Structural model of HLA-DR1 restricted T cell antigen recognition

Cell. Vol

52. 515-523.

February

7li

1988

CopyrIght

CC 1988 by Cell Press

Structural Model of HLA-DRI T Cell Antigen Recognition Jonathan B. Rothbard,’ Robert Kevin Howland,’ Vineeta Bal,t Rafick Sekaly,il Eric 0. Long.11 and Jonathan R. Lambt * Laboratory

of Molecular

lmperral

Cancer

Lincolns

Inn

London

WC2A

Our

Immunology

Research 3PX

t Department

Fund

England

of Immunology

Tuberculosis

Royal

Postgraduate

Hammersmith

and

Medical

lnfectrons

Unit

Milwaukee,

Wrsconsin

fr Laboratory

National

Institute

National

Institutes

Bethesda, p National

Maryland Institute

London

Mill

NW7

class

II molecule

and

rium

dialysis

subsequently

Infectious

method

has

(Buus

et al..

receptor

and

subsequently

for

al., 1987), have cules examined addition.

England

on

ability

Summary

The

Two human helper T cell determinants in influenza have been identified, one in the hemagglutinin and the other in the matrix protein (Mt). Both were shown to be DRl restricted by using transfected L cells to present antigen. Comparison of the sequences of the two peptides revealed a similar pattern that could account for their DRl specificity if the peptides adopt a helical conformation. The model was supported by the demonstration that hybrid peptides, composed of the amino acids that interact with DRl from one determinant and the residues that interact with the T cell receptor from the other, were recognized by each clone. The generality of the motif was confirmed by the finding that DRl individuals respond to a ragweed peptide containing the defined pattern.

A unified veloped

model for T cell recognrtron from recent experimental

laboratories. are

lysed

fluenza

The when virus

(Townsend as well tures helper

teractions

(Bjorkman

residues proposed

with

genetic

populations

that with

human

histocompatibte

peptrde

and

has deseveral

murine

cells

fragments

ever, of the

helices

T cells

and

cells

preferentially

we

Berzofsky, have noted

toxic

T cell

residues appears

and

structure.

amino-terminal

regions

proteins

acting

DeLisi

have

have

that

amphipathic

helices

epitopes

contain

two

adjacent

by a charged

to be necessary differing

residue

for recognition

in emphasis mutually

similarities

in the

two and

of CTLs together

elements of lymphocytes

to peptides with with the shared

by the

T cell (Davis.

those usage

receptors 1985)

of of

on the argue

that

Several the

been

groups

sequences

segregated

demonstrated

have of

The

1986).

observations provide

details

molecules.

by their

commented

the

determinants

by restriction

element,

that

[Rothbard,

of the merit ability

on

antigen

bind-

of each

analy-

to identify

similar

when which

are

additional

ously undefined T cell determinants successfully et al , 1987; Lamb et al.. 1987; Rothbard and Taylor,

sis has

of ?he MHC

the

exclusive,

hydrophobic or a glycine

The

fea-

T

(DeLlsi

a MHC

similar

the

helper

rng sites

many

as that struc-

analyzed

postulated

that cytotoxic T cells processed antigens in

molecule.

How-

of residues

the areas in proteins a number of common

recognize

preceded

for

sheet

be modeled

1985; Spouge et al., 1987). Independently. that the majority of both helper and cyto-

not necessarily evidence

p-pleated can

of MHC

and

epitopes

In-

Polymorphic

IS composed

of the

et

T cells. structure

a similar

site

13 subunits. the model

Berzofsky

helper

1986)

are concentrated in a sides are composed of the

to adopt

strands

re(Guillet

of MHC-peptide

II molecules

the

et

I molesate In

fragments

helper

1987b).

strands

peptide antigens, by Tcells have

features.

known

antigen-specrfic

1987a,

class

case,

u and with

receptors for are recognized

to bind

with

details

al..

the

MHC

latter

of both the Consistent

tural

et

homology

in the

proteins

et al..

ini1986)

(Gulllet class

three-dimensional

further

with

base.

by sequence

Although

of in-

cytotoxic

the

II proteins

class I molecules binding site whose

u-helices.

forming

et al.,

(Guillet

of both

of the

revealed

of MHC peptide

of two

level

(McMichael

solution has

MHC

peptrde by the

1986) demonstrated T cells recognize

in the response T cells taken

identical two

by both

et al., as helper

conjunction

observatron incubated

of proterns results from

recent

II and

recognized studtes,

generation

cytotoxic

of HLA-A2

and Introduction

and

class

Competition

to correlate

for the

1987)

class

class

purrfied

of the

appears

If al-

MHC

implied that the class II and have a single antigen-combrning

sponsiveness,

al.,

cellular

wrth

the

of proteins

T cell.

the

original

class

data).

specifically

the

the

human

between

entity

to be et al..

