monoclonal antibody (b27m2) subdividing hla-b27

Monoc lona l Ant ibody (B27M2) Subdiv id ing HLA-B27

F. Carl Grumet, Brian M. Fendly, Linda Fish, Steven Foung, and Edgar G. Engleman

J A B S T R A C T : Because HLA~B27 shows strong association with ankylosing spondylitis, it u'as of inrer¢st

to produce routine mbnodonal anttbodies to study this antigen in detail. The first such anti-B27 antibody, anti-B2 7M l. reacted u'ith 100% of B27 + cells and cross-reacted with homoz,g~us B7 cell~. In the present report a second monoclonal antibody, anti-B27M2, is described which subdirides HLA-B27 into two variants. Among health)' B27 ~ indirMuals. 87% were B27MI " and B27M2 ~. and 13% were B27MI + but B27M2 by standard lymphocytotoxicity ass~:~s. The specificity of B27M2 antibody for the HLA-B27 molecule was confirmed u'ith blocking studies using F(ab'J, fragments of HLA alloantibodies. Both the B27M2* and B27M2 rariants of HLA-B27 bred true in family studies. Unlike B27M1, B27M2 antibody did not react wttb B7 but did react with the rare Bu'47 allele.

For a,tibody binding studies Epstein-Barr rirus-tran~fomued B ~,mphoblastoid cell line~ w~r~ derired from normal donors KCA (by lymphocytotoxicity shown to be B27". B27M I ". B27M2 ~ ~. VC (B27 ' . B27M 1 ' . B27M2 - ). and WH ¢B27 . B27MI -. B27M2- J. Each cell line bound equiralent amounts ofW6/32 emonoclonal anti-HLA-ABCJ: KCA and VC bound similar am~unt~ of anti-B27M 1. but only KCA bound substantial anti-B27M2 antibody. These data are consistent with a model in which all B27 antigens possess a B27MI epitope: wherea~ most. but not all. possess an additional and distinct epitope, B27M2. Although the relation of these genetic rariants to disease susceptibility remains to be detertnined, the arailability of epitope-specific monoclonal antibodies should help to refine our understanding of the structure and function of HLA molecules.

A B B R E V I A T I O N S BSA bovine serum albumin IEF isoelectric fo=using CML cell mediated lympholysis MHC major histocompatibility com- DMEM Dulbecco's modified Eagle's plex

medium NCS newborn calf serum EB Epstein-Barr PBL peripheral blood lymphocytes HAT hypoxanthine, aminopterin, PBS phosphate buffered sa~ne

thymidine RT room temperature HBSS Hanks balanced salt solution

The human MHC*, or HLA system, has an extensive polymorphism currently defined by alloantisera [1]. Finer discrimination among alleles of the HLA loci would be of interest because of the possibility for improving transplantation and

From the Department of Pathologs. Staptford Unirersity School of Medicine. Stanford. California. Address requests for reprints *o F. Cad Grumet. M.D.. Department of Patholo~'. L-235. StanJ~rd

Unirersity Medical Center. Stanford. CA !)4305. ReceiredJanuar[' 18. 1982: accepted March 8. 1982.

Human Immun:)lo~y 5, 61-72 (1982) © Elrcvicr Science Publiflfing Co., Inc., 1982 52 Var~tcrbilt Ave., New York, NY 10017

61 I) 198-8859182/O5~V,161 - [ 2S 2." 5

62 F.C. Grumet et al.

transfusion compatibility testing [2,3] and for providing better markers tbr de- tection of possible human immune response and disease-susceptibility genes [4,5]. In this latter ree;rd, because of its striking association with ankylosing spondylitis and Reiter's syfMrome, the HLA-B27 alloantigen of the MHC has attracted a great deal of attention [6-8]. Further study of the B27 antigen itself may help clarify whether this cell surface protein plays a direct role in disease pathogenesis or if it merely serves as a readily detectable genetic marker for some nearby, specific disease-susceptibility gene. Because murine monoclonal antibodies produced by somatic ceil hybridization (hybridomas) offer the promise of finer discrimination among antigenic markers, numerous attempts to produce hybridoma anti- B27 antibody have been undertaken, but only limited success has been reported [9]. The first anti-B27 hybridoma, B27M1, reacted with all B27-positive cells but also demonstrated cross-reactivity to the B7 antigen. In the present report we describe a new monoclonal anti-B27 antibody that is not cross-reactive with B7 and that has the unique characteristic of subdividing the current B27 Mloan- tigen.

