Effects of localized HLA class II β chain polymorphism on binding of antigenic peptide and stimulation of T cells

Effects of Localized HLA Class II/3 Chain Polymorphism on Binding of Antigenic Peptide and Stimulation of T cells

Debra K. Newton-Nash and David D. Eckels

ABSTRACT: The relationship between HLA-DR1 polymorphism and recognition of antigen by T cells was investigated. Two allelic variants of HLA-DR1, which differ by amino acid substitution at positions 85 and 86 of the/3 chain, were characterized for the effect of substitution on recognition of foreign antigen by DR1-restricted T cells. Substitution of alanine and valine for valine and glycine residues at positions 85 and 86 of the DRI /3 chain resulted in deficient T-cell stimulation as demonstrated by the requirement for higher concentrations of antigen to induce maximal levels of T-cell proliferation, induction of lower levels of proliferation at optimal anti-

gen concentrations, and slower kinetics of formation of stimulatory peptide-DR1 complexes. Direct binding studies employing both biotinylated and radioiodinated forms of antigenic peptide demonstrated quantitatively lower levels of peptide bound to substituted DR1 molecules and low levels of site-specific binding as assessed by competitive inhibition analyses. The effect of MHC class II polymorphism on peptide-binding affinity as opposed to induction of appropriate peptide conformation and the impact of polymorphism at DR1/3 chain positions 85 and 86 on allorecognition of HLA-DR1 are discussed. Hu- man Immunology 33, 213-223 (1992)

A B B R E V I A T I O N S B-HA306_320 biotinylated peptide B-LCL B-lymphoblastoid cell line FITC fluorescein isothiocyanate HA306-320 peptide corresponding to residues

306-320 of influenza hemagglutinin

HLA human leukocyte antigen complex

125I-HA306_320 LMFCN

MHC NHS-LC-biotin

TLC

125I-conjugated peptide linear mean fluorescence channel

n u m b e r major histocompatibility complex long-chain derivative of

N-hydroxysuccinimide-conjugated biotin

T-lymphocyte clone

I N T R O D U C T I O N

Recognition of foreign antigen by T-lymphocytes is ge- netically restricted by molecules encoded within the major histocompatibility complex (MHC), H-2 in mice, or HLA in humans. The structural basis for T-cell antigen recognition is the formation of a trimolecular complex involving T-cell receptor, the self MHC molecule, and a peptide fragment of foreign antigen. In this context, it is apparent that MHC polymorphism can influ-

From the Blood Center of Southeastern Wisconsin, The Blood Research Institute, Milwaukee, Wisconsin.

Address reprint requests to Dr. D. K. Newton-Nash, Blood Center of Southeastern Wisconsin, The Blood Research Institute, 1701 West Wiscon- sin Avenue, Milwaukee, WI 53233, USA.

Received October 10, 1991," accepted February 21, 1992.

ence the ability of a given T-cell receptor to recognize the p e p t i d e - M H C complex by altering the MHC molecule's interaction with either T-cell receptor or antigenic peptide. Because alloreactive T-lymphocytes may also depend upon peptide for recognition of alloantigenic determinants, as proposed by Matzinger and Be- van [1], MHC polymorphism may also affect alloantigen-specific T-cell stimulation in similar ways.

Our laboratory has been interested in characterizing the relationship between MH C polymorphism and recognition of antigen by HLA-DRl-rest r ic ted T cells. Polymorphism associated with HLA-DR is limited to the DR /3 chain as the DR ce chain is invariant and whereas most HLA-DR haplotypes express two DR/3

Human Immunology 33, 213-223 (1992) 2 13 © American Society for Histocompatibiiity and Immunogenetics, 1992 0198-8859/92/$5.00

214 D.K. Newton-Nash and D. D. Eckels

chains encoded by both the/31 and the 33 loci, HLA- DR1 haplotypes express a single/31 product [2]. Re- cently, two Dw subtypes or alleles (Dwl and Dw20) associated with HLA-DR1 have been defined by alloreactive T-cell clones, mixed lymphocyte reactivity, and cDNA sequence analyses [3-6]. The DRBI*0101 allele codes for valine and glycine residues at positions 85 and 86, respectively, whereas the product of the DRBI*0102 allele differs only by the substitution of alanine and valine, respectively, at these positions [4- 6]. Based upon low-resolution x-ray crystallography as well as sequence and functional similarities with class I molecules for which the crystal structure is known [7], models of class II structure predict that amino acid residues 85 and 86 of the DR1 /3 chain would lie at the extreme carboxy-terminal end of the a-helical region of the DR1/3 chain first domain [8]. Although these substitutions represent a very localized and conservative polymorphism, previous studies in our laboratory have established that recognition of HLA-DR1 by some alloreactive T-cell clones is dramatically affected [6]. The extent to which these effects depend upon alteration of the interaction of MHC residues at these positions with antigenic peptide or with the T-cell receptor remains to be established.