MHC

recently,

unpublished

on

of Health

and,

stable (Buus

to extend

different

complex

to be the

complexes

techniques

used

that

Babbitt murine in equilib-

II-peptide

be sufficiently

been

1987)

the

performed

Research

to filtratron

antrgen

peptrdes. a purified peptide

class

several

(Jardetzky, formed,

trally

Medical

Diseases

Hill

1AA

gel

to include

antrgen and

shown

of

by

by the frnding

a lysoryme the

by using This

Once

enhanced

experiments;

IS believed

of Allergy

Rrdgeway.

MHC

II proteins

of Immunogenetics

greatly

antrgens

basis

specrfrcally bind this by usng

Wisconsin 53233

molecular

II proteins demonstrated

leles

of Southeast

of the been

protein

MHC class et al. (1985)

observations

Section

Center

recognize

has

1986). Road

England

Blood

understanding

separated

School

Ducane

cytotoxic T cells mechanism.

recognition

were Related

Hospital,

§lmmunogenetics

The

helper and a common

Fields

r MRC

London,

I. Lechler,t David D. Eckels,§ William R. Taylor,”

Restricted

prevr(Cease 1988).

features the

may

latter be respon

In are

G I

1

ANTIGEN

ANTIGEN

Figure Clone

Figure 1. Deflnltlon Clone TLC 72

of Matrix

Protein

Determmant

Recognlred

by

(A) Localization ot antigenic determnant to sequence 17-31 wrthln matrix protein. IB) Defimtion of ammo termmus of the minimal peptide capable of maximum stimulation (C) Determination of the carboryl termmus of the peptide capable of mawmum stlmulatlon. T cells (5 x lO”/ml) of clone TLC 72 were cultured with rrradxated hlstocompatlble PEMC (1.25 x 105/ml). wth antigen at the concentrations Indicated. Prohferation as correlated wtth (3H]TdA incorporation was determined after 72 hr The control prollferatwe responses of the T celfs to Amexas Influenza virus (5 HAU/ml) and to presenting cells alone were determined

sible for their specific binding to the MHC protein (Guitlet et al., 1986; Buus et al., 1987; Rothbard and Taylor. 1988). However, as provocative as such allele-specific subpatterns are, they have yet to be demonstrated experimentally. In this paper we have defined two DRl-restricted helper T cell epitopes in influenza. one in the hemagglutinin and the other in the matrix protein. The restriction was established by demonstrating that each clone was stimulated when the peptide antigens were presented by L cells transfected with the a and B chains of DRl. Comparison of the sequences of the two peptides revealed a similar pattern that could account for their specific binding by DRl. The model was successfully tested by demonstratrng that hybrid peptides stimulated the appropriate T cell clone. The hybrid pepttdes were composed of the amino

2 Definition HA 1.7

1

CONCENTRATION

CONCENTRATION

of Hemagglutlwn

10

(UG,ML,

(“G:ML)

Determinant

Recognlred

by

(A) Deflmtion of the amino terminus of the mammal hemagglutinin peptide capable of maximum st!mulatlon. (B) Determlnatlon of carboxyl terminus of the mbnlmal hemaggfutlnin peptlde capable of maximum strmulation. T cells (5 x 104/ml) of clone HA 1.7 were cultured with IIradiated hlstocompatible PBMC (1.25 x 105/ml), with antigen at the concentrations indicateo. Proliferation was determlned as described in the legend lo Figure 1 The control prolrferatlve responses of the T cells to A/Texas influenza virus (5 HAUlml) and to presenting cells alone were determmed

acids that interact with OR1 from one determinant and the residues thai interact with the T cell receptor from the other. Finally, the generafity of the pattern was examined by analyzing the ability of OR1 individuals to respond to a ragweed peptide containing the motif. Results Definition of Two DRl-Restricted T Cell Determinants Seven peptides containing motifs characteristic of the majority of T cell epitopes (Rothbard and Taylor, 1988) within the sequence of the matrix protein (M1) of influenza were screened for their ability to stimulate a matrixspecific human helper T cell clone. The specificity of the clone, TLC 72. for the matrix protein has been previously described (Lamb et al., 1982b). Clone TLC 72 was stimulated only by the peptide corresponding to residues 17-31 (Figure 1A). The minimum sequence necessary for maximum stimulation was defined by testing nested sets of peptides that differed in length. Peptides with a common carboxyl terminus, but differing at their amino terminus, were used to stimulate the clone. Residues 18 and 19 were