MATERIALS A ND METHODS

Anticoagulated whole blood was obtained from normal volunteer blood donors. From these samples PBL were isolated b7 Ficoll-Hypaque density gradient cen- trifugation and were used for test procedures either immediately after separation or after storage in liquid nitrogen.

An immunization protocol was devised that was biased towards generating cytotoxic lgM antibodies [10-13] and that harvested the lymphoid cells most likely to be exposed to a focused antigen c h a l i c e . For immunization, 4{) x 10 c' PBL obtained from a Caucasian male patient ~HLA phenotype A2; B27, Bw44, Bw4; Cw2) with Reiter's syndrome were susper~ded in PBS ~ and administered into each footpad of a 6-week-old BMb/c femM¢ mouse. Three days postimmun- ization, the inguinal and axiil~'y lymph m~des ~ d the spleen were harvested, and the lymphoid cells suspended in DMEM* {Gilxo, Grand Island, NY). For fusion the lymphoid cells were mixed at ~, ratio of 5:1 with SP2/08~2 mouse myeloma cells, centrifuged at 400 × g for l0 rain ~ 22~C, and the pellet resuspended in 1 mi of 50H polyethylene glycol gLBDH, Poole, England) in DMEM. This mixture was graduMly resuspended in,o 7 ml DMEM over a 6-rain period at RT*, centrifuged, and ultimately resuspended in DMEM supplemented with 15t~ NCS ~ g lrvine Scientific, Santa An& CA) in • finid concentratMn of l0 million viable cells per cubic centimeter. One tenth cubk centimeter of th,s cell suspension was then phted into e~'h of 60 wells of a 96-well tissue c~leure plate (Flow Laboratories, McLean, VAL Following overnight incubation at 37°(: in 6H CO2, each well received an equal volume of 15~ NCS in DMEM supplemented with 8.0 x I0~" M hypoxanthine, 2 × l0 ~° M aminopter/n, and i.3 x 10 -~ M thymidine (HAT*) (Sigma, St. Louis, MO) for selection of fused cells. On each of the following 3 days, b~df of the vo|ume of each well was ;eplaced with fresh HAT medium. By day 10 those wells showing contiiJued gro~gh in the HAT medium had supertl~tant samples drawn off to test for antibody activity by a standard microlymphocytotoxkity assay [ t4].

Alloantisera used for HLA typing consisted of reagents obtained from the authors' own labo~'atories, from the NIH serum bank, or by exchange with other investigators. Monoclonai antibody reagents included B27M l, a lympho~=ytotoxic IgG2a anti-B27 antibody [9]; W6/32, a lymphocytotoxi6 Ig~2~ ami-HLA-ABC antibody [ 15] kindly-provided by Dr. Peter Parhgm, Stanford University; L368, a noncytotoxic lgG-I anti-13,~-mkroglobuiiti m3tibody [16J kindly provided by

Monoclonal Antibody Subdividing HLA-B27 6~

Dr. Ronald Levy, Stanford University; and A2,28M 1, an lgM mom~chmal an,i* body with anti-A2,A28 specificity (r = 1.00) produced in the authors' l ~ r ~ - tories. HLA typing was performed as previously described [14].

For cytotoxicity and binding titrations alloantisera were diluted in m~rmM, male donor, pooled AB serum; hybridoma antibodies were diluted in 3~; BSA ° (Miles Laboratories, Ilkhart, IN) in HBSS ~ (Gibco, Grand Island, NYL Lym- phocytotoxicity blocking experiments were performed essentially as previously described [ 16]. Preparation of noncytotoxic F(ab')2 blocking fragments generally followed the method of Madsen and Rodkey [ 17] using pepsin (Sigma Chemk'A Corp., St. Louis, MO) digestion of ammonium sulfate-precipitated lg from "A- loantisera. The only change from their procedure was that the final step of the F(ab'), preparation consisted of dialysis against PBS, rather than column purification, prior to concentration to one-fifth the original volume. Briefly, target cells which had been prelabeled with the vital fluorescent dye carboxyfluorescein diacetate (Molecular Probles, Piano, TX) were preincubated with blocking antibody or F(ab'), preparations for 30 rain at RT. Cells, now precoated with blocking reagent, were then distributed into tissue typing trays which had been preloaded with cytotoxic test sera in the usual manner for standard HLA typing. After incubation, first with antiserum alone and then with added complement, test results were read by a microscope-mounted photomultiplier tube detecting residual intracellular fluorescence as a quantitative measure of cell viabilit2.,' [ 17,18}. Quantitative cytotoxicity titrations were also read with the same photomultiplk-r system.

