Monoclonal antibody 1a3 recognizes a monomorphic epitope unique to DQ molecules

Ionoclonal Antibody l a3 Recognizes a Monomorphic Epitope Unique to DQ Molecules Linda Shookster, Takami Matsuyarna, * Gerd Burmester, and Robert Winchester

ABSTRACT: Monoclonalantibody la3 reacts with a unique monomorphicepitopepresent only on DQ molecules as determined by immunoprecipitation, two-dimensionalgel analysis and partial Nterminal amino acid sequencing. This monoraorphicepitope is found on DO. molecules of all homozygous B lymphoblastoidcelllines tested, including those that type as either DQwl, l ~ w 2 , DQw3, or DO. "blank." Analysis of thepredictedsecondarystructure of class H moleculesindicates that the regions most likely to constitute an antigenic determinant are variable in DQ molecules, possibly accountingfor the rarity of antibodies to monomorphic DQ epitopes. ABBREVIATIONS MHC MoAb IEF

major histocompatibility complex monoclonal antibody isoelectric focusing

SDS-PAGE sodium dodecylsulfatc po!yacrylandde gel electrophoresis

INTRODUCTION The genes encoding the human class II or major histocompatibility complex (MHC) molecules are grouped into three subregions: DR [1], DO. [2], and D P [3]. The molecules encoded by these regions are similar in their a, ~ subunit configuration, yet structurally distinct [4]. The heavier a chains differ among subregions in their overall variability: DR a chains are invariant, whereas DQ and DP a chains are polymorphic, containing variable residues scattered throughout the first domain. DQ a chains contain many such residues and DP c~ chains, very few. On the other hand, the/3 chains of all three regions contain amino acid substitutions within their first domains. DR/3 chains have three principal hypervariable regions, D Q / 3 chains possess four discrete hypervarhble regions, and DP B chains contain a small number of scattered variable residues [5]. Between hypervariable regions, each a and ~8chain possesses invariant framework sequences, some of which are shared among molecules of each subregion, while

From the Department of Rheumatic Diseases, Hospital for Joint Diseases, New York University School of Medicine, New York, N Y 10003. Addross reprint requeststo: Linda Shookster, M.D., Department of Molecularlmmunobiolo~, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, N Y 10021. Received May 7, 1987; acceptedJuly 10, 1987. ®Present address: Laboratory of Tumor Immunology, Dana Far3*rCancer institute, Harvard Medical School, Boston, MA 02115. Human Immunology 20, 59-70 (t987) © Elsevier Science Publishing Co., Inc., 1987 ~2 Vanderbilt Ave., New York, NY 10017

59 0198-8859/87/$3.50

60

L. Shookster et al. others are specific for molecules of each subregion. The second domains of both a and ~ chains in ~l three subregions are minimally polymorphic. Monoclonal antibodies (MoAbs) recognizing MHC molecules are divided into two categories: those directed to monomorphic determinants representing framework regions of the molecules, and those directed to polymorphic, or variable, epitopes. Many MoAbs have been described m monomorphic epitopes on DR molecules, whereas only a small number of MoAbs to polymorphic epitopes have been reported. In contrast, there is only one report of a MoAb directed to a monomorphic determinant located on a class II molecule other than DR: MoAb B7/21, which recognizes a determinant found on DP molecules [6]. All reported antibodies directed to DQ molecules recognize polymorphic epitopes and may be placed into four groups. The first contains MoAbs that recognize epitopes that occur in parallel to those recognized by alloantisera, such as IVD12 [7] and Genox 3.53 [8], directed to DQw3 and DQwl molecules, respectively. The antibodies of the second group recognize polymorphic epitopes expressed on more than one, but not all varieties of DQ molecules, like Leu 10 [9] or 106al [10], both of which recognize epitopes expressed on D Q w l and DQw3 molecules but not on DQw2 molecules. The third group contains antibodies directed to isoallotypic epitopes that are monomorphic for one isotype but polymorphic on DQ molecules, e.g., SG171 [11], which recognizes all DR molecules but only the DQw2 molecules encoded by DR7 haplotypes. The last group of existing MoAbs, such as Tu22, recognizes monomorphic specificities located on D Q molecules as well as either mono- or polymorphic determinants on DR molecules [12,13]. The present article is a description of MoAb la3 directed to an epitope restricted to TI~Qmolecules. Seconda~.y protein structure of class lI molecules is estimated as a means of explaining the rarity of described MoAbs to monomorphic DQ epitopes.

