HLA serological cross-reactivity: HLA-B15 has two public antigens

HLA Serological Cross-Reactivity: HLA-B 15 Has Two Public Antigens

Benjamin D. Schwartz, Lisa K. Luehrman, Tsung Lee, and Glenn E. Rodey

A B S T R A C T : On the basis of their serologic cross-r'acth,ity, HLA antigens can be organized int6 cross-reactive groups or CREG's. We hare recently defined immunochemically two public alloantigenic determinants X and Y u,hich ca ~ account for the serological cross-reactil'ity of the B7-CREG and B5-CREG. respectively. O~.'e of the smaller of these CREG's consists of HLA-B 15 and B 17. Using microcytotoxicity testing, a fluoresceinated Protein A binding assa~. and chemical immunoprecipitation techniqut~, u,e have defined a net, public alloantigeuh determinant, tentatively designated "Z," w h i h is present on the 44,000 dalton glycoproteiu chains of HLA-B 15 and HLA-B 17, but dist;nct from the B 15 and Bi 7 determinants. Since HLA-BI 5 is also a member of the B5-CREG, ¢nd therefore bears allo, teterminant Y. this repoct constitutes the first immun~chemical demonstration of :u,'o public dete~'minants. Y and Z. on a singk HLA-B molecule. HLA-B 15.

A B B R E V I A T I O N S 0 . , / ~ 02-microglobulin PAGE polyacrylamide gel electro- CYNAP cytotoxicity negative, ab- phoresis

sorption positive SaCI Staphylococcus aureus Cowan IgG lmmunoglobulin G strain 2-ME 2-mercaptoethanol SDS sodium dodecyi sulfate NHIgG normal human immuno- TBS tris-buffered saline, pH 7.4

globulin G TSN tris-buffered saline contain- NHS normal human serum ing 0.25e/~ NP-40 NP-40 Nonidet P-40

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

The role of HLA antigens as the predominant factors in determining the success or failure of tissue and organ transplants was the initial stimulating event le~.dir~g to the delineation of the HLA system. This delineation of the HLA antigens is dependent on the analysis of the reaction patterns of various alloantisera on panels of human cells [1, 2, 3] and has led to the recognition of a number of antigenic determinants by the International Nomenclature Committee [4]. Analysis of the reaction patterns has also frequently shown that an alloantiserum

Fro~ the Howard Hughes Medical Institute Laboratory and Departments ,~f Medicine. Pathulog~ and Microbiology and Immunology. Washington Uniz'ersi(~ School of Medicine. and the Missouri-h'linais Reg/onal Red Cross Blood Program. St. Louis, Missouri.

Address requests/or reprints to Dr. Benjamin D. Schu'artz. Washington Unirersity School of Medicine. Department of Internal Ntedicine. Division of Rheumatology, 660 South E~,clid Are.. St. Louis. MO 63 t 10.

Received 1980.

Human lmmunoloigy 4, 331-345 (1980} ~) Elsevier North Holland, Inc., 1980 'J2 Vanderbilt Ave., New York./qY 10017

33t 0198-8859180/0433 I- 1552.25

32 Benjamin D. Schwartz et al.

produced after exposure to a given HLA antigen may also react with other HLA antigens as well This second set of reaction patterns has allowed the assignment of particular HLA determinants into cross-reactive groups or CREG's [5 ].

We have recently reported two studies which nave immunochemically defined two antigenic determinants, tentatively designated X and Y, that can explain the basis of cross-reactivity in two of the CREG's [6, 7]. Antigen X is a determinant found on the 44,000 dalton glycoprotein chain of HLA-B7, Bw22, B27, B40, and Bw42 molecules, and can adequately account for the cross- reactivity of this group, the B7-CREG. Its presence on several different HLA molecules led us to call it a "public antigen" [6]. Similarly, antigen Y is a public antigen on the heavy chain of HLA-BS, B15, B18, and Bw35 molecules, and can explain the cross-reactivity of the B5-CREG [7]. Since B15 and BiT have also been found to be cross-reactive, the absence of Y on the B 17 molecule i~ noteworthy [7].

Recendy, however, we have obtained a serum which reacts with cells bearing B15 and BI7. This report demonstrates that another public antigenic determinant common to B17 and B15, but not to other members of the BS-CREG, is responsible for this cross-reaction. Furthermore, since BI5 also bears antigen Y, this report constitutes the first demonstration that two distinct public antigens detected by alioantibodies, and defining different cross-reactive groups, are present on the same HLA heavy chain.