Cell surface immunoglobulin binding was determined by an indirect radioim- munoassay. A 50A volume of 105 cells suspended in PBS with lq~ BSA and 0.2c?; sodium azide were incubated with 100A of each antibody dilution fi~r 1 hr at RT in flexible, round-bottom microtiter plates (Dynatech Corp , Alexandria, VA) precoated with 2 ~ BSA. The cells were washed three times with PBS- BSA and then resuspended in 50A (1 x 10 ~' c p m ) o f a 1:I mixture of 12~i-go~¢ anti-mouse-lgG and anti-mouse-IgM (Tago, Burlingame, CA). The goat anti- mouse antibodies were iodinated by the chloramine-T method (Amersham, R~- dioiodination Techniques, 1979). After 1 hr at RT, the cells were washed three time.~ with PBS-BSA. Cell pellets were transferred by cutting and placing individual wells into tubes, and counted for bound radioactivity in a gamma scin- tillation counter.

lmmunoglobulin subclassing was performed by Ouchterlony analysis with com- mercial antisera (Miles Laboratories, El!:hart, IN).

EB* virus transformation was graciously performed by Mr. David Buck, Cetus Corporation, Berkeley, California. Using the technique of Sly et al. [19], 2 × l ip PBL were incubated with 0.2 ml of EB virus suspension derived from a culture of marmoset line B95-8, with a feeder layer of irradiated rabbit fibro- blasts. The transformed B cell lines were subsequently HLA typed and compared with the typing of the original cell donor to confirm identity.

RESULTS

One of the hybridomas, selected because 10 days after fusion its antibody-containing culture fluid showed differential cytotoxicity to B27 + compared to B27 PBL, was repeatedly subcloned by the method of limiting dilution. The subclone, secreting only an IgM immunoglobulin and designated B27M2, was expanded in culture and was also grown as ascites in syngeneic host mice. The results of lymphocytotoxicity screening of B27M2 supernatant fluid against a panel of PDL from 43 unrelated blood donors, representing almost all HLA-ABC antigens,


are shown in Table I. B27M2 antibody reacted with most, but not all, B27 + individuals as well as with an individual possessing the Bw47 antigen, but not with any of the six cells that were Cw2 +, B27- , Bw47-. To confirm the B27 and Bw47 specificities, supernatant fluid was further tested and shown to be lymphocytotoxic to PBL from 84/96 additional individuals with B27. These included Caucasian, Negro, Mexican-American, Japanese, and Chinese donors among the 84 reactive cells and Caucasian,Japanese, and Chinese among the nonreactive cells. Among the small number of B27 +, B27M2- cells, no A, B, or C antigen occurred at an unusually high frequency. On further testing, the B27M2 antibody also reacted with 3/3 cells with Bw47, and 0/4 B7/B7 homozygotes. These latter cells were selected because of the previously observed cross-reactivity of the earlier B27M I antibody with B7. B27M 1 antibody reacted with all of the B27 * and the B7/B7 cells, but not with the Bw47 cells. Based on the population data, B27M2 thus defines a subset of the B27 antigen and also recognizes all Bw47 ÷ cells. Although family member cells were not available from the Bw47 + cell donors, families from several of the B27 + individuals were. As seen .:n Table 2, both the B 2 7 M 2 0.e., reactive with B27M2 antibody) and the B27M2- (i.e., unreactive with B27M2 antibody) variants orB27 bred true. From the population and family studies it was also clear that B27M2" vs B27M2 - reactions did not correlate with the Cwl or Cw2 ant/gens so often associated with B27.