MATERIALS A N D M E T H O D S Cell lines. The following human homozygous B-lymphoblastoid cell lines were used: LG2 (DW1, DRI, DQwl) consanguineous, GM3107 (DW2, DR2, DQwl) comanguineous, GM6823 (DW3, DR3, DQw2) consanguineous, GM3162 (DR3, DQw2, DQw3), 85TO (DR4, DQw3), GM3103 (DW4, DR4, DQw3) consanguineous, GM3105 (DWS, DR5, DQw3), GM7254 (DR6, DQwl), Burkhardt (DR7, DQw2), 3163 (DWT, DR7, DQw2) consanguineous, GM6815 (DR9, DQw3), KT3 (DWI5, DR4, DQ"blank"), Has 15 (DW15, DR4, DQ"blank"), and Madura (DW8, DR8, DQ"blank"). Those with a GM designation were obtained from the Human Mutant Cell Repository (Camden, NJ). Burkhardt was a gift of Dr. R. Karr, LG2 was a gift of Dr. R. Gatti, and KT3 was obtained from Dr. Go Nepom. Has 15 was a gift of Dr. Nancy Reinsmoen, and Madura was origillally obtained from Dr. A. Svejgaard.

Monodonal antibodies. MoAb la3 was initially prepared by Dr. G. Burmester in this laboratory. It is of 72a class as determined by radioimmunoassay using defined reagents provided by Dr. Patricia Mongini, also of this laboratory. MoAb gVD12, obtained from Dr. R. Giles, and Genox 3.53, from ATCC (Rockville, MD), have been previously described [7,8]. They recognize polymorphic DQ determinants corresponding to DQw3 and DQwl, respectively. MoAbs SG157 and 8G171, obtained from Dr. S. Goyert, are directed to DR monomorphic determinants present on all B cell lines. In DR7 cell lines, MoAb SG171 also binds to DQ molecules [1 ;.].

A Monoclonal RecognizingAll DQ Molecules

61

Internal labeling and immunoprecipitation. This was done as previously described [14], except that cells were initially suspended at 4 x 106 cells/ml in medium lacking methionine. One millicurie of [3s$] L-methionine was then added to each culture and the cells incubated for 15 hr at 37*(;. Once lysod in buffered 0.5% Triton X-100, each cell suspension was incubated with the appropriate MoAb for 2 hr at 4*(2, and then incubated with sheep anti-mouse IgG for 1 hr at 4*(; if the MoAb was ~/1 (IVD12 & Genox 3.53). Thirty microliters of 50% v/v protein A-Sepharose (Sigma, St. Louis, MO) was then added to each mixture and the mixture rotated for 1 hr at 4°C.

lsodectric focusing. Isoelectric focusing (IEF) was performed according to the method of Shackelford and Strominger [15], with the modLfications previously described [14]. Briefly, the first dimension consisted of discontinuous sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) cm ! 2% gels as described by Iaemmli [16]. AII samples were run ~mder nonreducing conditions. Regiop.s of the gel encompassing the desired molecular weight range for a and B chains were localized using dansylated molecular weight standards and were excised as strips from the SDS gel. IEF was then performed using an LKB 2117 Multipbor flat bed gel apparatus (LKB Instruments, inc., Rockville, MD), with the following amounts of individual ampholytes (LKB Instruments, Inc. Rockville, MD) added per gel: pH 2.5--4, 0.3%; pH 3.5-5, 0.3%; pH 5-8, 0.4%; pH 7-9, 0.4%; pH 3.5-10, 1.1%. Gels were electrofocused at 1000 V for 7V2 hr at 8°C, fixed in 7.5% acetic acid and 5% methanol, incubated wlth EN3HANCE (New England Nuclear, Boston, MA) for 1 hr, and then dried and autoradiographed on Kodak XAR-5 film at -70°C.