Studies of antigenic peptide binding to MHC molecules in detergent solution [9, 10] have established a correlation between MHC restriction and the level of binding of a peptide to its appropriate restricting element [ 11]. Although the precise intracellular site within which antigenic peptides bind to class II molecules has not yet been elucidated, the cellular environment in which protein-derived peptides normally interact with class II MHC molecules is likely to differ substantially from the conditions under which peptide binding to detergent-solubilized MHC molecules is studied. For example, recent demonstrations of enhancement of binding ofpeptides to class II MHC molecules in detergent solution by supplementation with phospholipids suggest that membrane lipids may play an important role in facilitating peptide binding to class II molecules [12]. These differences may account for the failure of MHC molecules in detergent solution to bind antigenic peptides with the rapid kinetic characteristic of formation of peptide-MHC complexes on live cells [13, 14]. Alternative experimental approaches that employ intact cells for analysis of peptide binding to MHC molecules [ 15, 16], therefore, may be more informative than those that address peptide binding to purified MHC molecules and may enable elucidation of other cellular components involved in formation of stimulatory peptide- MHC complexes.

In the present study, we carefully examine the bio- logic role of a limited polymorphism within the HLA-

DR1/3 chain as it pertains to both T-cell receptor interaction and peptide binding. These studies are important in consideration of the physiologic function of MHC proteins and suggest that there may be areas on the surfaces of MHC molecules that differentially affect either or both types of molecular interaction.

MATERIALS AND METHODS

T-cell proliferation assays. B-lymphoblastoid cell lines (B-LCLs) homozygous for DRBI*0101 (workshop nos. 9006, 9004, and 9005) or DRBI*0102 (nos. 9002, 9078, and 9079), previously characterized (a) for their abilities to stimulate alloreactive T-lymphocyte clones [3] and (b) by cDNA sequence analysis and hybridiza- tion analysis using specific oligonucleotide probes [6] were maintained in continuous culture in our laboratory. A peptide corresponding to amino acid residues 306-320 of influenza hemagglutinin (HA306-320, CPKYVKQNTLKLATG) was synthesized by standard solid-phase methods and purified by high-performance liquid chromatography. T-lymphocyte clones (TLC) HA1.7 or HA3.41 (10-20 × 103 cells/well) were cultured with irradiated B-LCLs (50-100 × 103 cells/well) and HA306-~20 in a total volume of 200 /~l of RPMI media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 IU/ml heparin, 50 /~g/ml gentamicin, and 25 mM Hepes buffer. Monoclonal antibody (mAb)-blocking studies were performed with L243, Genox 3.53, B7/21, and W6/32, which recognize HLA-DR, -DQ, -DP, and HLA-A,B molecules, respectively [ 17-21]. Antibodies were purified from hybridoma supernatants or ascites fluid using recombinant protein G-sepharose (Gen- zyme) chromatography. Cultures were incubated for 72 hours at 37°C in a humidified 5% CO2-air atmosphere, pulsed with 1.0 ~Ci of tritiated thymidine (~H-TdR) for the final 18 hours of culture, and harvested onto glass fiber filter strips. Incorporation of ~H-TdR was measured by liquid scintillation spectroscopy. Proliferation is expressed as mean counts per minute (cpm) -+ standard error of the mean (SEM) for triplicate cultures.

For analyses of the kinetics of formation of stimulatory complexes of HA306-320 with HLA-DR1 variants, B-LCLs (106 cells/ml) were pulsed with appropriate concentrations of HA306-320 for time intervals up to 4 hours and extensively washed prior to addition to stimulatory cultures. To inhibit cell division, cells were fixed with 0.1% paraformaldehyde for 60 minutes at room temperature prior to peptide pulsing. T cells (104 cells/ well) were cultured with pulsed B-LCLs (5 x 104 cells/ well) in a final volume of 200/~l of supplemented RPMI medium and T-cell proliferation was determined by ~H- TdR incorporation as described above. Percent maximal

Peptide Interactions with HLA-DR1 Allelic Variants 215

responses were determined by comparing proliferative values obtained in the presence of a continuous source of HA~06-320 at optimal concentrations.