DRl 517

B~nd~rlg of Antigen

Table

1

T Cell HA

Prollferatlve

Clone ..~__._

1 7a

Response

of DRJ-Restricted

Antigen-Presenting

T Cell Clones ~___~ Cells

72L

Presented

by L Cells

+ B + B L L

cells cells cells cells

OR1 + PBMC DRl+ PEMC EBV-transformed DRI + cells EBV-transformed DFtl + cells DRla DRlP-transfected L cells DRla DRl[\-transfected L cells L ceils L cells

Expressing

DR1

.__

Response (cpm 2 % SEM)

Antigen

DRl+ PRMC DRl + PBMC EBV-transformed DRl EBV-transformed DRl DRlu DRlp-transfected DRlu DRlb-transfected L cells L cells

TLC

to Antigen

HA 306-324 HA 306-324 HA 306-324 HA 306-324 MAT

18-30

MAT -

18-30

MAT -

18-30

MAT

18-30

520 304 19,051 732 11.771 253 9,396 200 285

-c * % k 2 t k ‘k

3ib IO 2 73 7 23 2 15 12

112k 176i 6.022 k 633 + 8,668 k

5 9 8 3 5

607-t

4

15,751 k 281 2 144,

4 4 9

* Clone HA 1 .7 (5 x lOa cells/ml) was cultured with and without HA peptide 306-324 (0 3 uglml) I” the presence of irradiated PBMC (1.25 x lOs/ml), autologous EBV-transformed 13 cells (lO”lml), or mltomycln C-treated munne L cells transfected with tl Proliferation was measured as described in Figure 1. ‘ Similar culture conditions were used for clone TLC 72 except that matrix 18-30 (3 pg/ml) was the stimulating antigen -.

shown to be required for maximal stimulation (Figure 1B). Serine-17 is not essential. Similar experiments examining the carboxy-terminal residues reveal that glutamic acid-29 is necessary but residues 30 and 31 are not. defining the minimum epitope to be 18-29 (Figure 1C). Previous experiments with panels of antigen-presenting cells have suggested that clone TLC 72 was restricted by DRl (Eckels et al., 1982). Another clone, HA 1.7. reactive with the hemagglutinin of influenza and isolated from the same individual, was shown to be restricted by the class II determinants DRl/DQwl on panel studies (Eckels et al., 1984). The determinant for clone HA 1.7 had been partially characterized in earlier experiments to be present between residues 306 and 329 (Lamb et al., 1982a). This sequence contained two potential T cell recognition motifs (Rothbard, 1986), between residues 308 and 311 and 316 and 319. To test whether either or both of these areas are necessary for stimulation of the clone, we rnrtrally synthesized 306-324. This peptide contains all possible decamers containing the patterns As seen in Figure 2A, 306-324 was fully stimulatory. The cysteine at 306 was not necessary for recognition because 307-324 was equally recognized. However. removal of prolineand, more effectively, lysine-308 results in the loss of stimulation (Figure 2A). The carboxyl terminus could be reduced to include alaninewithout any loss of recognition (Figure 26) allowing us to localize the determinant to residues 307-318, which contained the pattern from 308-311. Presentation of Peptide Antigens to DRl-Restricted T Cells by L Cells Expressing Human Class II Antigens The identification of restriction elements for human T cell clones is complicated because of the microheterogenelty of the HLA-D region products and the shortage of locus-

...~~

hlstocompatible DRlu DRlp.

specific monoclonal antibodies. To overcome this problem. we used murine fibroblasts transfected with the u and 13 chains of DRl to prove unequivocably that both clones are stimulated by the appropriate antigen in the presence of DRl (Table 1). The matrix-specific clone TLC 72 proliferated when the peptide was presented by autologous 6 cells transformed with Epstein-Barr virus, DRl-positive peripheral blood mononuclear celts, and DRl-transfected L ceils (Table 1). Similar results were obtained with the hemagglutinin-specific clone by using 306-324 of the hemagglutinin (Table 1). The proliferative responses were in all cases inhibited by monoclonal antibodies directed at monomorphic DR determinants (R. Lechler, V. Bal. J. Rothbard, E. Long, R. Sekaly, R. Germain, and J. Lamb, submitted). From the results of these experiments, we conclude that both clones are DRl restricted. Analysis, Synthesis, and Testing of Hybrid Hemagglutinin-Matrix Peptides Previous analysis of known T cell epitopes has revealed the presence of both general and allele-specific subpatterns that have been used for successful prediction of several previously undefined determinants (Rothbard and Taylor, 1988). The two DRl-restricted epitopes can be aligned in a simrlar manner based on the two adjacent hydrophobic residues (Figure 3A). Such an alignment reveals additional similarities at other positions. Most strikingly, tysine-21 IS aligned with lysine-311. Also, a third pair of hydrophobic residues occurs at a fourth position. leucine-28 and alanine-318. These residues, whose relative positions are 1, 4, 5, and 8, would be juxtaposed if the peptides were to adopt a helical conformation, and would form a conserved face of the helix (Figure 3B). We have previously postulated that the similar residues

