A > G) - ~ . . . . . + 150 (A > D) - ~ . . . . . +

151 ( H > D ) ' --' + + + + + + 152 V > E ~ . . . . . . 152 (V > A) * --> + + + + + +

154 E > K ~ . . . . . +

154 (E > A) - ~ . . . . . +

155 Q > K ~ + + + + + +

155 (Q > L)* ~ + + + + + + 156 L > F ~ + + + + + +

157 R > G ~ + + + + + + 160 L > M ~ + + + + + + 161 E > V ~ + + + + + + 162 (G > D) A -~ - - + + + +

166 (E > G) ̂ ~ - - + + + +

170 R > G 1 + + + + + + 170 R > S ,L + + + + + + 171 Y > F ~ + + + + + + 174 N > T ~ + + + + + + 174 (N > I)" ~ + + + + + +

174 (N > K)" J, + + + + + + 175 (G > V)' $ + + + + + + 176 K > M ~ + + + + + + 176 (K > R)" ~ + + + + + +

177 E > D --> + + + + + + 177 E > Q -~ + + + + + + 181 (R > G)" ,L + + + + + +

The results of four experiments for each mutant cell l ine are shown. The effects of the amino acid substitutions shown in Table 1 are presented. BoM type is used to indicate substitutions that led to alteration in MA2.1 and/or T-cell recognition. Reduction in MA2.1 staining is indicated by a down-pointing arrow. The symbol + indicates a stimulation index > 10 and the symbol - indicates a stimulation index < 3. Responder cells were either T-cell clones (K7, K20, and C8.1) or T-cell lines (TvG, PvG, and RvR).

4 0 "

30-

20-

10-

0~" 1

K20

A

3 10

80 - . ~

60-

40

2o !

0 ~ ~ ~" 1 3 10

stimulator cell number x 10 "4

Fig. 2. T cell responses to different numbers of transfectants expressing altered A2.1 molecules. Proliferative responses to different numbers of A2.1-transfected stimulator cells are shown as c p m × 10 3. Responder T cells were anti-A2.1 alloreactive clones K20 and C8.1. The stimulator cells were C1R cells expressing A2.1 wild type ( A ) , the A2.1 mutants V152E ( 0 ) , L156F (O) , R157G (O), and A140D E148K H151D (A).

a consequence of substitutions at three positions at the car- boxy (COOH)-terminus of the a 2 domain a-helix. This can be interpreted in two ways. If the binding site of this antibody is located on the a 1 domain, as has been sug- gested previously, the results obtained here would suggest that the COOH-terminus of the a-helix of the a2 domain interacts with and influences the conformation of the oq domain. There are precedents for interdomain interac- tions influencing the conformation of MHC class I molecules (Darsley et al. 1987; Kanda et al. 1987). In ad- dition, Braunstein and Germain (1987) described the loss and gain of serological epitopes in mouse Aa i domains due to assembly with haplotype-mismatched AB chains. The residues responsible for these effects mapped to the floor of the ~3 t domain. The alternative explanation for the findings reported here is that the MA2.1 epitope spans

154 G. Lombardi et al.: Mutational analysis of anti-A2 allorecognition

Fig. 3. Schematic diagram of HLA-A2 indicating the effects of amino acid substitutions. Mutations resulting in loss of A2.1-allorecognition are shown as filled circles, the ones that give a variable loss of allorecognition are stippled, and mutations that had no effect on allorecognition are open. Mutations that led to loss of allorecognition, but were mutated in conjunction with other residues are hatched. The boxed section on the o~ 1 domain a-helix represents the site of the MA2.1 epitope as mapped previously (Kahn-Perles et al. 1987).

both of the amino-terminal domains of A2, and involves the direct binding of residues at the COOH-terminus of the a 2 domain. The testing of additional anti-A2 monoclonal antibodies may help to distinguish between these possibilities. It is interesting to note that in the study of Hogan and co-workers (1989), substitutions at posi- tions 149 and 152 lead to some loss of MA2.1 reactivity. This may futher illustrate the distant effects that can result from very limited sequence changes.