Peptide-binding assays. To prepare biotinylated HA306-~20 (B-HA306-~20), HA306-320 and a twofold molar exess of the long-chain derivative of N-hydroxysuccinimide-conjugated biotin (NHS-LC-biotin) (Pierce) were dissolved in 0.1 M sodium bicarbonate, pH 7.4, and incubated for 2 hours at 4°C. Unreacted biotin was separated from B-HA306-~20 by dialysis against phos- phate-buffered saline (0.01 M NaH2PO4 and 0.15 M NaC1, pH 7.2) using a Pierce Microdialyzer System 100 equipped with a dialysis membrane (1000-Dalton cut- off). Biotinylation of lysine side chains, particularly that of the lysine at position 3 11 of the peptide, has been shown to affect the ability of HA306-320 to bind to HLA- DR1 [22]. Consequently, the reaction was performed at pH 7.4 to favor biotinylation at the amino terminus of the peptide (pKa = 6.8-7.9) as opposed to the side chains of lysine residues (pKa = 10.4). Furthermore, at concentrations used for assessment of peptide binding, biotinylated and unbiotinylated forms of the peptide revealed no significant differences in the induction of peptide-specific T-cell proliferation confirming the assumption that extensive biotinylation of lysine side chains had not occurred (Table 1). B-LCLs were suspended in RPMI media supplemented with 1% fetal bovine serum (FBS) at a concentration of 2 × 106 cells/ ml and cultured with B-HA306-320 for 18 hours at 37°C in a humidified 5% CO2-air atmosphere. Surface-associated B-HA~06-320 was detected by flow cytometric analysis following labeling with fluorescein isothiocyanate (FITC)-conjugated avidin D (Vector Laboratories). Cells cultured in the absence of B-HA306-320 and stained with FITC-conjugated avidin D were used for the deter- mination of background fluorescence. In competitive inhibition studies, unbiotinylated HA~06-320 was added 1 hour prior to the addition of B-HA306-320 and was present throughout the duration of culture. Levels of fluorescence intensity, measured on a log scale, were quantitatively compared following conversion to linear mean fluorescence channel numbers (LMFCNs). For competitive inhibition studies, the percent of uninhibited B-HA306-~20 binding observed at each concentration of unbiotinylated HA306-320 was calculated according to the following formula:

[ inhibi ted- background] LMFCN [uninhibited- background] LMFCN! × 100

Flow-cytometric analysis of levels of HLA-DR expression on B-LCL surfaces was performed following indi- rect staining with L243 and FITC-conjugated goat anti- mouse immunoglobulin (Becton Dickinson). Results

TABLE 1 Comparative functional activity of unbiotinylated and biotinylated HA306-320 peptides

Peptide concentration

(~*M)

Peptide derivative

Unbiotinylated Biotinylated

0 76 ± 10 a 90 ± 51

1.0 14,462 ± 606 12,695 ± 388 10.0 14,075 ± 235 10,764 ± 1015

°Tritiated thymidine incorporation by T-lymphocyte clone HA3.41 is expressed as mean counts per minute _+ standard error of the mean calculated from triplicate wells.

are expressed as mean LMFCN + standard deviation calculated from 2-3 independent experiments.

125I-labeled HA306-320 (125I-HA306-~20) was prepared by radioiodination according to the method of Hunter and Greenwood [23] and was separated from free Na125I by Bio-gel P2 (BioRad) chromatography. Appro- priately pooled column fractions were assayed for the presence of radioactivity by gamma scintillation spectroscopy and for peptide concentration by comparison of the optical density at 220 nm against a standard curve prepared with unlabeled HA306-320. The specific activity of *~5I-HA306_320 generally approximated 5/,Ci//,g peptide. B-LCLs were suspended in RPMI media supplemented with 1% FBS at a concentration of 2 × 106 cells/ml and cultured with 125I-HA306_320 (1.0/*M) in a humidified 5% CO2-air atmosphere for 18 hours at 37°C. Following culture, B-LCLs were washed extensively, resuspended in lysis buffer (0.05 M Tris, 0.15 M sodium chloride, and 0.5% Nonidet P-40) containing a protease inhibitor cocktail (0.01 M ethylenediamine te- traacetic acid, 20 mM iodoacetamide, 1 mM phenyl- methylsulfonyl fluoride, 1.3 mM 1,10-phenanthroline, 6 mM N-ethylmaleimide, and 0.2 mM N-tosyl L-lysyl chloromethylketone) and incubated at 4°C for 30 minutes. Cell lysates were cleared of nuclei and debris by centrifugation at 100,000 g for 30 minutes at 4°C. Ly- sates were precleared of nonspecifically precipitable ma- terial by incubation with recombinant protein G - Sepharose overnight at 4°C. ~25I-HA306_320 bound to HLA molecules was detected by immunoprecipitation with monoclonal antibodies specific for HLA-DR (L243), -DQ (Genox 3.53), -DP (B7/2 1), and for HLA- A and -B (W6/32) molecules. Lysates were incubated with one of the antibodies listed above for 2 hours at 4°C. Antibodies were precipitated by centrifugation following incubation with recombinant protein G - Sepharose for 1.5 hours at 4°C. 125I-HA306_320 present in immunoprecipitates was detected by 3' scintillation spectroscopy.