N-terminal amino acid sequencing. Cells were labeled in the same manner as above, this time using 2.0 mCi of [3HI L-tyrosine per cell line. After incubating with a MoAb as above, a and B chains were separated by electrophoresis on 12% polyacrylamide tube gels. The gels were frozen on dry ice, cut into l-ram slices, soaked overnight in 0.01% SDS, and assayed for radioactivity corresponding to a- and B-chain proteins. The slices containing/3 chains were filtered through 0.45-~m Millex-Ha filters (Millipore Corporation, Bedford, MA), d/alyzed against 0.001% SDS, and lyophilized. The samples were then applied to a model 470A protein sequencer (Applied Biosystems, Foster City, CA) for 30 or 31 automated Edman degradations. The 02PTH program was used. The fractions were then dried in a Speed-Vac centrifuge (Savant Instruments, inc., Hicksville, NY), reconstituted in acetonitrile, and then a:sayed for radioactivity on a Beckman LS3133T beta counter (Beckman Instruments, inc., Berkeley, CA) as previously described [14].

Western blotting. Western blotting was performed according to the method of B. Cohen et al. [17]. Unlabeled $STO B lymphoblastoid cells were lysed as above, except I ml of buffered 0.5% Triton X-100 was used to lyse 5 x 10~ cells. SDSPAGE was performed on 12% slab gels using 1.5 × 106 cell eqmvalents of this whole-cell lysate per lane. Electrophoretic transfer was then performed as described by Towbin et al. [18]. The nitrocelldose strips were incubated with neat culture supernatants and then washed with Western buffer consisting of 0.4% Tween 20 [19]. Each lane was developed using an ELISA consisting of alkalinephosphatase conjugated sheep anti-mouse hnmunoglobulin followed by Fast Violet B salt (Sigma, St. Louis, MO) as substrate.

62

L. Shookster et al.

acid ---> (-) SG,57

Genox 3.53

bose d

o ~,~

SGI57

ORa

~

Genox3.53

~B ~OQB

¢

o

f la3a (+)

~ra~3B J ~ Io3Q

lo3

FIGURE 1. Two-dimensional gel profiles of DR (a) and DQ molecules (b and c) from a DR1 homozygous cell line, LG2, and of DR (d) and DQ molecules (e and f) from a DR2 homozygous cell line, GM3107. DR and DQ molecules were isolated with MoAb SG157 (DR) and MoAbs Genox 3.53 and la3 (DQ). The x and y axes represent the IEF and SDS-PAGE dimensions, respectively.

Secondary structure analysis. The method of Garnier [20] was used to estimate secondary structure of the first and second domains corresponding to several allelic products of each of the three class II loci. A decision constant corresponding to an a helix content o f less than 20% was used because the second a and/8 domains [21], and ver:~,possibly the first domains as well [22], have strong amino acid sequence homology to the immunoglobulin-constant domains, which are predominantly/8oplemed sheets. Hydrophilicity analysis was performed by using the method o f H o p p and Woods [23], averaging six amino acids for each estimate, approximately the length of an antigenic determinant [23,24]. Since balb/c mice are the source of the present anti-MHC MoAbs, estimates of the potential to generate an antibody to a determinant were made based on the following criteria. For an amino acid sequence to be considered antigenic, it should be estimated to be surface prominent on the molecule as well as nonhomologous to any regions on I-E or I-A molecules. Accordingly, such a region must be hydrophilic and coincide with a/8 turn, a coil, or an a helix. Surface prominent framework regions unique to only one o f the three human class II molecules were considered as possible sources of MoAbs to unique monomorphic epitopes on DP, D Q or DR. Surface prominent variable regions unique to one o f the three class lI molecules were considered as possible sources of MoAbs to unique polymorphic epitopes. Surface prominent regions shared among combinations o f DP, DQ, and DR molecules or shared with I-A or I~E molecules have been excluded. These models, of course, cannot estimate which areas o f the

A Monoclonal Recognizing All DQ Molecules

(-)

acid - - >

SGIS?