ATERIALS A N D M E T H O D S

Cells. Peripheral blood cells were obtained from healthy caucasian volunteers who had previously been typed for HLA-A and -B determinan~.s. Table 1 lists the HLA types of individuals whose cells were used as a source of various HLA-A and HLA-B antigens. In collecting cells, approximately 50 ml of blood was immediately heparinized with 0.5 ml ammonium hepari,, (100 /Mini) (Sherwood Medical Industries, St. Louis, MO). For all assays except the Protein A binding assay (see below), 25 ml of heparinized blood in each of two tubes was mixed with 5 ml 6% Dextran in normal saline (McGraw Laboratories, Glendale, CA) and allowed to stand for 60 rain at 37°(:. At the end of this time, the top 15-20 ml were collected, washed, and used as the source of white cells in subsequent steps [8]. For the Protein A binding assay, peripheral blood mononuclear cells were purified from the heparinized blood by centrifugation

TABLE ! HLA-types of individuals used as a source of various HLA antigens

HLA Antigen HLA-type of Individual A2 A2,A-,B 12,B40 BI5 AI,A9,BI2,BI5

A2,Aw24,B 15,Bw35 BI7 AI,A2,BI2,BI 7 B5 A2.A-,BS,Bw2 l,(Bw49)

A2,A-,BS,Bw2 l(BwS0) Bw35 A2,A-,B7,Bw35

A2,A3,B7,Bw35 B18 Ag,A-,BI4,BIL Bw21 A2,A-,BS,Bw2 l(Bw49)

A2,A-,B5,Bw2 l(BwS0) Controls A2,A28,B7,B40

HLA-B15 Has Two Public Antigens 333

over Ficoll-Isopaque (Pharmacia Fine Chemicals, Piscataway, NJ) for 15,000 x g-min [9].

Alloantiserum. Serum MH was obtained from a multiparous caucasian female (HLA-A3, A-, B8, B14) whose husband was typed as HLA-A2, Aw30, B7, BIT. When analyzed by microcytotoxicity [10], this serum reacted with HLA-A2 cells at a titer of 1:1, with HLA-B17 cells at a titer of 1:4, and with HLA-BI5 cells at a titer of 1:2, but had no titer on any other member of the BS-CREG (Table 2). In order to separate the various antibody populations within the set-am, a seri,~ of platelet absorptions was performed. The serum was first absorbed with HLA-A2, A31, BI2 , 1340 platelets [11] as follows. The platelets from one platelet transfer pack ( - 7 - 8 x 10 to platelets; 2-ml packed volume) were used for each absorption. The platelets were thoroughly washed with acid citrate dextrose solution-Formula A (Fenwall Laboratories, Deerfield, ILL diluted 1:5 in normal saline. The packed platelets were then resuspended in 60 ml (ff mrum MH, and incubated at 37°C for 2 hr. The platelets were removed by centrifugation at 12,000 x g for 15 rain. This absorbed serum was absorbed twice further with HLA-B17- , B15 + platelets (actual typing of platelets was HLA-A2, A-, BS, B15 and HLA-A2, A-, BT, B15) using the procedure described above. This three-t imes-absorbed serum when concentrated four-fold and retested by microcytotoxicity retained activity with HLA-B17 cells at a titer of 1:4, but had completely lost its reactivity on HLA-BI5 cells and had no reactivity on HLA-A2 cells (Table 2). This absorbed serum was used as a functionary monospecific anti-HLA-B 17.

The antibody which had reacted with the B17-, B15 + platelets in the first absorption step was eluted by resuspending the placelets in 250 lal o f physiologic saline, which had been brought to pH 3.0 with HCI ~11]. The platelets were incubated for 3 min with shaking, and then removed by centrifugation at 12,000 x g for 10 rain. The pH of the eluate was adjusted to 7.0 with NaOH. When retested in a microcytotoxicity assay, the eluted antibody reacted with both HLA-B15 and HLA-B17 cells at a titer of 1:8, but had no reaction against HLA-A2 cells or other members of the B5-CREG (Table 2). "!his antibody was tentatively designated anti-Z. Evidence of its functional monospe- cificity in this system is g ive , in the Results.