To test that B27M2 was reacting with the B27 molecule and not some other possibly non-HLA cell surface antigen in linkage disequilibrium, further studies were perfi)rmed. First, target lymphocytes from a B27 + individual, against which B27M2 supernatant fluid was cytotoxic to a titer of 16, were precoated with anti- 32-microglobulin antibody L368 for testing in a technique known ~o block cytotoxicity of HLA-ABC alloant/bodies but not other alloantibodies {e.g., DR) [16]. After precoa6ng, the iymphocytes were no longer lysed by B27M2 supernatant at any dilution, ~emonstrating that anti-B27M2 reacts with a ~32-microglobulin associated molecule. Second. B27 ÷ target lymphocg'tes were precoated with noncytotoxic F~ab')2 preparation of and-B27 alto~mtiserum Lehr, As shown in Figare 1, following precoating, the cells were shown to be relative!y resistant to lysis by Lehr native antibody and by anti,B27M2, while cytotoxicity of anti- A2,28M 1 was equivalent for both uncoated and for precoated cells. Additional controls with F(ab')2 preparations of allo~ti-HLA-A2,28~serum Harb)-antibody did not affect B27M2 cyto~oxicity0 but effectively blocked anti-A2,28M l lysis.

TABLE 1 Lymphocytoto~:ic geactivity of B27M2 (supernatant) antibody to PBL from unrelated donors"

A ~ 6 g e n / B 2 7 M 2 react iv i ty :

Ant igen + / + + / - - / + - / -

B27 (, 1 ~ 35 B w 4 7 | () 7 35 B27 + B w 4 7 " ~ O 35

"Panc-I intEnded two +~+r rmn'+ c,+..Ih idcm~lk'd ~+ e~ch f++~ ~J+" m' HLA-A~+C ,+pet-if~ty ¢~<+cel~t +',+mr d ~ ft.~lo,w+ng: one cell eaxh fi~r Aw~6. Bw4 ~. B~59. B*v6L ~ Te*~; ~o +d|+ with A w ~ ~r Bw58+ Spedfu: ccJL~ w~h B2 ~ cross- reacting .antigen group ar~/gen~ weft: three L~'. tbr¢~e B~-4 L three ~ 2 . three- Bw4~. two BwSL three. 13w55, two Bw56. two Bw60. three B ~ 6 | . ~ a five ( ~ 2 ", B2 "~ e e ~

numerator, and . ) anti-B27M2 beir~g re~cdve ~ ¢ ) ~ r n~nue*xdv*: ~ - ) i~ ~he d e ~ i ~ r .

M o n o c l o n a l Ant ibody Subdiv id ing H L A - B 2 7 65

TABLE 2 Lymphocytotoxic reactivity, in families, of B27M2 (supernatant) antibody

Family (Ethnic group)

Parents Children's B27M2 r e a , rivi,9"

haplotype B27M2 reactivity + -

Lau Caucasian a A 2 , C - ,Bw38 -

b A 3 , C w 7 , B 7 t A w 2 4 , C w 4 , B w 3 5 + _ d A 3 , C w 2 , B 2 7

Tat Caucasian a A I °Cw7,B8 -

b Aw24 ,CwT,B7 c A 2 , C w 2 , B 2 7 +

d A 2 8 , C w 8 , B 14 Del" Caucas ian a A 1 , C - , B 8 -

b A 3 , C - , B I 8 c A 2 , C w 1,B27 +

d A 2 9 , C - , B w 4 4 T h , Caucas ian a A 2 , C w 6 , B 17 +

b A I I , C w I , B 2 7 c A 2 , C w 5 , B w 4 4

d A 3 , C w 2 , B w 5 1 Sai Mexican-American a A I , C w 7 , B w 4 9 --

b A 2 , C w 7 , B w 4 4 c A w 2 4 , C w 2 , B 2 7 +

,t A 2 6 , C - , B w 4 8 Ben ~ Caucas ian a A2 ,C - ,B7 -

b A w 2 4 , C w 4 , B w 3 5 c A I , C w 2 , B 2 7

d A 2 6 , C - , B 3 7 O k a J a p a n e s e a A w 2 4 , C w 3 , B w 6 2

b A w 3 3 , C w l , 3 , B w 4 6 c A2 ,C - ,B27

d A w 2 4 , C - ,Bw52

a/d,b/d,b/d d c

a/c a /d ,a /d ,b 'd

b/c a /d ,b/d ,b/c - d ~

~ d , ~ d ~ d

a/c a/d

t i c , t i c

a/d,Wc,b/c

"Cw6-Cw8 not tested fi)r.