63

base

57

?i

Io3

(+)

FIGURE 2. Two-dimensional gel profiles of DR (a) and DQ molecules (b) from a DR3 homozygous cell line, GM6823, and of DR (c) and DQ molecules (d and e) from a DR7 homozygous cell line, Burkhardt. DR molecules were isolated with MoAb SG157, and DQ molecules with MoAbs SG171 and la3. The "a's" correspond to acdn at molecular mass 44,000 Daltons. The brackets in a and b represent the distances of the a and subunits from the invariant actin molecules, the further distance corresponding to the lower molecular mass° molecule are involved in cx-//chain interactions or form conformational determinants. RESULTS Isoelectric Focusing Figure 1 demonstrates that MoAb la3 detect~ the same molecules as MoAb Genox 3.53 on DR1 and DR2 DQwl homo~:ygous cell lines. O f note is that each DR homozygous B cell line is electrofocused on a single gel, allowing optimal comparison of isoelectric points. Different amounts of each of the//-chain glycosylation intermediates are recognized by MoAb's Genox 3.53 and la3, but the isoelectric points are identical. O f interest i,, that MoAb la3 reacts well by immunofluorescence with B cells incubated ~,vernight in medium to which 2 /zg/ml of tunicamyc/n was added (data not sk :~wn), diminishing the likelihood that it reacts with a carbohydrate epitope. T~ .~ apparently double a-chain precipitated by MoAb la3 in Figure I f is curre~:gy under investigation, with preliminary N-term/hal sequencing of both chaiv.t~ revealing tyrosines in positions corresponding to D Q ~ chains only. Figures 2a and 2b show that MoAb 1a3 recognizes a molecule distinct from the DR chains on the homozygous DR3 ceil line, GM6823. No MoAb recognizing DQ molecules on DR3 ceil lines was

L. Shookster et al.

64

acid

base d

• 0

SG157

~Q

DRa

SGI57

e

e

b

DQa

[VOl2

-

I'V' D 12

OQa

J6~

OOB ¸ f

la3 c ~lo3a

la3

{+}

|¢$a

06. la3~

FIGURE 3. Two-dimensional gel profiles of DR (a) and DQ molecules (b and c) from

a DR4 homozygous cell line, GM3103, and of DR (d) and DQ molecules (e and f) from a DR5 homozygous cell line, GM3105. DR molecules were isolated with MoAb SG157, and DQ molecules with MoAbs IVD12 and la3.

available as a control, but amino acid sequencing (see below~ provides conclusive evidence tho- bIoAb la3 recognizes D Q molecules on this cell line. O f interest is that the majority of D Q a chains that have been molecuh,dy sequenced contain very few methionine residues [5]. Because of this, it is often difficult to discern [3~S]L-methionine-labeled D Q a chains on two-dimensi¢3al gels, such as those shown in this figure. Although the isoelectric points of the ~: and fl chains d~,~ected by MoAb la3 and SG157 are similar, the chains recog~fized by MoAb 1~3 are lower in molecular mass than are the DR subunits, as can be seen by their positions (bracketed) in relation to actin molecules (labeled a) as a :~.ference point. As a control for DQ molecules on the DR7 ceil line, MoAb SG171 was used, as shown in Figure 2d. This MoAb recognizes both D Q and DR molecules on DR7 cell lines [11]. As Figure 2e demonstrates, bIoAb la3 recognizes a molecule identical to the DQ subunit shown in Figure 2d. Figure 3 shows that bIoAb la3 appears to isolate predominantly lateT, or more acidic, DQ fl-chain glycosylation intermediates than those precipitated by MoAb IVD12 on DR4 and DR5 DQw3 cell lines. Figure 4 demonstrates that MoAb la3 recognizes a molecule distinct from DR molecules on the DR4, Dw15, DQ"blank" cell line, KT3. Once again, the methionine-poor D Q a chain is barely discernible on this gel.