Quantitative immunoprecipitation of HLA-DR1 was 35 performed using lysates of surface-iodinated B-LCLs. 30 Cells ( - 3 x 107) were added to Iodobeads (Pierce) in ~ 25 the presence of 0.5 mCi of Nal25I (Amersham) for 15 'o minutes at room temperature after which cells were re- ~ 20

z moved from the beads and washed extensively in RPMI a 15 medium containing 10% FBS. Detergent-solubilized ly- o_

o 10

sates of B-LCLs (4 x 107 cells/ml) were prepared as "-" c 5 O described above. HLA-DR-specific immunoaffinity .~

columns were prepared by coupling of L243 [17] or ? 0 O

DA6.147 [24] to cyanogen-bromide-activated ~ 30 Sepharose (Pharmacia) according to manufacturer's in- o o

c 25

structions. Cell lysates were applied to antibody-cou- ~" 20 pied Sepharose in roughly equal volumes (13-20 /~1) F-

I 1 5 calculated to yield equal amounts of radioactivity (5 × z 106 cpm) and cell equivalents (1.25 × 106 cells) per ~ 10 immunoprecipate. HLA-DR1 was bound to antibody- 5 coupled Sepharose for 2 hours at 4°C and eluted by

0 boiling directly into electrophoresis sample buffer containing 2%/3-mercaptoethanol. 125I-DR ~ and/3 chains in eluted samples were resolved on a 10% SDS-polyacrylamide gel and identified by autoradiography.

RESULTS

Effect of HLA-DR1 allelic variation on T-cell stimulation. The methods used for derivation of influenza-specific T- lymphocyte clones have been described in detail else- where [25, 26]. Previous studies have characterized CD4 + human T-lymphocyte clone (TLC), HA1.7, which is restricted by HLA-DR1 [27] in its recognition of an antigenic peptide derived from influenza hemagglutinin [28]. The TLC HA3.41, derived under identical circumstances and characterized similarly, demon- strates peptide specificity and restriction patterns identical to HA 1.7 (present data and D. D. Eckels, un- published observations). As demonstrated in Fig. 1, recognition of both HA306-320 and intact hemagglutinin was affected by amino acid substitution at positions 85 and 86 of the DR1 /3 chain. Specifically, relative to cells homozygous for DRB 1"0101, B-LCLs homozygous for DRBI*0102 required 100-fold higher concentrations of HA306-320 to induce proliferative responses by both HA1.7 (Fig. la) and HA3.41 (Fig. lb) and induced peak proliferative responses that were -2- fo ld lower in magnitude. Recognition of intact hemagglutinin was even more dramatically affected by HLA-DR1 allelic variation. For DRB 1"0101-homozygous B-LCLs, equimolar concentrations of intact hemagglutinin and HA306-~20 elicited superimposable proliferative response profiles from both HA1.7 (Fig. lc relative to a) and HA3.41 (Fig. ld relative to b). With DRBl*0102-homozygous B-LCLs, however, superoptimal concentrations of intact hemagglutinin relative to HA306-~20 failed to induce any

a

Workshop # . • 9 0 0 6

• 9o,o tT . • 9 0 0 5 0 9 0 0 2

9 0 7 8 [] 9 0 7 9 /

C

r-

- 4 - 3 - 2 - 1 0 1 o z

b

d , ,

- 4 - 3 - 2 - 1 0 1 o

Log [Antigen] (uM)

FIGURE 1 Proliferative responses ofT-lymphocyte clones HA1.7 (a and c) and HA3.41 (b and d) to increasing concentrations of HA306-320 (a and b) or intact influenza hemagglutinin (c and d) presented by B-lymphoblastoid cell lines homozygous for DRBI*0101 (closed symbols) or DRBI*0102 (open symbols). The Tenth International Histocompatibility Workshop designations for the B-lymphoblastoid cell lines used are indicated. Results are expressed as mean tritiated thymidine (3H-TdR) incorporation determined as described in Materials and Methods.

significant TLC proliferation (Fig. lc and d relative to a and b, respectively).

To ensure that deficiencies in the recognition of antigen and antigenic peptide demonstrated by TLCs HA1.7 and HA3.41 were not the result of antigen presentation in the context of an alternative HLA-D region-encoded product on DRB 1"0102-homozygous B- LCLs, monoclonal antibodies specific for the various class II and class I isotypes were assessed for their ability to block TLC proliferation. Proliferation of TLC HA1.7 in response to HA~06-320 was blocked by a monoclonal antibody specific for HLA-DR, but not by monoclonal antibodies specific for either HLA-DQ, -DP, or HLA- A,B molecules when B-LCLs homozygous for either DRBI*0101 (Fig. 2a) or DRBI*0102 (Fig. 2b) were used as antigen presenting cells. Interestingly, complete inhibition of the DRBl*0102-restricted response was observed at a tenfold lower concentration of HLA-DR- specific monoclonal antibody than was required for complete inhibition of the DRBl*0101-restricted response (Fig. 2). Identical results were obtained in studies of the restriction pattern of TLC HA3.41 (data not shown).


2 5 I O

x 2O

E Q. (o

~ J

E 15 0

LC, c3

0

c

5 rY" x~ I--- i

-r- 0 r,3 O

I

~ , O O O ' O O

A n t i b o d y S p e c i f i c i t y

None HLA-DR HLA-DO H L A - D P H L A - A , B

EMonoclonal Antibody] (ug/ml)

FIGURE 2 Effect of increasing concentrations of monoclonal antibodies specific for HLA-DR, HLA-DQ, HLA-DP, or HLA-A,B on the proliferative response of T-lymphocyte clone HA1.7 to HA306_320 presented by B-lymphoblastoid cell lines homozygous for DRBI*0101 (a) or DRBI*0102 (b). Cells were cultured with 1 /~M HA306-320 in the absence of monoclonal antibodies or in the presence of antibodies at the indicated concentrations throughout the duration of culture. Results are expressed as mean tritiated thymidine (3H- TdR) incorporation determined as described in Materials and Methods.