Anti-Y was prepared by a similar procedure to that above as described previously [7].

The anti-HLA-B12 (Upson) and anti-HLA-B15 (Malotsky) were obtained from the American Red Cross, St. Louis, MO. The anti-HLA-B15 is functionally monospecific so that it gives no reaction on HLA-B17, BS, BI8 , Bw21, or

T A B L E 2 Titers of serum MH, anti-Z and anti-B 17 on cells bearing various HLA antigens

Cells Tested

A2 BI5 BI7 B5 BI8 Bw21" Bw35

Serum MH 1: i 1:2 1:4 NR b NR NR NR .~ nti-Z NR 1:8 1:8 NR NR NR NR Anti-B17 NR NR 1:4 NR NR NR NR

(absorbed)

"Identical results obtained for the Bw49 ~.ad Bw50 splits of Bw21. )NR = No reaction with undiluted serum or antibody.

Benjamin D. Schwartz et al.

Bw35 cells. The anti-HLA-Bl2 gives no reaction on HLA-B15, B17, B5, or Bw35 cells, but is known to react with HLA-Bw21.

Absorption of anti-Z with leukocytes. Fifty microliters of anti-Z was incubated with 2 x l0 T leukocytes bearing B5, B15, BI7, B18, Bw21, or Bw35 for 60 rain. at 370C, and the cells removed by centrifugation at 1100 x g for 10 rain. This procedure was repeated once, so that in all cases where absorption was necessary, the anti-Z was absorbed twice.

Preparation of an lgG fraction from normal human serum. In cases where the human fl.-microglobulin present in normal serum might interfere with the reaction of rabbit anti-fl2-microgiobulin in the test step of sequential immunoprecipitation experiments, a fl2-microglobulin-free IgG fraction of the serhrn was used. This fraction was prepared as follows [12]. One volume of serum was incubated with 10 volumes of a 10% suspension of Protein A bearing Staphylococcus aureus Cowan l strain (SaCI) for 30 rain at 4°C. After thorough washing, the IgG was dissociated by the addition of one volume of 3.5 M MgCl., for 15 min at room temperature [13], and the SaCI pellet was removed by centrifugation. The supernate containing the purified IgG was dialysed against tris-buffered saline, pH 7.4 (TBS). This dialysate was used as the IgG fraction in subsequent experiments.

Xenoantiserum. Rabbit anti-human az-microglobulin was purchased from Accu- rate Scientif/.c Co., Hicksville, NY.

Radiolabeling cf cells and preparation of solubilized antigens. The procedure used has been previously described [6, 7, 14]. In brief, peripheral blood leukocytes were resuspended at 2-5 x 107 cells/ml in modified Eagle's medium without serum in which the sole source of leucine was aH-leucine (170 Ci/mM, Amersham Corp., Arlington Heights, IL) at a concentration of 200 tzCi/ml and incubated for 4 - 6 hr at 37°C in a humidified 5% COo atmosphere. The cells were harvested, washed, and then solubilized in TBS containing 0.5% of the nonionic detergent Nonidet P-40 (NP-40; Particle Data Laboratories, Elmhurst, IL). Insoluble debris was removed by ultracentrifugation at 100,000 "< g for 60 rain. The glycoprotein molecules containing glucose or matinose were then purified 10- to 20-fold by allowing them to bind to an affinity column of Lens culinaris lectin covalently attached to Separose 4B (Pharmacia Fine Chemicals, Piscataway, NJ) and eh.,ting them from the column with 0.1 M ,v-methyl mannoside [15]. This purified glycoprotein fraction was incubated with 300- 500/zl packed SaCI for 30 rain at 40C to remove any endogenously radiolabeled IgG. The SaCI were removed by centrifugation, and the supernate was used as the source of solubilized antigen in subsequent steps [14].

lmmunoprecipitation and analysis of solubilized HLA molecules. An aliquot of glycoprotein fraction containing 100,000-200,000 cpm was incubated with 100 tzl of alloantiserum (or eluted alloantibody) for 2 hr at 4°C. One milliliter of a 10% suspension of SaCI was then added to pellet any antigen-antibody complexes formed and the incubation was continued an additional 30 rain [14]. The pellets were centrifuged at 1000 x g for 10 rain, and washed three times in TBS containing 0.25% NP-40 (TSN). Radiolabeled antigen wa~ then dissociated from antibody and the staphylococci by boiling in 100/xl 0.0625 M tris, pH 6.8 containing 2% sodium dodecyl sulfate (SDS) and 2% 2- mercaptoethanol (2-ME) for 1.5 rain, and the staphylococci removed by centrifugation. One hundred microliters of 2% 2-ME, 20% glycerol, and 0.004%