Tdble 2 the Two

Sequences DFI-Restricted

of Wyixd PeptIdes Epitopes

Composed

of Residues

from

~--..~_ A The HAMAT scr~es Matrix 1i-29 HAMAT ! HAMAT 2 IiRMAT 3 HAMAT 4 HAMAT 5 HAMAT 6 HAMAT 7 HAMAT 8 HAMAT 9 HA 307-3 19 E The

MATHA

SGPLKAEIAQRLE S G Y, L K A N, S G !Y L K ‘Cl IN S G ‘Y C Y 0 ‘N S K Y, L K Q N, S K IY L K ‘Qi Nl

of the Known

DRl.

E”. and

(*)

sequences

to vnprove

As I” (C). certain

sequences

.K] R IKI R ‘K !L ‘K’ L lKl,L,

L L I L L

F E E E T

SGPLKAEIAQRLE

PKYVKQNTLK-LAT

hemagglutinln I” the MATHA

sequence series

E” T

:Ai Alignment of the two DRi-restrtcted peptldes based on two adjacent hydrophobic ammo acids. Boxed residues are those postulated to interact wth DRl (B) The two sequences displayed in a heluzal wheel (C) Altgnment of the deftned E”-restricted determinants. revealmg their slmilarQ with the DRl-restricted sequences. In the lower portlon of the flgure. the staphylococcal nuclease determinant has been reversed (-) to improve the ahgnment (D) Allgnrnent of the defined E”-restrlcted determinants. revealing their slmllarity with the DRl-

restrIcted

A A A A A

PKYVKQNTLKLAT

Resrdues enclosed I” boxes correspond to the ifi the HAMAT serres and the matrix sequence and Alignment

I I I I I

series

Matrz 17-29 MATHA 3 MATHA 4 MATHA 5 MATHA 6 MATHA 4 A-17 MATHA 4 A-t9 MATHA 4 V-l 9 HA 307-319

Flguw 3. Sequence Cell Determtnants

~

have Seen reversed

alignment.

rn the two determinants constitute the principal residues interacting with DRl, while the amino acids composing the opposite face of the helix would interact with the T cell receptor. If true, this hypothesis would predict that those residues that interact with the restriction element could be exchanged between the epitopes without affecting recognltion by either clone. Only those residues that are bound by the T cell receptor will be clonally specific. In Figure 38. the area recognized by the restriction element would be the lower half of the helix, whereas the residues composing the upper facade would interact with the antigen receptor of the T cell. To test this hypothesis and to determine exactly which residues interact with OR1 and which are bound by the T cell receptor, two sets of peptides were synthesized and tested for their ability to be recognized by the T cell clones. In one set, the HAMAT series, amino acids from the hemagglutlnln epitope were substituted Into the matrix sequence In the other set, MATHA peptides, amino acids from the matrix epitope replaced corresponding residues in the hemagglutinin determinant. The HAMAT series begins with three substitutions (HAMAT 1) and continues to HAMAT 9, which is identical to the original HA epltope except for one residue (Table 2A). The MATHA series is less extensive (Table 28). MATHA 3 con-

tains five substitutions, while MATHA 4 and 5 contain six and seven, respectively. The seven substitutions within MATHA 6 differ from those of MATHA 5 at two posltions. The peptides were individually titrated with each clone and analyzed for their ability to stimulate. As seen in Figure 4A, HAMAT l-3 were not recognized by HA 1.7, even at 100 ug/ml. In contrast, HAMAT 4 did stimulate the clone as well as the natural peptide at high concentrations, but was less efficient as the concentration of the peptlde was decreased. Interestrngly, HAMAT 5, 6, and 7 were not recognized even though they contained more of the HA sequence than HAMAT 4 HAMAT 8 and 9 were both recognized almost as well as the natural sequence. To confirm that this result was not simply characteristic of HA 1.7, the reciprocal experiment was performed. However. TLC 72 did not proliferate in response to MATHA 3-6, including MATHA 4, which contains the reciprocal exchanges with HAMAT 4. One possible explanation for the failure of MATHA 4 to stimulate TLC 72 is that it contains the sequence of proline, glycine, proline. at the ammo terminus. Proline is often stated to be incompatible with a helical conformatron because the side chain is bonded to the backbone nitrogen, preventing its participation in hydrogen bonding and sterically preventing the correct conformational angles from being adopted (Richardson, 1981). This is not completely correct, because in the first three positions of a helix these hydrogen bonds are not formed Therefore, although not favored. a proline can exist in the first turn of an u helix. However, in MATHA 4, the introduction of two prolines in the first three residues may have been sufficiently destabilizing to prevent an n-helical conformation from being adopted. To test this hypothesis, we synthesized three analogs of MATHA 4. One contained an