Assessment of levels of peptide binding to products of HLA- DR1 allelic variants: Biotinylated peptide-binding studies. DRBl*0101-homozygous B-LCLs (Fig. 4a)gener - ated dose-dependent shifts in fluorescence intensity upon culture with increasing concentrations of biotinylated HA~06-~20 that were greater in magnitude than those generated by DRBl*0102-homozygous B-LCLs (Fig. 4b). The data shown in Fig. 4 are those of a representative experiment. The mean _+ standard deviation calculated from four independent determinations of the linear mean fluorescence channel number (LMFCN) observed upon binding of 50 t~M B-HA306-~20, controlled for background fluorescence, revealed significantly greater levels of binding to DRBl*0101-homozygous (58.51 -+ 5.44) relative to DRBl*0102-homozygous (36.39 -+ 9.05) B-LCLs. In contrast, flow-cytometric

FIGURE 3 Effect of amino acid substitution at positions 85 and 86 of the DR1 B chain on the rate of formation of stimulatory complexes involving HA306-320 and HLA-DR1. (a) B- LCLs homozygous for DRBI*0101 or (b) DRBI*0102 pulsed at 37°C with 1, 10, or 100 gtM HA300-~20 for the times indicated, as described in Materials and Methods, were used an antigen-presenting cells. Results are expressed as the percent at each time point of maximal responses observed in cultures of pulsed presenting cells provided with optimally stimulatory concentrations of HA306_320 throughout the duration of culture.

Analyses of B-LCLs homozygous for DRB 1"0101 or DRBI*0102 as stimulators revealed dramatic differ- ~ 100

C ences in the kinetics of formation o f p e p t i d e - D R 1 com- o plexes stimulatory for T-cell proliferation. At the lowest ~- 80 concentration of peptide tested (1 tzM) DRBI*0101- ~: homozygous B-LCLs (Fig. 3a) generated maximally E 60 stimulatory levels o f p e p t i d e - D R 1 complexes within 60 "~ minutes of peptide pulse, whereas no stimulatory com- :~

40 plexes were formed on DRBl*0102-homozygous B- LCLs within 2 hours (Fig. 3b) or even after an overnight

00 2 0 pulse with peptide (data not shown). Increases in the 0_ pulsing peptide concentration correlated with decreases in the time required for formation of maximally stimula- 0 tory levels of pep t ide -DR1 complexes on both loo DRBI*0101- (Fig. 3a) and DRBI*0102- (Fig. 3b) ~'

c homozygous B-LCLs; however, dramatic differences be- o 80

£2_

tween the two allelic forms of DR1 were still observed, o For instance, DRBl*0101-homozygous B-LCLs pulsed ~" 6o with 10 and 100 ~M peptide for only the period of time E required to pellet them by centrifugation stimulated T- a 40 lymphocyte proliferation at 50% and 90% of maximal levels, respectively (Fig. 3a). In contrast, comparative ~d

2 0

levels of T-lymphocyte proliferation were induced by ~0 DRBl*0102-homozygous B-LCLs only after 2 hours of o_

0 pulsing with peptide at these higher concentrations (Fig. 3b).

b

0 0.25 0.5 1.0 2.0 Time (Hours)


4 0 - -

a

[B-HA3o5_320] A ~ 50.00 uM 30 - -- 25.00 uM

: ~/ ~ - - - 12.50uM . " , - I , h ~ . . . . . 6.25 u .

• " i " ~ ,1" ~ . . . . ~ . 1 2 o ~ • / ' l ~ ~ - - 0 uM 20 I : 6 ',

/. ,,,, : v - ." : 'I~

I : , " ; ~% / : : ,I : ,~ " . * 1 % 10 : r :~ ,, |

: 'I : ~% L_ ; I k " %

," , I .. , , , , \ E ~ 4 0 ~

Z 4 0 b - - D R m * o l o l

. . . . . D R B I * O I 0 2 (.9 30

3 0 .- : '- s" 20

.,:.. 'I ', ,,/% ,,i !