[LA-B15 Has Two Public Antigens 33~

phenol red were added, and the sample analyzed in a modification [161 of the discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) system originally described by Laemmli [17] and Maizel [18]. Both lOT and 15(~ polyacrylamide gels were used. The gels were cut into 2-ram slices; each slke incubated overnight at 37°C in 1.5 ml of toluene containing 7(¥ Protosol ~lqes,- England Nuclear) and 4 g/l Omnifluor (New England Nuclear) and the radioac- tivity of each slice determined in a Beckman LS 8000 scintillation counter (Beckman Instruments, Palo Alto, CA). Molecular weights were estimated from the position of marker proteins electrophoresed at the same time [19].

Fluoresceinated Protein A. Fluoresceinated Protein A was purchased from Phar- macia Fine Chemicals.

Fluoresceinated Protein A binding assay. Anti-Z and normal human immunoglobulin G (NHIgG) were ultracentrifuged immediately prior to use to remove material greater than 10S [20]. Three microliters of anti-Z (an amount known to be in excess) or normal human immunoglobulin were incubated with 30,0OO leukocytes for 30 min at 4°C in Terasaki plates. The cells were washed three times with Hank's balanced salt solution containing 2% ovalbumin, 5 /zg of fluoresceinated Protein A were added, and the incubation continued for 30 rain at 4°C. The cell pellets were then wa~Led three times, and the fluorescence emitted by each cell pellet was quantitatively determined using a Zeiss pho to microscope 3. Each test was done in triplicate, and average values and standard deviations are reported.

RESULTS

herum M H Contains Th ree Funct ional ly Defined Ant ibody Populat ions

The reactions of serum MH with A2 cell.~ and B17 cells would be expected from the HLA types of MH and her husband. However, the additional reaction of this serum with HLA-B15 cells, particularly at a different titer from the reaction with HLA-B17 cells, suggested that serum MH might contain an antibody to a determinant common to B15 and B17. To dissect out the various antibody populations, serum MH was subjected to a series of platelet absorptions [11]. The first set of platelet absorptions with HLA-A2 +, B15-, B I 7 - platelets was done to remove the anti-HLA-A2 antibody. The absorbed serum was then twice absorbed with B17-, B15 + platelets. This absorbed serum was demonstrated to react only with B17 cells (Table 2). In contrast, the anri- body(ies) eluted from the B17-, B15 + platelets reacted equally well with both B15 and B17 cells, suggesting that a single antibody population designated anti-Z was responsible for both reactions.

Ant i -Z Is Funct ional ly Monospecif ic

As determined by cytotoxicity testing, anti-Z lysed both B 15 and B 17 cells at an equa! titer. To determine if we were in fact dealing with a single antibody population, a series of absorptions was performed. Anti-Z was twice absorbed with HLA-B15 cells, HLA-B17 cells, or control cells, and then tested for residual activity against B15 and B17 cells. In both cases, absorption of anti-Z with either B15 or B17 cells completely removed activity for both cell types (Table 3). This result indica!ed tha t anti-Z contained only a functionally monospecific antibody population reactive with both B15 and B17 cells.

,6 Benjamin D. Schwartz et al.

T A B L E 3 Absorption of anti-Z by B 15 and B 17 cells

Percent Cells Staining with Eosin Undiluted Anti-Z Absorbed Twice with Cells Bearing BI5 B17

No Abmrptiov >90 >90 B7/B40 (Control) >90 >90 B15 <20 <20 BIT <20 <20

n t i - Z Reacts w i th B15 and BI7 , But N o t w i t h O t h e r M e m b e r s o f t he 5 -CREG

As measured by direct cytotoxicity, anti-Z recognized cells bearing B 15 or B 17, but did not react with cells bearing any o ther member o f the BS-CREG (Table 2). In our two previous studies, HLA public determinants not detectable by direct cytotoxicity could occasionally be detected by absorption (the C Y N A P phenomenon) [21 ]. We therefore tested cells bearing BS-CREG HLA antigens for their ability to absorb the cytotoxic acti~,ity of anti-Z for BI5 cells. These studies indicated that cells bearing BS, B18, the Bw49 or Bw$0 splits of Bw21, and Bw35 could not cffectively absorb anti-Z cytotoxic activity for BI5 cells.