A Monoclonal RecognizingAll DQ Molecules

acid --->

65

base

(-) ORa : SG157

FIGURE 4. Two-dimensional gel profile of DR (top) and MoAb la3-precipitated molecules (bottom~ on the DR4, Dwl5, DQw'blank" cell line, KT3. DR molecules were isolated with MoAb SG157.

~t

Io3

sa3Q

la3B N-Terminal Amino Acid Sequencing Further evidence that MoAb la3 recognizes DQ molecules was obtained by Nterminal amino acid sequencing of the/3 chains predpitated by MoAb la3 on cell lines corresponding to DQwl, DQw2 (including DR3 and DR7), DQw3, and DQ"blank." As shown in Table 1, DQ/3 chains always contain tyrosine residues at position 16 and sometimes at positions 9 and/or 30 [25-27]. DR chains, on the other hand, contain tyrosine residues in at least one of the following positions: 10, 13, 26, and 30 [25,27], thus distinguishing them from DQ/3 chains on the DQ alleles studied here. DP ~ chains are minimally variant and almost always contain tyrosine residues at positions 7, 9, 16, and 30 [28]. Table 1 indicates that from cell lines GM3107 (DR2,DQwl), GM6823 (DR3,DQw2), GM3163 (DR7,DQw2), GM3105 (DR5,DQw3), Has 15 (DR4, DQ"blank"), and Madura (DRS, DQ"bhnk"), MoAb la3 precipitates molecules that have tyrosine residues at positions corresponding to those of known DQ//-chain amino acid or DNA sequences, thus distinguishing them from DR or DP ~ chains.

Western Blotting Western blotting, performed to localize the epitope recognized by MoAb la3, revealed that this MoAb did not recognize the denatured, dissociated molecule (results not shown). This suggests that a conformadonal determinant was recognized.

66

L Shookster et al.

TABLE I

Comparison o f N-terminal amino acid sequences o f la3-reactive/3 chains with class I I / 3 chains. Y, single-letter code for tyrosine. Haplotype

la3-predpitate or sample sequence MoAb la3 DQ sequencea DR sequence" MoAb la3 DQ sequence~ DR. scq~icl'tce~' MoAb la3 DQ sequence" DR sequence" MoAb la3 DQ sequence" DR seuqence" MoAb la3 DQ sequencer DR sequence" MoAb la3 DQ sequence~ DR sequenced DP sequencer

Position

Cell line

DR DQ

GM3107 GM3107

2 wl 2 wl 2 3 w2 3 w2 3 7 w2 7 w2 7 5 w3 5 w3 5 zi blank zi blank 4 8 blank 8 blank 8 (invariant)

GM6823 0.127 GM3163 GM3163 GM3105 ~ GM3105 Has 15 KT3 MADURA GI

7

9

10

13

16

26

Y Y Y Y

30 Y Y Y

Y Y Y

Y Y

Y

Y

Y

Y Y'

Y Y Y Y Y Y Y

Y

Y

Y Y Y

Y Y

Y Y Y Y Y

Y Y

Y(F~

q~.ef.25. ~"quenced to position24 only. 'Ref. 26. '~gef.27. 'Heterogeneous,accordingto ref. 27. fRef. 28. Secondary Structural Analysis Figure 5 shows the results o f this analysis for sample sequences in D R , D Q , and DP. Only surface-prominent regions on each molecule, consisting o f at least four amino acids that potentially form the core o f an antigenic determinant, are displayed in this figure. Outlined boxes containing " x s" represent variable regions, while n o n o u d i n e d regions w i t h o u t " x s" indicate framework areas. T h e candidate antigenic regions for recognition by o u r present M o A b s contain residues that are predominantly framework on the D R a and fl chains, whereas those on D Q a and/3 chains almost entirely consist o f variable residues, Potentially antigenic regions o n D P molecules resemble comparable regions found o n D Q , D R , o r routine M H C sequences, leaving very few unique variable o r framework antigenic sequences. Table 2 tabulates the n u m b e r o f estimated variable and framework antigenic epitopes in each domain from Figure 5. This is in excellent agreement with Colombani and Lepage's tabulation o f the k n o w n M o A b s to m o n o - and ............ ,-:~a.Ja.~. *m.~a~ ..a r~Q op;mno~ t~ulya~.aultpttal, ~ ~ -'--r -- fTO1. -- . . which . . is presented in Table 2 u n d e r "actUal n u m b e r found." DISCUSSION M o A b la3 recognizes an epitope located on the products o f all D Q alleles studied so far. This M o A b is unique for two reasons: first, it appears to recognize de-