DRl 519

BInding

of Antigen

eral blood mononuclear cells (PBMC) from three DRl tndividuals. We selected this peptide because not only did it contain the pattern, it also was Immunogenic in H-2K and H-2D mice. As seen in Table 3. each of the individuals who recognized antigen E also responded to this peptide. PBMC from DRl individuals whose T cells fail to proliferate in response to this protein also did not recognize the peptide, demonstrating that T cell proliferation was not due to a nonspecific mitogenic effect of the sequence. Discussion

Figure 4. Response of Clones HA 1.7 [A) and TLC 72 (6) to Hemagglutlnln and Matrix Hybrid Peptides T cells (5 x lW/ml) were cultured with irradlated histocompatlble PBMC (1.25 x lO”/mlj, with antigen at the concentrations mdvzateo Prohferatlon was determIned as described r the legend to Figure 1.

alanine in place of proline-17, while the other two substituted an alanine and a valine for proline-19. If the peptide binds DRl in the orientation we propose, then the substitution of an alanine at 17 will allow the peptide to adopt a helix and still retain the proline at 19, which we predict is recognized by the T cell receptor. As seen in Figure 48, only MATHA 4 with alaninewas stlmulatory. As with HAMAT 4. it was not as efficient as the natural matrix sequence but it was, nonetheless, clearly recognized. In addition, the failure of the peptides containing alanine and valine at 19 is consistent with the proposed model. Finally, none of the HAMAT peptides were recognized by the matrix-specific T cell clone, nor did any of the MATHA peptides stimulate the hemagglutinin-specific clone (data not shown). Identification of a DRl-Restricted T Cell Epitope in Ragweed Antigen E To test the generality of the motif. we used a peptlde from antigen E of ragweed, residues 54-61. to stimulate periphTable Donor

3

Proliferative

Response .RaE

of PBMC

from

HLA-DRl

54-65

575 DR1.3 3,993 f 21 224 DRl _p 2,524 e 10 086 DRI .7 2.571 k 5 .._ --.~ -PBMC (5 x 1O”iwell) were cultured with Ra peptide incorporation was determined at 6 days --

54-65

Indlv;duals

The recent demonstration that MHC class II molecules specifically bind peptide antigens has provided a molecular explanation for empirical analyses of the defined T cell epitopes. revealing that they share several features. Taken together, these ideas imply that there will be allele-specific patterns that correspond to the regions in each determinant that Interact with the MHC proteins. To test these ideas, we have identified two DRl-restricted epitopes in tnfluenza. The restriction element to which they bound was demonstrated by using L cells transfected with both chains of DRl to present antigen. The details of the MHC-peptide interactlons were examined by comparing the ability of mutated peptides to stimulate the clones relative to the natural sequences. Assuming that the peptides share a common binding site, we examined the two DRl-restricted epitopes for common structural features. As seen in Figure 3, alignment of the two epitopes by the two adjacent hydrophobic residues revealed a similarity in the flanking residues. If the two lysines are considered to be residue 1. then there is similarity at relative positions 1, 4, 5, and 8. If the peptide adopted a helical conformation. these residues would be juxtaposed and would compose a common facade that could be characteristic of a DRl binding site. To determine whether the similarity in the epltopes had any structural validity and to analyze the conformation in which the peptide might bind MHC, we synthesized a family of peptides composed of residues from one determinant substituted into the other. The successful use of this strategy to produce a stimulatory peptide for each clone provides strong support for the proposal that both peptides bind DRI in a helical conformation. The recognition of the hybrid peptides, which contained six modiflcations in each determinant, IS especially remarkable considering the sensitivity of these and other peptide determinants to point mutations (data not shown: Sette et al., 1987; Allen et al., 1987). The three-dimensional structure of the proposed antigen-combining site of histocompatibility antigens can acto Ragweed

Peptide

54-65

RaE

Medium

PHA

1.489 Yk 23 8 545 i 16 6,869 f 26

285 A 5 353 & 26 229 k 29

150,037 82,864 79,808

(10 tlg;mli, ~~~___--~

RaE (5 tlyiml),

PHA, or medum.