To further test cells bearing BS-CREG HLA antigens for possible Z reactivity, we utilized a fluorescent Protein A binding assay to quantitate the binding of anti-Z antibody to (and therefore the amount o f available Z antigen on) the various cells [22]. While after incubation with anti-Z, BI5 and BI7 cells bound substantial and approximately equ~,l amounts of fluoresceinated Protein A, B5, B18, Bw21, and Bw35 cells bound little more than control (Table 4). We therefore concluded that only B15 and B17 cells had substantial reactivity with anti-Z.

knt i -Z Reacts w i t h an H L A - l i k e Molecu le

The serological data strongly suggested that Z was most likely an HLA public antigenic determinant .distinct from the public determinants previously described. To confirm this suggestion, we performed a series of chemical studies.

T A B L E 4 Reaction o f Ant i -Z with cells as assessed by fluoresceinated Protein A binding

[~uorcKcn¢¢'

Cells Anti-Z NHlgG Net b

BI5/BI2 62.30 ± !.5 18.6 4- 0.77 43.7 B17/BI2 61.0 ± L7 20.1 ± 0.63 40.9 BS/Bw21 (Bw49) 29.4 ± ~.8 21.1 ± 0.14 8.3 BS/Bw21 (Bw50) 29.5 ± ~.2 18.8 ± 1.4 10.7 BI8/B14 15.85 ± 4.5 16.1 ± 1.4 -0.25 Bw35/B7 28.0 ± 2.5 17.3 ± 1.5 10.7 BT/B40 (control) 24.65 ± 2.1 17.9 "*" 2.5 7.15

"Mean of triplicates ± standard deviation. tNet fluorescence equals mean fluorescence obtained with anti-Z minus mean fluore~:ence obtained with

NHIgG on each cell type.


The immunochemical reactivity of the absorbed ant i -Bl7 and the eluted anti-Z with an antigen preparation derived from B15 or B17 cells is shown in Figure 1. Whereas, on the B17 antigen preparation (bottom row), anti-Z and the absorbed anti-B17 both react with a molecule of approximately 44,000 daltons (~2-microglobulin cannot be resolved on a 10% polyacrylamide gel), en the B t5 antigen preparation (top row), only anti-Z and an anti-HLA-B15 serum react with such a molecule. This result indicates that an t i -Z is reacting with a molecule that could well be an HLA molecule, arid that the platelet absorption was efficacious in yielding a functionally monospecitlc anti-B 17.

A n t i - Z and Anfi-~.2tz Recogn ize the Same M o l e c u l a r Complex

When analyzed on a 15% polyacrylamide gel, anti-Z reacted with a molecule which migrated as two components of 44,000 and 12,000 daltons, (Figure 2, top row, middle panel), identical to the components r,eactive with rabbit anti-~,/z (top row, left panel). To prove that both a,~ti-Z and aati-~2tz were reacting with the same complex (and therefore that the 12,000 dalton component reactive with anti-Z was in fact ~z~t), we performed a sequential immunoprecipitation experiment [19] (Figure 2).

FIGURE 1 Discontinuous SDS-10% PAGE patterns of the molecules solubilized from aH-leucine labeled HLA-.B15 positive cells (~op) or HLA-BI7 positive ceils (bottom), and reactive with normal hum~Ln serum, the eluted anti-Z, the twice absorbed anti-Bl7, and an anti-Bl5. The molecules detected all migrate with an apparentmolecu- lar weight of 44,000 d.dtons. On 10% polyacrylamide gels, #2~ itself ca,'mot be identified.

100 - J J 12.1 u

~ 5 0 O. u

u D 15G

I

.1-

~ m 100 ._1

u

5C

NHS ANTI-Z ANTI-B17 ANTI-B15

2 4 6 8 10 2 4 6 8 10 2 4 6 8 10 2 4 6 8 10

CM OF GEL


900

6OO 0

:I: Z 300

9OO

=E

iii

U ~ =~ 600

Z z i i i i

. N

900

6OO N

I

Z 300

A N T I - j3~.u ~NTI - Z

3OO

6 .