~c-d haplotype = A2.C- .Bw44.

Unless a n e w class I antigen is proposed that is in strong linkage disequil/br/um with B27 and that reacts with noncytotoxic antibodies in the B27 alloandserum, these experiments are most consistent with anti-B27M2 reactivity directed aga/nst the B27 molecule.

To further investigate the difference between B27M2-defined subdivisions of B27, B cell lines were derived from EB virus transformation from a selected set of cells: KCA, (phenotype A I 1, Aw24; B27, Bw44, Cwl, Cw5)which was (by cytotoxicity) B27 +, B27M1 +, B27M2+; VC (phenotype A3, Aw24; B7, B2% Bw4, Bw6; Cw2) which was B27 +, B27M1 +, B27M2-; and WH (phenotype A l l , A28; Bw35, Bw35, Bw6, Bw6; Cw4, Cw5) which was B27-, B27Ml- , B27M2- (negative control cell). PBL from each cell donor were tested for cytotoxicity with both anti-B27M 1 and -B27M2, and each of the cell lines was then tested for binding of antibodies B27M1, B27M2, and W6/32. The results of these experiments are shown in Figures 2 and 3. The cytotoxicity curves show that both B27 + cells are effectively lysed by B27M1. The slightly greater cy-


too

a

~. s o

2 5 ' "

ANT IBC~DY mLUTiON

100

i 7S

50

o

25

ANTIBODY DILUTION

lOO

>- 75

!- 2 S

c . - '~ ~@ ~ ,oo

2~

ANThOnY ID I~JT~

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ANT~0D¥ D~UTION

FIGURE t F(ab')2 cyt,~toxkity bJ~xking ex~rimcnts with target lymphocytes of phenotype A2, A3: B27, Bw39, Bw6; Cw2. C¢ll~ were pr6~t6M with the Fiab')2 fragment of anti-B27 allo~ntiserum Lehr (-'[~'-L F{~,b')¢ fremont of anti-A2,A28 M|oantlserum Harb (--9)--), or tissue culture medium ¢ontro| (-A-L exposed m v~ying dilutions of either cytotoxi¢ (~) lJative ai~antibody Lehr. ~b) B27M2 super~ant. (c) B27M2 ascites, or (d) A2,28M 1 li~ites before fin~d ~ddition ofcom#e~nt . Fa~h p~int is the me~n, gith vertical bars representing the range, of t r i p l i ¢ ~ e t e s t s .

totoxicity of B27MI against VC, compared to KCA, is most likely due to the weak cytotoxic cross-reactivity of ,hat antib~|y lot" the B7 antigen present as ceil VC's other B locus specificity. The VC ce|l is not |ysed by B27M2 antibody even when used ~s ascites fluid with a 10" titer ~gainst the KC cell. The binding experiments yielded similar results, with the two B27" cells VC and KCA binding equal amounts of the W6/32 and B27MI antibodies. The KCA |ine binds substantial amounts of B27M2, whereas VC binding f~r that andbody is almost indistinguishable from nonspccific binding to the negative control WH cell. The rising background binding of B27M2 ~ B27M1 to WH with higher antibody concentrations is a common problem with monockmal ~dbodies [20], at~d the nonspecificky of this b ~ c ~ noise is indkated by the binding observed with high antibody concentrations tested ~ s ¢ the HLA-ABC- Daudi cell.

Monoclonal Antibody Subdividing HLA-B27 67

mo i i

75 / 0 t

50 /

2 5

ANTIBODY DILUTION

50 ' •

0

d o d ~ ~ d o 6 o o o ¢~ o m o o

A.TIBODY o,.UT,O.

IO0 r .~t'- &

! C

o so / -

2 ~

. . . . . . . .

oJ .

A N T I B O D Y D I L U T I O N

FIGURE 2 Monoclonal antibody cytotoxicity titrations against lymphocytes with B27M2 variant antigens. Target PBL of donors KCA (B27 ' , B27M1 +, B27M2 ~, - 0 - ) , VC (B27 +, B27MI ~, B27M2-, -.I--1.- and WH (negative control, B27-, B27MI , B27M2 . -A-) were tested for percent cytotoxicity with varying dilutions of (a) B27MI ascites, (b) B27M2 ascites, or (c) B27M2 supernatant before addition of complement. Each point is the mean, with vertical bars representing the range, of triplicate tests.