Prollferalion

k 10 f 4 + 6

as correated

,til!h ‘HTdR

Figure

5

Space

Fllllng

Models

of the Defined

DRl-RestrIcted

Determinants

and the

Sttmulatory

Hybrid

Peptldes

rn Helical

Conformations

(A) Comparison of hemagglutinin 307-319 (left) with HAMAT 4 (righl) vlewed along the helical axes (z axis]. with ammo terminl in the foreground. The orientation of the side chains 1s Identical to that dlsplayed in the helical wheel in Figure 38. The resldues proposed to Interact with DRl compose the lower facade of the helix, while those that interact with the T cell receptor are above (see text) (B) An orthogonal view of hemagglutinln 307-319 (left) and HAMAT 4 (right) to that of (A) (y axis). emphaslzlng the areas of the two peptldes proposed to Interact with the antigen receptor of HA 1 7 (see text) Relatwe to the orlentatlon of the peptldes in (A] and in Figure 48, th1.s wew IS from above. (C) Comparison of matrix 17-29 (left) with MATHA 4, Gth alanine (yellow] at positlon 17 (right). wewed along the hellcal axis (z axis) as in (A) (D) An orthogonal vfew of matrix 17-29 (left) and MATHA 4. with alanlne (yellow) at posltlon 17 (right), to that ot (A) (y axIs) As in (8). this view reveals that the residues necessary to Interact wth the antigen receplor of TLC 72 compose the face of the helix in the foreground In each picture. residues corresponding to the hemagglutinin detennlnant 307-319 are red, while those of the matrix epltope, residues 17-29. are blue

commodate a peptide in a helical conformation without any significant conformational changes in the protein (Bjorkman et al., 1987b). If we Interpret our data in this context, the residues composing the described pattern would interact with the side chains of the amino acids of the HLA molecule pointing upwards from the p-pleated sheet, while the residues on the opposite face of the helix would point outward from the cleft and would interact with the T cell receptor. The amino acids of the epitope that are on the sides of the helix may. or may not. interact with the correspondtng residues on the MHC helices depending on their length, polarity, and the complementary residues in the HLA protein. Consequently, even though the stimulatory hybrid peptide for each clone contained an identical number of substitutions on the upper facade of the helix, they do not necessarily make similar molecular contacts when bound by both receptors. For instance. the hemagglutinin-specrflc clone requires lysine-308 for stimulation, which had to be substrtuted by glyctne in the stlmulatory

MATHA peptide However, from its positlon on the helical wheel and space filling models (Figures 3B and 5). we believe that glycine-19 interacts with the restnction element and not the T cell receptor. Lysme is of sufficient length to extend out of the binding site and interact with the T cell receptor, while glycine’s lack of a side chain would prevent any interactron The space filling models also provide a possible explanation as to why glutamic acid-29 and threonlne-319 are not required for recognition even though from their position on helical wheels (Figure 38) they appear to have a suitable orientation to interact with the T cell receptor. Both are the carboxy-terminal residues of the peptide and can be seen to be remote from the other clonal-specific residues. The diameter of an u helix is approximately 10 A. If we assume that the T cell receptor combinmg site is geometrically similar to that of an antibody, and the area of interaction is equivalent to that defined by the cocrystals of lysozyme and an antibody (30 x 20 A) (Amit et al.

DHI

BIndIng

uf Anr gen

521

1986), then the receptor could contact the upper facades of all three helices, that of the epitope and the two of the MHC molecule. The length of the interaction along the helices would then be of the order of 20 A. consistent with the results with the hybrrd peptides (Figure 5). The space fillrng models also reveal that the shapes of the areas proposed to be recognrzed by the two receptors are distinctly different. As expected from the sequences of the two peptides, the faces of the helices interactrng with the restriction element are quite srmilar (Figure 5). We expect, as with other instances of helices packing onto [l-pleated sheets (Cohen et al., 1982). that the turns of the helical peptide will interdigitate with the four central strands of the MHC molecule. Consequently, we expect that variations in brndrng will occur, parttcularly if peptrdes can bind in either direction (vide infra). However, the two independently derived determinants described In this report appear to interact with the identical residues in DRl, suggesting that vartattons in binding might not be as large as one would initially expect. That the hybrrd peptides are not as effective in stimulating the clones is not surprising, primarily because residues from one helix cannot necessarily be substituted into another and still guarantee a helical conformation. This is vividly demonstrated by the failure of MATHA 4 to be recognized by the matrix-specific clone. Even though each peptide contained a proline in its ftrst three amino acids, the hybrid synthesis resulted in two prolines in the same sequence which, as we discussed earlrer, is unlikely to form a helix In addition, in such an unprecedented situation where two macromolecular receptors bind the same ligand, subtle sequence-specific interactions may be important. These experiments also emphasize the potential diversity of molecular contacts with different T cell receptors. For although we predict a constant MHC-peptide interaction, the T cell receptor is still free to interact to varying degrees with residues exposed on the upper facade of the bound helical peptide. This would be consistent with the known differences and the fine specificity of T cell clones recognizing the identical peptide(Finket al., 1986). We have also provided preltminary evidence on the generality of the pattern of a positive residue-and three hydrophobrc amino acids will be characteristrc of DRl responsiveness (Table 3)-by demonstrating that a ragweed peptide can stimulate peripheral blood lymphocytes from DRl individuals. We were attracted to this peptide for several reasons. Ragweed was a useful system because a large number of individuals are known to respond to it. In addition to containing the pattern. this peptide was previously shown to be strmulatory in BALB/c and CBH.OH mice. A similar analysis of the defined I-Ek and I-Ed restricted epitopes (Rothbard and Taylor, 1988; Figures 3C and 3D) revealed a potential similarity to the two DRlrestricted determinants These sequences could also be aligned to have a positively charged residue (either a lysine or an arginine) and two residues from a pair of hydrophobic amino acids, and be separated by six residues from a third. Conceivably, the similarity in the sequences of the determinants reflects common features In the antigen-combrmng site of the DRI, I-E”. and I-Ek mole-