Turning to the results of the functional studies with the anti-A2.1 T ceils, in response to the A2 subtypes, only the C8.1 clone proliferated when stimulated by A2.3- expressing cells. This subtype differs at only three posi- tions 149, 152, and 156, all located in the a2-domain a- helix. The contribution of these residues is further ex- plored by use of the mutants reported in this study. The functional effects of the a2 domain a-helical mutations on anti-A2 allorecognition are presented in a diagram- matic form in Figure 3. Several points can be made as a result of this analysis. First, a large majority of the amino acid substitutions examined in this study (18 out of 24) had no detectable effect on A2.1 allorecognition by any of the T cells tested. This implies that the deter- minants for which these alloreactive T cells are specific are located in, or controlled by, limited regions of the a2 domain of the MHC molecule.

A second observation is that the single most influential change, of the 24 amino acid substitutions that have been examined, was the introduction of glutamic acid in place of valine at the naturally polymorphic position 152. Previous studies have made similar observations for A2,

A3, and B27 allorecognition (Healy et al. 1988; Hogan et al. 1989; Santos-Aguado et al. 1989; Calvo et al. 1990). The orientation of the side chain of the amino acid at this position is into the peptide-binding site of the A2- molecule, as deduced from the crystallographic studies of Bjorkman and co-workers (1987). These results can be interpreted in three ways: 1) the importance of residue 152 allorecognition may reflect the specificity of these an- ti-A2.1 T cells for peptides whose binding to A2.1 is in- fluenced by valine at position 152. A second possibility is that the side chain of the amino acid at this position is contacted directly by the alloreactive T-cell receptor. A third mechanism that might be responsible for the effects of introducing a charged glutamic acid at this position is the induction of a conformational alteration in the a 2 do- main a-helix. This caveat must always be borne in mind when interpreting the results of mutagenesis experiments, and the testing of other A2-specific mAbs will be required in order to test this possibility. It is interesting to note that the C8.1 clone did respond to A2.3, which has the glutamic acid substitution at position 152, together with changes at positions 149 and 156. This emphasizes the interplay between neighboring residues, and the compen- satory effects that combined amino acid substitutions can have.

The third conclusion derived from these results is that substitutions at three positions which are predicted to point up towards the Tcr had substantial effects on the anti-A2 response of the T cells used in this study. Substitutions at position 154 led to a loss of response for five of six T-cell populations, and changes at positions 162 and 166 abolished the response of two of the three clones. Taking note of the fact that the HLA-A alleles of the responder who gave rise to these clones differed at these positions, these results suggest that these polymorphisms constitute part of the ligand for which the K7 and K20 clones are specific.

In contrast, substitutions at two other in-pointing posi- tions, 156 and 160, had no influence on the response of these anti-A2 T cells. From these data, it appears that residues in this region of the molecule which are available for contact with the alloreactive T-cell receptor may be more important in anti-A2.1 allorecognition than in-poin- ting peptide-binding residues. This does not mean that these T cells are not specific for A2.1-bound peptide. Other aUelically polymorphic residues in the floor and/or in the a-helix of the a 1 domain of A2.1 may well control the binding of key endogenous peptides. Nonetheless it can be inferred from these results that T-cell receptor-con- tacting residues play a crucial role in determining the specificity of alloreactive T cells.

In a parallel study, the same transfectants were tested as antigen presenting cells for A2.1-restricted, influenza matrix-specific T cells (Moots et al. 1991). A great deal of overlap was seen in the substitutions which abolished

G. Lombardi et al. : Mutational analysis of anti-A2 allorecognition 155

A2.1-restricted recognition of peptide. This provides fur- ther evidence that MHC-restricted and allospecific T cell recognition are structurally very similar phenomena.

The final point of note is that, despite the influence of the changes at the COOH-terminus of the a-helix of the a 2 domain on the display of the MA2.1 epitope, they did not have any effect on allorecognition. Whichever of the explanations for the serological change is correct, this discrepancy further suggests that certain regions of the A2.1 molecule are less critical in the anti-A2.1 response. In addition to the area where the substitutions were made, this may apply to the region of the a-helix of the a a do- main to which the MA2.1 epitope has previously been mapped.

In conclusion, the small fraction of total substitutions which lead to loss of allorecognition implies that limited regions of the A2.1 molecule are responsible for the ex- pression of the allodeterminants for which anti-A2.1 T cells are specific. The orientation of the residues which abolished allorecognition fits with the concept that the specificity of alloreactive T cells is determined both by direct interactions between the Tcr and polymorphic residues in the allogeneic MHC molecule, and by interac- tions with peptides that are differentially associated with the responder and stimulator MHC molecules.

Acknowledgments. Giovanna Lombardi and Sid Sidhu acknowledge the support of the Medical Research Council of Great Britan.

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