D I S C U S S I O N

Because B27 is a genetic marker for susceptibility to certain spondyloarthro- pathies [6-8] , it was of interest to determine if this antigen could be further subtyped with hybridoma antibodies. Although the first monoclonal anti-B27 antibody, anti-B27M1, recognized an epitope common to all B27 + ce~s, the second monoclonal anti-B27 antibody, anti-B27M2 (described here), was more selective by both cytotoxicity and binding studies, identifying a variant of the B27 antigen. This variant, termed B27M2-, comprised 13% of the normal cells recognized as B27 + by alloantisera but was not distinguishable by conventkmaJ allogeneic typing sera. Both the B 2 7 M 2 and B27M2 + variants were present in different ethnic groups, and each bred true in family studies. B27M2- PBL were neither susceptible to lysis, even by exceptionally high titered B27M2 ascites


m ~)

o

o

~a | ~6

// ,'.1

11

_._ I . . . . . !

ANT~iODY D~UT~N

7O

15

K so

u_

|

I /

I I

. . , . ~ . f °..d~

A N T I ~ ¥ ~ U T I O N

FIGURE 3 Binding ~ffmon,gbn~d ~fibodie~ to B cell i/nes derived froth normal donors ~ith B27 variant antigens a~d to the f.t~mdi ceil line. Antibody binding is expressed on tlxe vertical axis a, counts per minute !cpm) × IO ~. T~rget B cell l/n,., KCA (B27 ", B27M1 ¢, B27M2*, -4~-L VC (B27-, B27MI ". B27M2 , - 'O'-L WH mngadve control, B 2 7 , B 2 7 M I . B27M2, -A-) ~ D~di ( A l ~ . -O-) were incubated first with varying dilmions of(a) B27Ml as~ite,. ~b) B27M2 ~i tes . or (c) W6/32 gscites, and then (after appropriate wash steps) with ~ o n d ~ re'4ent a2*l-hbek'd go~t ami-mous¢ (IgG + lgM) antibody. Each lx~int represents the m¢~, with vertkM bars the r~ge, of triplkme tests.

fluid, nor capable of specific binding of the B27M2 ~qdbody. The cytotoxic cross- reactivity of B27M2 with Bw47 but no, BT, ¢ompm-ed to that o f B27MI with B7 but not Bw47 (i.e., eomplc¢l~entmT c¢oss-rc~£dve ~t te rns) , strongly suggests qualitative rather than qu~qtitarice d/fferences ~ the B27 ~n t i~n in B27M2 + vs B27M2 - i n d i v i d ~ Further, both B27M2 ~ ~ B27M2- cells were effectively lysed by anti-B27Ml antibody. ~ ¢ ~ r g ~ d d~,~ ~ s t thai ~1 f.~,27

Monoclonal Antibody Subdividing HLA-B27 69

~" 1~

x

D 1( Z

/ / /N / I t

I l i i i i

% 0,:, o ',o ~ o

ANTIBODY DILUTION

FIGURE 3 ¢continuedJ

molecules possess a B27M1 epitope and most, but not all, also possess a second distinct epitope, B27M2.

All three B cell lines (KCA, VC, and WH) bound essentially equal am~mms ofW6/32, indicating equivalent cell surface densities of total HLA-ABC antigens. The two B27 + cell lines, KCA and VC, bound similar amounts of B27MI, suggesting approximately equal cell surface densities of B27 molecules. The cross-


reactivity of B27M1 with the B7 antigen of VC is estimated to contribute less than 20~ of the binding of B27M1 to VC, based on results of B27M1 binding assays on many different established B27 ÷ and B7 ÷ cell lines (P. Parham, personal communication; J. Shively, personal communication.) This weak cross-reactivity problem ultimately can be circumvented by testing binding against a cell line derived from a B27 +, M2- , B7- donor; development of such a cell line is currently underway. Again in contrast, B27M2 showed substantial binding to KCA, whereas binding to VC was similar to binding to the WH B27- control cell. These data are consistent with the concept that cell surface density of B27 molecules is similar on cells from both of the B27 variants, but that the B27M2 specific epitope is present (or sterically accessible) only on the B27 molecule of B27M2 + cells. Whether the differences between the B27M2 ÷ and the B27M2 - variants is due to HLA region encoded, primary protein sequence differences or to HLA-linked posttranslational differences must await structural studies or amino acid sequencing, or both, of the two different molecules.