cules. However. in order for the murine class II proterns to bind each of the known determinants in a manner srmilar to DRl. not only must each peptrde adopt a helrx. but in several cases the direction of the helix in the binding sate is reversed. Such a situation would not necessanty violate the chiral nature of other known ligand-receptor rnteractions, because in a helical conformation the main chain amide nitrogens and the carbonyl oxygens are internally hydrogen bonded. Consequently. they would not interact with either the MHC molecule or the T cell receptor. tn addition, the flexrbility of the amino acid side chains on both the antigen and the two receptors could be sufficiently great to allow similar interactions to occur regardless of the relative orientatron of the helix. Our results on the recognition of this peptide by DRl individuals support these ideas and have prompted us to investrgate further the srmilarities between DRI and la Ed and Ek (Lechler et al submrtted). We would not have identified many of these features if we had adopted the expertmental strategy of pornt mutations used by the other two groups that have published analyses of peptide-MHC-T cell receptor Interactions (Allen et al.. 1987: Sette et al , 1987). As useful as these modifications have been to identify critical residues in MHC and T cell receptor binding, their interpretation IS difficult. Ambiguity can arise because the two receptors bind a structure that results from the interactrons between the am!no acids of the antigen, and those interactions are not necessarily independent of each other As we have shown, a single substitution at one position can remove all recognition, but a compensating change at another residue will restore bmdmg. Our assumption that the peptide can be divided Into two halves. one that is critical for binding to class II and one that binds to the T cell receptor, appears to be valid. Either half can be viewed as a recognrtion sate that may be moved between determinants; however, mutations within each set often result in dramatrc reduction in binding to either receptor. The strategy of substituting alanine for the individual residues of the determinant has an additional shortcoming: only if the replaced amino acid dramatically differs In either size or polarity from alanine will a clear-cut distincnon be seen (Allen et al., 1987; our unpublished data). For instance, with the hemagglutinin determinant, alanine can be successfully substituted at each position except for lysine-311 without significantly affecting recognition (,data not shown). Consequently, certain residues appear not to interact with either receptor by this analysis. In Contras!. our results indicate that each amino acid is involved in binding to DRI. the T cell receptor or both. We are currently attempting to Identify The exact interactions the peptides make with DRI by measuring brnding constants of these and other analogs to purified DR1 and by crystallographrc studies of both DRl and DRl-peptide complexes Detailed structural information from an HLA atomic model combined with similar sequence analyses of both helper and cytotoxic epitopes will lead to similar allele-specific subpatterns that can be used to develop logical strategies to modulate the immune system.

Experimental

Procedures

Preparation

of Lymphocytes

Pertpheral bload mononuclear cells (PBMC) obtarned from healthy adults were rsolated by centrrfugation on a drscontrnuous gradrent of Ftcoll-Hypaque (Pharmacra) and were cryopreserved. For use rn experiments, lymphocytes were resuspended rn complete medium RPM1 1640 supplemented with A* serum 7 mM L-glutamrne, and 100 fU/ml of perricill~nlstreptomycin

lsolatlon

of Antigen-Reactive

T Cell Clones

The isolatron and characterrzatton of human T lymphocyte clones HA 1 7 and TLC 72 have been descrrbed previously (Lamb et al 1982a. 1982b). Brrefly, PBMC were strmuiated for 6 days with rnfluenza hemagglutrnm (H3) or Intact vrrus (H3N2) for HA 17 and TLC 72, respectively. Lymphoblasts were enrrched on a Percoll densny gradient (Pharmacia. Uppsala) and cloned by limrting dtlution (0.3 cells par well rn Mtcrotest II plates, Falcon) rn the presence of autologous lrradtated (3000 rads) PBMC, viral antigen, and rnterleuktn 2 At day 7, growing clones were transferred to 96well flat bottom mrcrotlter plates and subsequently, to 24-well plates At each transfer the clones recetved filler cells, antigen, and IL-2 The clones were expanded and marntatned by the addttron of fresh IL-2 every 334 days, and filler cells together with specific antrgen every 7 days. Prior to use rn proliferation assays, the T cell clones were rested for 6 to 8 days after the last additron of filler cells.