/ / / , " " I ~ ", I I 101 102 103 10 4

/i ". I, \ Log Ruoresce e

10 l . l "

10 ~ 102 10 ~ 10

Log Fluorescence

FIGURE 4 Flow-cytometric analysis of levels of dose-dependent binding of biotinylated HA30c._320 (B-HA~0c.-320) to the surface of B-LCLs homozygous for DRBI*0101 (a) or DRBl*0102 (b). B-LCLs were cultured overnight with B- HA30(,_320 at the indicated concentrations, stained with FITC- conjugated avidin D and analyzed for fluorescence intensity by cytofluorimetry as described in Materials and Methods. Equivalent levels of expression of HLA-DR1 on all DRBI*0101- and DRBl*0102-homozygous B-LCLs was demonstrated by staining with FITC-L243 (inset).

light scatter of cells cultured in the presence of such high concentrations of peptide. The demonstration of 50% inhibition of the binding of B-HA306-320 to DRBl*0101-homozygous B-LCLs by a tenfold molar excess of unbiotinylated peptide is consistent with competitive inhibition studies of Busch and Rothbard [15]; however, failure to inhibit binding of biotinylated peptide to DRB 1"0102-homozygous B-LCLs has not previously been reported. We conclude from these data that both saturable and nonsaturable components contribute to the measurable binding of biotinylated peptide to cells expressing both forms of HLA-DR1. The inhibition pattern observed with DRBl*0101-homozygous B-LCLs reveals a clearly measurable saturable binding comPonent that was less apparent on DRBI*0102- homozygous cells• These results emphasize the importance of performing competitive inhibition analyses when relative binding affinities are estimated from studies of biotinylated peptide binding.

Assessment of levels of peptide binding to products of HLA- DR1 allelic variants: Iodinated peptide-binding studies. The HLA-DR1 specificity of the binding of HA306-~20 to cells homozygous for allelic variants of DR1 was estab-

FIGURE 5 Dose-dependent inhibition of binding of biotinylated HA306_320 to products of HLA-DR1 allelic variants. Following preincubation for 60 minutes with indicated molar excesses of unbiotinylated HA~0c~-320, cells were cultured overnight with 12.5 tzM biotinylated HA306-320, washed, and stained with FITC-conjugated avidin D. Inhibi- tion of binding is expressed as a percentage of the level of binding observed in the absence of unbiotinylated peptide as described in Materials and Methods.

100

analysis following staining with a monoclonal antibody specific for HLA-DR revealed no significant differences in levels of expression of HLA-DR1 on the surface of o ° 80

c

DRBl*0101-homozygous relative to DRBI*0102- o~' homozygous B-LCLs (Fig. 4, inset).

The ability of unbiotinylated HA306-~20 to compete o 60 with biotinylated peptide for binding to HLA-DR1 was "

-O used as a measure of the saturability of peptide binding. In three independent experiments, unbiotinylated E 4o

c HA30~-320 present at up to a tenfold molar excess rela- "E tive to B-HA~06-320 inhibited binding of the biotinylated peptide to DRB 1"0101-homozygous B-LCLs in a dose- 2O dependent manner whereas inhibition of B-HA30c~320 binding to DRB 1"0102-homozygous B-LCLs was con- siderably less extensive (Fig. 5). Inhibition by greater

0 excesses of compet i tor peptide could not be assessed in these experiments because of gross alterations in the

/X

z5 /% /X

/X

/k

Hoplo type • DRBI*OIO1 /k DRBI ,O102

i , , 2.5X 5X 7 5X

[Cold C o m p e t i t o r ] (MoiGr Excess)

l l0x


600 a b

E 5OO o_ o

-A 400 [D

Q~

"G 300 03

C~

200

E ~oo

IHLA-DR ~HLA-DQ ~HLA-DP ~HLA-A,B

0

M o n o c l o n o l A n t i b o d y S p e c i f i c i t y

FIGURE 6 Assessment of complex formation involving HA306-320 and HLA-encoded gene products. HLA-DR, HLA- DQ, HLA-DP, or HLA-A,B-encoded molecules were im- munoprecipitated from detergent lysates of DRBI*0101- (a) or DRBI*0102- (b) homozygous B-LCLs exposed to usI- HA306-320 in overnight culture as described in Materials and Methods. Counts per minute (cpm) in immunoprecipitates were determined by T scintillation spectroscopy. Results are expressed as the mean -+ standard deviation calculated from 2-3 separate binding experiments.

lished by immunoprecipitation of 125I-HA306-320 from detergent lysates of cells cultured with radiolabelled peptide. 125I-HA306_~20-DR1 complexes were detected by immunoprecipitation with an HLA-DR-specific monoclonal antibody from lysates of DRBI*0101- homozygous B-LCLs (Fig. 6a) but not from lysates of B- LCLs homozygous for DRBI*0102 (Fig. 6b). Com- plexes of uS1-HA306_320 with HLA-DQ, -DP, or HLA-A,B molecules were not observed in lysates of either DRBI*0101- or DRBl*0102-homozygous B- LCLs (Fig. 6a and b) as evidenced by an inability to immunoprecipitate radiolabeled peptide with monoclonal antibodies specific for these determinants.