Over the past two decade~ the polymorphism of the HLA system has been extensively defined by alloanfisera. Xenoantisera have been of limited use [20- 23L It has been generally anticipated, however, that the availability of hybridoma antibody techniques would provide a new source of useful xenogeneic HLA typing reagents. From studies of hybridoma anti-H-2 alloantibodies in mice, it also appears that because of their purity and rest:icted specificity, hybridoma reagents have the potential of defining new MHC antigenic relationships [24- 28L Many of the antibodies produced, however, recognize only supertypic specificities, shared by different alleles and occasionally even by antigens of different H-2 or HLA loci [25-27]. Akhough some of the rodent hybridoma antibodies reactive with HLA antigep.s recognize particular HLA allospecificities [29,.30], most appear to be either supertypic or fail to correlate with any known antigens or cross-reactive groups as currently identified by alloantisera. With r¢~pect to the abilit~ ' of xecogeneic hybridoma antibodies to improve HLA antigen characterization by "splitting" or subdividing well-defined private allo',pecificities, hybridoma antibodies recognizing possible A2 and B40 variants (each present on single cell lines) or DR4 using rosetting technique have been reported [20,31,32]~unfortunately without sufficient family and population da~a to prove the genetic determination of the v~iant antigens. B27M2 and l~ ty described in the present report is thus the first hybridonm antibody to both identify and definitivdy subdivide a well-defined, private HLA antigen, B27~ into two genetically determined subtypes or v~iants beyond those identifiable with alM- antisera. One of the variants, B27M2, is M~o now shown to share ~t identical or cross-reactive epitope with the Bw47 ant/gen.

Biddeson et M. [33] have reported an HLA-A2 variant in an individual which is detectable by CML* but not serologicMly. This A2 v~riant was also demon- strable by IEF* of purified antigen. Studies are currently underway to determine whether or not the vafant B27 molecules described here also ea~q be distinguished by CML or IEF; however, the availability of a cyr;otoxic monoclonal antibody makes the B27 variants substandaUy easier to detect them those requiring the more difficult CML or IEF technolog~s.

Given the establishment of the vat/ant B27 subtypes, an obvious application is to determine if this subtyping Mfects correktdon of the 1327 antigen with susceptibility to spondyloasthropmhy. ~ o f t ~ low freq~e~.y of the B27M2- variant, a relatively large popuhtion of patients must be carefully studied to reliably establish such a correlation, gn this contest it wiR be ofimerest to evaluate the role of the B27M2 epitope0 pa~kuhrly in 1~47" individ~s, ~ a ,lise~e- susceptibility marker. Finally, the availability ~ff different monodonM antibodies

Monoclonal Antibody Subdividing HLA-B27 7l

d i rected against different epi topes on the same B locus molecules should permi t mapping o f epi topes on HLA molecules [ 16].

ACKNOWLEDGMENTS This wor' was supported by grants and contracts from the NIH (Al21662, NOI-AI-82558, CA24607, HL-07127), the Cetus Corporation, a~ld the State of California.

The authors gratefully acknowledge the generous gifts of reagents from Drs. Ronald Levy and Peter Parham; the EB transformation of cells by Mr. David Buck; the technic~d assistance of Ms. Karen Watson and Dolly Ness, and the assistance of Ms. ChaHene Butte and Mrs. Susan Booth in the preparation of this manuscript.

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3. Yankee RA, Grumet FC, Rogentine GH: The selection of compatible platelet donors for refractory patients by lymphocyte HL-A typing. N Engl J Med 281:1208, 1¢X)9.

4. Sasazuki T, Grumet FC, McDevitt HO: The association between genes in the ma~r histocompatibility complex and disease susceptibility. Ann Rev Med 28:425, 197".

5. McDevitt HO: Regulation of the immune response by the major histocompatibi[i~- system. N EnglJ Med 303:1514, 1980.

6. Brewerton DA, Albert E: Rheumatology. In: J Dausset and A Sveggaard, Eds. HLA and Disease. Copenhagen, Munksgaard, 1977, pp. 94-108.

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monoclonal antibody (b27m2) subdividing hla-b27

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