3O0

NHIoG

2 4 6 8 10 2 4 6 8 102 4 6 8 1 0 CM OF GEL

FIGURE 2 Discontinuous SD$-15% PAGE patterns of the molecules solubilized from 3H-leucine labeled HLA-B 17 cells, and reacted in a :~equential immunoprecipitation experiment with normal human IgG (NHIgG), anti-/~h/z, or anti-Z. After pretreatment with NHIgG (top row), anti-/~/z and anti-Z both react with molecules consisting of a 44~000 dalton chain and a 12,000 dalton chain. Pretreatrrent with anti-/~/z (middle row) removes all molecules reactive with itself and anti-Z. Pretreatment with anti-Z (bottom row) removes all subsequent activity for itself, but leaves molecules reactive with anti-/32/z.

An antigen preparation from B17 cells was divided into three aliquots. The first was pretreated with normal human IgG ( N H I g G ) as a control: the second aliquot was pretreated with an t i - /~ t to remove all molecules conta in ing /~ t t ; and the third aliquot was pretreated with anti-Z to remove all molecules bearing determinant Z. Each of these pretreated samples was in turn divided into three aliquots and tested with anti-/~/z for the presence o f /~ / z containing molecu!(.s,

LA-B15 Has Two Public Antigens 3~9

200

150

1- 100 Z

50

20O

e, tw U ~ to

tub.= co

Z ' 100

Y~ 5o ~m 20O

150

r q !

- 100

ANTI-BI$

TEST SERUM ANTI-Z NHS

I I

Z

50

2 4 6 8 10 2 4 6 8 I0 2 4 6 8 10 CM OF GEL

FIGURE 3 Discontinuous SDS-IO°~ PAGE patterns of molecules solubilized from aH-leucine labeled HLA-B15 ce.lls and reacted in a sequential immunoprecipitation experiment with NHS, anti-B15, and anti-Z. Pretreatment with NHS (top row) leaves molecules reactive with both anti-Bl5 and anti-Z. Pretreatment with anti-Bl5 (second row) or with anti-Z (bottom row) realoves virtually all molecules reactive with both antibodies.

with anti-Z for the presence of Z-bearing molecules, or with N H l g G as a control. After pretreatment with N H I g G (top row), both anti-fl._,V and anti-Z react with the molecules containing a 44,000 dalton chain and a 12,000 dalton chain, as noted above. Pretreatment with anti-fl2~ (middle row) removes all molecules reactive with both itself and anti-Z, indicating that anti-Z was indeed reacting with ~ molecular complex containing flzlZ. In contrast, pre t rea tmem with anti-Z (bottom cow) removes molecules reactive with itself, but leaves

340 Benjamin D. Schwartz et ai.

behind molecules reactive with anti4~/z. This, of course, is the expected result, since anti-//2tz reacts with all HLA-A, HLA-C, and non-Z-bearing HLA-B molecules. Thus, anti-Z is reacting with an HLA-like molecule consisting of a heavy chain and ~2~. Since no polymorphism is known for human/32/z, the Z determinant is almost undoubtedly on the heavy chain.

Anti-Z Reacts with Molecules Bearing HLA-BI5 and HLA-BI7

The findings that anti-Z reacted with a molecular complex similar to that bearing the HLA antigens, and that anti-Z reacted with ceils bearing HLA-B15 and HLA-B17 suggested that Z wa~ most likely a determinant on the heavy chains of B15 and B17. To confirm this suggestion, we ag'~in utilized sequential immunoprecipitation.

After pretreatment with normal human serum (NHS) (Figure 3, top row) of an an-.igen preparation derived from B15 cells, anti-Bl5 and anti-Z both reacted with a 44,000 dalton component (~gain, /3z/z cannot be discerned on 10% polyacrylamide gels). Pretreatment with either anti-B15 (middle row) or anti-Z (bottom row) completely removed all molecules reactive with both antibodies. This rest>.lt indicates that Z is a determinant on the h i 5 heavy chain.