A MonoclonalRecognizingA~ DQ MolecuLes 1 st O O M A ~

2 nd D O M A I ~ as ~

~s;~ ~'~%~

cccc

O~O~elH~ 14 17

S3

39

65

tsgs ~

nccs

70 ~

~

~ 96 l

w

~03 e

~s ~

~40 ] 4 5 !

]63166 ~c¢c~

xxxgxxxgxg.~-xgxxxg 34

38

s~|o

I~ ' . 1

I ."2:1L-'-., I~l

I~Jl

.....

.H,~j.~s I,:o::~

. . . . . . . . .

IS.ll

88 8g

SCtC OP(~NH:

P,I_e

73 75

SSCC~! 102

108

CC~S 133 137

SCCC~S

164

172

FIGURI/5. Surface-prominentvariable and frameworkregions of DR, DQ, ~'ld DP a and/3 chains. Both first and second domainsare depicted, with amino termini at extreme left. Outlined boxes containing "x s" repre~nt variable regions, while straight lines correspond to framework regions. "+ s" represent areas of high hydrophiliciv/,while " - s" indicate less hydrophificregions. The letters t, c, h, and s correspond to predicted ~ turns, coils, a helices, and//sheets, respectively. The following amino acid sequences were examined to generate this figure:DRa (DR1,1) [28], DR~I (DR2,2) [34], DRy2 (DR4,6) [34], DQal (DR4) [25], DQa2 (DR4,6) [34], Dg~l (DR3,6) [25], DO.~2 (DR3,6) [34], DPa (Dw6,Dw6) [28], DP/31 (DRw6) [25], and DPB2 (DR4,w6) [34]. terminams on all homo- and heter~:ygous B cell lines ces~ed, and second, it recognizes only DQ molec~:I¢s as determined by immunoprecipitation and twodimensional gel an~!~sis and confirmed by N-terminal sequencing dam. MoAb la3 also re~et~ with DQ molecules that express "blank" specificR/es, among them the one described and designated DQwa [27], which is seen on B cells v/ping ~s DR4, DW15 & DR8, DWS. Previously described MoAbs m DQ molecules recognize either polymorphic determinants [30] or monomorph/c determinants shared with DP and/or DR [31,32]. A few MoAbs, such as those listed in Table 2 under "actual number found," have been suggested, but not proven, to recog~.:ze mo~omorph;c DQ a~ - .,. ~ ,. .,. .,. .;. . ... . .. . . . r-m Thc .~^A~. q-.,~ _.L:_L L - - s , , . ' -221 'J.A,.,aJ,~ J,L~.~, wa~t.aa £'ccognizes a monomorph~c determinant on DQ molecules [ 12], is reported to react with epitopes ~iso present on DR molecules [13]. There has been no conclusive explanation for the paucity of MoAbs that recognize monomorphic DQ epitopes. Assessment ofsecond0xyprotein structure by available computerized methods, however, provides an estimate of surface

6~

L. Shookster et al.

TABLE 2

Total Actual number found~

Total estimates o f framework and variable determinants (from Figure 5) located in each dcmain o f DP, DQ, and D R a and/3 chains. DR

Framework DQ

DR

Variable DQ

3

0

3 2

1 0

2

0

4

0

0 0

0 3

3 3

0 1

1

2

3

9 58

3 4

5

1

0

0

4 13

10 27

1

DP

Ratio DQ/DR

0.33 0.07

DP

Ratio DQ/DR

2.5 2.1

~Re£ 29. p. 211.