Proliferation

Synthesis,

Analysis,

and

Purification

Peptrdes were synthesrzed by usmg solid phase technrques (Barany and Merrrfreld. 1979) on an Applred Blosystems Peptrde Synthesrzer with commercrally available Pam resrns t-Boc protected ammo acrds, and commerctally available reagents (Applred Btosystems, Foster City, CA). The peptrdes were cleaved from the resin and the side charn protecting groups were srmultaneously removed wrth anhydrous hydrofluoric acrd wrth anisole as a free radical trap. The side chain protecting groups were extracted with ether Subsequently, the peptides were dissolved in 15% acetrc acid, ftltered from the resrn, and lyophrlrzed. The crude peptrdes were analyzed by HPLC on a C-8 reverse phase column (Aquapore RP-300, Brownlee Labs) and by amtno acrd analysis. Peptides that were not of greater than 90% purity as judged by analyttcal HPLC analysts were purrfred by usrng a preparative RP300 column and a water-trtfluoroacetic acid-acetonrtrile gradient Pr~ncrpal peaks were collected. lyophrlrzed, and analyzed by ammo acid analysra

Cloned

Genes

The DFtl u and (i genes were full-length cDNA gene clones. descrrbed by Tonnelle et al. (1985). They were rnserted Into the pRSV.5 and pRSV.3 cDNA expression vectors, respectively (German et al 1983) The pRSV5 vector contarns the pSV2gp? gene (Mulltgan and Berg, 1981)

DNA-Mediated

Gene

Mitomycin

C Treatment

of Mouse

Fibroblast

(L) Cells

After trypsrnrration. cells were washed in serum-free RPM1 1640 Up to 10’ cells were suspended per ml of serum-free medrum. Mrtomycrn C (Sigma) was added to a final concentratron of 50 tlglml. Calls were Incubated at 37°C for 45 min, washed extensively in A’IRPMI, and used rn the proliferation assays.

Acknowledgments We thank Janet Thornton and Ted Jardetrky for helpful drscussrons We are also grateful to Dr M. Contreras of the Nattonal Blood Transfuston Center rn Edgeware for provtdlng blood donors. The costs of publrcatron of this article were defrayed in part hy the payment of page charges Thts article must therefore be hereby marked “adverrfsement” in accordance wrth 18 U.S.C Section 1734 solely to rndrcate this fact. Received

October

13, 1987: revised

December

21. 1987

References

Assays

Cloned T cells (5 x lo4 per ml) were cultured wrth soluble antigen in the presence of irradiated histocompatible PBMC (1.25 x 10s per ml), autologous EBV-transformed B cells (lOi per ml), or mrtomycin C-treated transfected murtne L cells (lo5 per ml) tn a total volume of 200 nl of complete medium in 96well round bottom plates. Where L cells were used as presenting cells, the assays were performed rn 96. well flat bottom plates. After 72 hr of rncubatron, trittated methyl thymrdine (1 ~CI. 13HJTdR; Amersham Internattonal, Amersham, U. K.) was added to the cultures for 8-16 hr and then harvested onto glass fiber filters. Proliferation as correlated with faH]TdR Incorporation was measured by liquid scrntillatron spectroscopy The results are expressed as mean counts per minute (cpm) plus or minus percentage error of the mean for triplicate cultures

Peptide

fully transfected celfs selected in meddum contarmng either MXH (mycophenolrc actd. xanthrne. and hypoxanthrne) orG418. Colonres of transfectants were pooled, stained with approprtate monoclonal antrbodies. analyzed by mrcrofluorrmetry. and repeatedly sorted by preparative microfluorrmetry to achieve high levels of cell-surface MHC class II expressron.

Transfer.

Selection,

and

Cell Sorting

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: i~i, ,.-7

BIndIng

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epl-

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o‘ protein

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A sequence

structure

response

common

Ann

to T cell ep~

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C

Demars

R

and Long.

E 11985)

Cease. K propensities

301:

Berrofsky enhance

ri n?w Ii chalrl

gene