A representative autoradiograph of HLA-DR1 purified by L243-Sepharose affinity chromatography from lysates of surface-iodinated B-LCLs homozygous for DRBI*0101 or DRBI*0102 is shown in Fig. 7. The major products isolated are HLA-DR1 a and/3 chains migrating at apparent molecular weights of 32 and 34 kD, respectively. The band corresponding to an apparent molecular weight of 66 kD represents unreduced heterodimer and has been observed previously in purified preparations of HLA-DR [29]. The minor band appearing at an apparent molecular weight of 44 kD is an unidentified artifact previously observed in preparations of HLA-DR purified with L243 [ 17]. Quantitative immunoprecipitation of HLA-DR1 from lysates of B- LCLs homozygous for DRB 1"0101 or DRB 1"0102 has been confirmed using a second HLA-DR-specific mono-

clonal antibody, DA6.147 (data not shown). These results confirm the results of flow-cytometric analysis revealing equivalent levels of expression of HLA-DR1 variants at the cell surface and maintenance of proper conformation and demonstrate that the failure to immunoprecipitate complexes of uSI-HA306__320 with HLA-DR 1 from lysates of DRB 1 *0102-homozygous B- LCLs was not due to a failure of the antibody to interact with HLA-DR1 on these cells, but a failure of the peptide to bind.

DISCUSSION

We have investigated whether precisely localized allelic variation within HLA-DR1/3 chain genes affects recog-

FIGURE 7 Quantitative immunoprecipitation of HLA- DR1 from lysates of B-LCLs homozygous for DRBI*0101 or DRBI*0102. HLA-DR1 was purified from detergent lysates of surface-iodinated B-LCLs by immunoaffinity chromatography using an HLA-DR-specific monoclonal antibody coupled to cyanogen bromide-activated Sepharose. Eluted uSI-HLA- DR1 was characterized by SDS-polyacrylamide gel electrophoresis and subsequent autoradiography as described in Ma- terials and Methods. The positions of DR a and/3 chains and of molecular weight markers with molecular weights given in kilo-Daltons are indicated.

D R o - - ~

D R ~ - - ~

DRB1 "0101 DRB1 "0102

t,D '¢ U3 04 O 0 0 ~

0 0 0 0 0 0

• ,=---'-- 97.4

66.2

• ,=-..--- 45

: 31

2 1 . 5


nition of foreign antigen by DRl-restricted cloned human T cells. The products of two alleles, differing only at positions 85 and 86 of the/3 chain, have been studied: DRBI*0101 (V85G86) and DRBI*0102 (A85V86). We have observed that conservative substitutions involving positions 85 and 86 of the DR1 /3 chain profoundly affected T-cell recognition of foreign antigen in that DR1 molecules possessing DRBl*0102-encoded /3 chains were inefficient in presenting antigen in peptide form and were virtually incapable of generating the appropriate peptide epitope from intact antigen. Appro- priate restriction of peptide recognition in the context of both forms of HLA-DR1 was verified by monoclonal antibody-blocking assays. Excluded as an explanation for deficient T-cell stimulation was a deficiency in the level of expression of the variant form of HLA-DR1 at the cell surface. Results of peptide-binding analyses using variant forms of HLA-DR1 were consistent with the hypothesis that amino acid substitutions at positions 85 and 86 of the DR1/3 chain dramatically influence T-cell recognition, at least in part, through an effect on the ability of the DR1 molecule to bind antigenic peptide.

Our observations, similar to those of Busch and Rothbard [15] and Busch et al. [30], of quantitatively lower levels of binding of antigenic peptide to products of the DRBI*0102 allele are consistent with the inter- pretation that the affinity of interaction of the substituted HLA-DR1 molecule with this peptide is altered. Specifically, we observed that cells expressing DR1 /3 chains that possess A85V86 demonstrate (a) a requirement for higher concentrations of HA306-~20 to induce maximal levels of peptide-specific T-lymphocyte proliferation, (b) saturation of T-cell proliferative responses at levels equal to one-half those observed using cells expressing V85G86 as stimulators, (c) a requirement for lower concentrations of HLA-DR-specific monoclonal antibody to inhibit proliferative responses completely, and (d) extremely slow but dose-dependent kinetics of formation of maximally stimulatory numbers of peptide-DR1 complexes. Peptide-binding studies are revealed lower levels of binding of peptide to DRBl*0102-homozygous cells, which coincided with virtually undetectable site-specific binding as demonstrated by failure (a) to competitively inhibit binding of biotinylated peptide with native peptide and (b) to im- munprecipitate complexes of radiolabeled peptide and HLA-DR1 from detergent lysates. Consequently, whereas HLA-DR polymorphism may indeed influence the conformation of bound peptide, that effect may be only secondary to an effect on the overall affinity of interaction dictated by one or a few residues.