prominent sites on class H molecules that may be recognized by present murinego.nerated MoAbs. O u r data predict that anti-DR MoAbs predominantly recognize framework determinants located on either ot or 13 chains. In contrast, MoAbs directed to D Q epitopes are predicted to react almost exclusively with variable regions on both a and ~ chains. The abundance o f potentially prominent a-chain sites implies that a chains may have an important role in intercellular interactions. By the theoretical structural analysis presented here, it is unlikely that M o A b la3 recognizes an epitope located in the first domain o f D Q molecules, as there appear to be no framework antigenic candidates in this domain. Since this M o A b does not appear to recognize a glycosyla~ion, it is possible that it binds to an epitope in the second domain. Indeed, by the model presented here, one antigenic candidate exists in the second domain o f D Q a chains, and two antigenic candidates are located in the D Q ~ second domain.

ACKNOWLEDGMENTS

This study was supported by U.S. Public Health Service Grants AI 19411 and AM 35404. The authors wish to thank Dr. Carolyn Hurley for her helpful comments on this manuscript. REFERENCES 1. Winchester RJ, Fu SM, Wernet P, Kunkel HG, Dupont B, Jersild C: Recognition by pregnancy serums of non-HL-A alloantigens selectively expressed on B lymphocytes. J Exp Med 141:924, 1975. 2. Goyert S, Silver J: Isolation of I-A subregion-like molecules from subhuman primates and man. Nature 294:266, 1981. 3, Shaw S, Johnson AH, Shearer GM: Evidence of a new segregant series of B cell antigens that are encoded in the HLA-D region and that stimulate secondary allogeneic proliferative and cytotoxic responses. J Exp Med 152:565, 1980. 4. Trowsdale J, Young J, Kelly A, Austin P, Carson S, Meunier H, So A, Erlich H, Spielman R, Bodmer J, Bodmer W: Structure, sequence and polymorphism in the HLA-D region. Immunol Rev 85:5, 1985.

A Monoclonal Recognizing All DQ Molecules

69

5. Korman AJ, Boss JM, Spies T, Sorrentino R, Okada K, Strominger JL: Genetic complexity and expression of human class II histocompatability antigens. ]mmunol Rev 85:45, 1985. 6. Watson AJ, DeMars R, Trowbridge IS, Bach ~:H: Detection of a novel human class II HLA antigen. Nature 304:358, 1983. 7. Giles R, Nunez G, Hurley C, Nunez-Roldan A, Winchester R, Stastny P, Capra J: Structural analysis of a human I-A homologae using a monodonal antibody that recognizes an MB3-1ike specificity. J Exp Med 157:1461, 1983. 8. Brodsky RM, Parham P, Bodmer WF: Monoclonal antibodies to HLA DRw determinants. Tissue Antigens 16:30, 1980. 9. Chen YX, Evans R, Pollack M, Lanier L, Phillips J, Rousso C, W~ner N, Brodsky F: Characterization and expression of the HLA-D antigens defined by and-Leu 10. Hum Immunol 10:221, 1984. 10. Seremeds S: Unpublished data. 11. Goyert ~, Shively JE, Silver J: Biochemical characterization of a second family of human h molecules, HLA-DS, equivalent to murine I-A subregion molecules. J Exp Med I56:550, 1982. 12. Paweiec G, Shaw S, Ziegler A, Muller C, Wernet P: Differential inhibition of HLAD or :gB-directed secondary lymphoproliferative responses with monodonal antibodie~, detecting human Ia-like determinants. J Immunol 129:1070, 1982. 13. NakatsujiT, Moriya K, Ando A, Inoko H, Tsuji K: HLA-DQ structural polymorphism in HLA-DR2 associated HLA-D clusters. Hum Immunol 16:157, 1986. 14. Matsus'ama T, Schwenzer.L Silver J, Winchester R: Structural relationships between the D R ~ and DRy2 subunits in DR4,7 and w9 haplotypes and the DRw53 (MT3) specificity. J Immunol 137:934, 1986. 15. Shackelford DA, Strominger J: Demonstration of structural polymorphism among HLA-DR light chains by two-dimensional gel electrophoresis. J Exp Med 151:144, 1980. 16. Laemmli UK: Cleavage of structural proteins during the assembly of the head of the bacteriophage "1"4.Nature 227:680, 1970. 17. Cohen B, Deane D, Moxley M: Analysis of antibody specificity by western blotting. Dis Markers 2:135, 1984. 18. Towbin H, Staehelin T, Gordon J: Electrophoredc transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nad Acad Sci LISA 76:4350, 1979. 19. Luka J, $ternas L, Jornvall H, Klein G, Lerner R: Antibodies of predetermined specificity for the NH2 terminus of a cellular protein p53 react with the native molecule: Evidence for the presence ofdifferet~r p53s. Proc Nad Acad Sci 80:1199,1983. 20. Gamier J: Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 120:97, 1978. 21. Travers P, Blundell TL, Sternberg MJE, Bodmer WF: Structural and evoluriona,~/ analysis of HLA-D-region products. Nature 310:235, 1984. 22. Norcross M, Kanehisa M: The predicted structure of the h at domain. Scand J lmmunol 21:511, 1985. 23. Flopp TP, Woods Kg: Prediction of protein at~dgenlc determinants from amino acid sequences. Proc Natl Acad Sci 78:3824, 1981. 24. Berzofsky, JA: Intrinsic and extrinsic factors i~. ~rotein antigenic structure. Science 229:932, 1985.