Several accumulating observations support proximity of DR /3 chain position 86 and position 309 of the HA~06-320 peptide. First, Jardetsky et al. [31] have dem-

onstrated that only the tyrosine residue at position 309 of HA307-319 appears to be important for binding of this peptide to HLA-DR1. Second, binding and T-cell recognition of HA307-319 is affected by polymorphism or mutagenesis resulting in amino acid substitution at positions 85 and/or 86 of the HLA-DR1 [15, 30, and the present study], HLA-DR4 [30, 32], and HLA-DR7 [33] /3 chains. Finally, deficient binding and presentation of unsubstituted HA307-3~9 by mutant DR7 molecules bearing amino acid substitution at position 86 of the/3 chain could be overcome by substitution within the peptide at position 309 [33]. These interpretations necessitate a reevaluation of the proposed structural model of the HA307_319-DR1 complex [22] in the context of the HA307-3~9-DR7 model proposed by Krieger et al. [33] favoring placement of the amino-terminal end of the peptide near the carboxy-terminal end of the DR1 /3 chain cx helix. Furthermore, modeling of HA307-319 within the DRl-binding groove in an ex- tended conformation similar to that proposed by Krieger et al. [33] would preserve DR1 and T-cell receptor contacts previously proposed to be important for recognition of this peptide in the context of DR1 [22, 34]. Verification of this model will require structural studies of purified HLA-DR1 molecules bearing the specific peptide.

The results of our studies are also relevant to proposed models for the structural basis of allorecognition that have been formulated to account for (a) direct interactions between the T-cell receptor and polymorphic residues of the alloantigen and (b) influence of polymorphism on binding of peptides that may be required for formation of the alloantigenic epitope. Several recent studies have documented effects on HLA-DR1- [5, 6] and -DR4- [35] specific alloreactive T cells that were attributable to substitution at positions 85 and 86 or position 86 alone of the DR1 or DR4/3 chains, respectively. Our present studies suggest that those alloreactive TLCs that were affected in their recognition of alloantigen by polymorphism at position 86 of the DR/3 chain may depend upon occupancy of the binding groove by a particular peptide in the proper orientation. Alloreactive T cells that recognize alloantigenic epitopes unaffected by DR/3 chain polymorphism at position 86 could be either peptide independent in their recognition of alloantigen or, more likely, dependent upon occupancy of the groove by another peptide whose interaction with HLA-DR is not influenced by polymorphism at DR/3 chain position 86. Studies of the binding and presentation of HLA-DRl-restricted peptides other than HA~06-~20 are currently in progress in our laboratory to determine the extent to which position 86 of the DR1/3 chain is important for interaction with a variety of antigenic peptides. These studies


should aid in the understanding of the relative importance of peptide involvement in recognition of MHC molecules by alloreactive T cells.

In our present study, the level of binding of antigenic peptide to its appropriate restriction element was evalu- ated at the surface of intact cells. One important advan- tage of the use of cellular binding assays is the potential for assessment of peptide binding to HLA-DR molecules under conditions that are more closely related to those under which peptides derived from whole antigens are complexed with MHC molecules; however, even these assays take issue with the problem of physiologic relevance. For instance, we characterized in this study the product of an HLA-DR1 allelic variant, DRB 1"0102, which demonstrated quantitative deficiencies in the levels of peptide to which it bound, qualita- tive differences in the saturability of peptide binding, and deficiencies in the rate of formation of stimulatory complexes with antigenic peptide. All of these deficiencies correlated not only with relatively inefficient peptide presentation to cloned T-lymphocytes, but also, interestingly, with virtual failure to present a naturally processed peptide derived from intact antigen. Failure of the HLA-DR1 variant to stimulate T cells with intact antigen at concentrations in vast excess of optimally stimulatory concentrations of peptide suggests that the peptide binding we observed may not directly correlate with the physiologic formation of complexes involving MHC molecules and peptide epitopes derived from intact antigens. A relationship between assayable peptide binding and peptide-MHC complex formation may, however, be understood in the context of the proposed importance of a rapid kinetic intermediate in the formation of stable peptide-MHC complexes in intact cells [13]. During antigen processing, failure of antigenic peptides to complex rapidly with MHC molecules may result in failure to protect them from complete proteolytic degradation [36]. Our observations that the product of the DRBI*0102 allele was associated with both slow kinetics of formation of stimulatory peptide- MHC complexes and an inability to generate the appropriate peptide epitope from intact antigen suggest that deficiencies in peptide binding may, indeed, be related to the ability of an MHC molecule to generate a complex with protein-derived peptide. Whereas formation of stable complexes may depend exclusively upon direct interactions between antigenic peptide residues and amino acids within the MHC molecule, third-party proteins such as invariant chain [37, 38] and members of the heat-shock protein family [39, 40] may play important roles in facilitating peptide binding to MHC molecules in intact cells. The assumption, therefore, that T- cell antigen recognition depends solely upon the formation of a trimolecular complex may be overly sim-

plistic, given the large array of molecular interactions that may be required to form a structure recognizable by the T-cell receptor. Ongoing efforts in our laboratory are aimed at further characterization of the physiologic requirements for interaction of antigenic peptides with MHC molecules within cells and establishment of the relevance of those interactions for the phenomenon of antigen processing and presentation.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the excellent technical assistance of Roxanne Pinzl, Deborah Albers, and E. Roger Knight. This work was supported grants AI22832 and HL44162. D.D.E. is the recipient of Research Career Devel- opment Award AI00799. D.N.-N. is the recipient of National Research Service Award AI08425.

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Documents

Effects of localized HLA class II β chain polymorphism on binding of antigenic peptide and stimulation of T cells