70

L Shookster et al. 25. Giles R, DeMars R, Chang CC, CapraJD: Allelic polvmorphism and transassociation of molecules encoded by the HLA-DQ subregion. Proc Natl Acad Sci USA 82:1776, 1985. 26. Gregersen PK, Shen M, Song Q, Merryman p, Degar S, Seki T, Maccari J, Goldberg D, Murphy H, Schwenzer J, Wang C, Winchester R, Nepom G, Silver J: Molecular diversi~? of HLA~DR4 haplotypes. Proc Natl Acad Sci USA 83:2642, 1986. 27. Endo T, Obata F, Ishikawa N, Kojima H, Kashiwagi N: A novel human class II specificity, DQ "wa," resides on DQ molecules of DR4, Dwl5 and DR8, Dw8 B cell lines identified as DQ "blank." Hum Immunol, in press. 28. Kratzin H, Gotz H, Thinnes F, Kruse T, Barnikol H, Wernet P, Hilschmann N: Structure of human class 1I antigens expressed by a homozygnus lymphoblastoid B cell line. In BG Solheim, E Moiler, S Ferrone (eds): HLA Class II Antigens. Berlin, Springef, 1986, p 49. 29. ColombaniJ, Lepage V: Monoclonal antibodies against HLA class II antigens. In BG Solheim, E Moiler, S Ferrone (eds): HLA Class II Antigens. Ber!in, Springer, 1986, p 204. ~'* Karr RW: Immunochemical analysis of the Ia polymorphisms among the family of DR7-associated HLA-D specificities. J Immunol 136:999, 1986. 31. Shaw S, Ziegler A, DeMars R: Specificity of monoclonal antibodies directed against human and murine class I! histocompatibility antigens as analyzed by binding to HLAdeletion mutant cell lines. Hum Immunol 12:191, 1985. 32. Spits H, Borst J, Giphart M, Coligan J, Terhorst C, DeVries J: HLA-DC antigens can serve as recognition elements for human cytotoxicT lymphocytes. EurJ Immunol 14:299, 1984. 33. Yamamoto K, Kumagai Y, Hiramatsu K, Okumura K, Tada T: I-A homologous antigen on human lymphocytes detected by a monoclonal antibody. Immunogenetics 17:101, 1983. 34. Kaufman JF, Auffray C, Korman A, Shackelford D, Strominger J: The class II molecules of the human and murine major histocompatibility complex. Cell 36:i, 1984.