Similar results were obtained from the sequential immunoprecipitation experiment on the antigen preparation derived from B17 cells (Figure 4). Thus, pretreatment with NHS (top row) left molecule~ reactive with both anti-B17 and anti-Z. Pretreatment with either anti-B17 (middle row) or anti-Z (bottom row) left no molecules reactive with either serum on retesting. Thus, Z is a determinant on the same heavy chain bearing B 17.

The B15 Heavy Chain~ Bears at least T w o Public Antigens

We previously demonstrated that the B15 heavy chain bears a public determinant provisionally designated Y which is common to HLA-B5, B15, B18, and Bw35 belonging to the B5-CREG, but is absent from B17 and Bw21 [7]. The finding that B15 also bears Z suggests that BI5 bears two distinct public determinants, Y and Z. To prove that this was in fact the case, we performed yet another sequential immunoprecipitation on an antigen preparation derived from B12, B15 cells (Figure 5).

After pretreatment with normal human serum (top row), anti-Z, anti-Y, anti-B15, and anti-B12 all react with a 44,000 dalton component. Pretreatment with anti-Z (second row), anti-Y (third row), or anti-B15 (fourth row) removes all molecules reactive with any of these sera, but leaves molecules reactive with anti-B12. Conversely, pretreatment with anti-B12 (bottom row) completely removes all molecules reactive with anti-B12, but leaves molecules detectable by anti-Z, anti-Y, and anti-B15. This result indicates that Y, Z, and B15 are in fact three determinants on the same HLA heavy chain.

Anti-Y Does Not React with All HLA-B Molecules

The demonstration that Z was a determinant on B15 and B17 molecules did not indicate whether or not Z might also be a determinant oa other HLA-B molecules. We therefore prepared labeled antigen preparations from cells of several individuals who did not possess B17 or B15. AlthouLh, the HLA-B molecules in question could be readily detected by appropriate anti-HLA alloantisera arid/or rabbit anti-/~/z, thus establishing a positive control, anti-Z did not react with BS, BT, B12, B14, B18, Bw35, B40, Bw49, or BwS0-bearing

HLA-BI5 Has Two Public Antigens ~ 1

200

150

Z

50

200

u t~

~ a~ 100

e ~ e ~

200

150

¢',4 t

100 Z

50

TEST SERUM ANTI-B17 ANTI-Z NHS

, t-

2 4 6 8 I0 2 4 6 8 I0 2 4 6 8 I0 CM OF GEL

FIGURE 4 Discontinuous SDS-10% PAGE patterns of molecunes solubilized from 3H-leucine labeled HLA-B17 cells and reacted in a sequential immunoprecipitation experiment with NHS, anti-Bl7, and anti-Z. Pretreatment with NHS (top row) leaves molecules reactive with both anti-B17 and anti-Z. Pre~reatraent with anti-Bl7 (second row) or with anti-Z (bottom row) removes essentially all molecules reactive with both antibodies.

molecules (data not shown). These results strongly suggest that Z is a determinant confined to B15 and B17.

D I S C U S S I O N

The studies presented in this report indicate that HLA molecules beating HLA-B15 and B17 share a distinct antigenic determinant which we have tentatively designated Z. Antigen Z is thus the third example o f an


TEST SERUM ANTI-Z ANTI-Y ,,tM'~'TI-.BL.5 AmP-m2 NHS

~OOI~ t 2O0

2OO

i

z i

n 400 0 2OO t.iJ Z > - ~ 20(1 w ._J !

w)

200 z <

. .A.x , . , , . , / ~ , , ~ ~ A J ~ 20O

40O [ 2OO

I z

2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 CM OF GEL

FIGURE 5 Discontinuous SDS-tO% PAGE patterns of the molecules solubilized from aH-leucine labeled HLA-BI2/BI5 positive cells and reacted in a sequential immunoprecipitation experiment with NHIgG, anti-Z, anti-Y, anti-B15, and aati-B-12. Control pretreatment (top row) leaves molecules reacfve with all four antibodies. Pretreatment with anti-Z (second row), anti-Y (third row), or anti-Bl5 (fourth row) completely removes all molecules reactive with any of these antibodies, but leaves molecules reacdve with anti-B12. Pretreatment with anti-B12 (bottom row) removes all molecules reacdve with itself on retesting, but leaves molecules reacdve with anti-Z, anti-Y, and anti-B15.

alloantibody-detected public alloantigen which can be immunochemically demonstrated and readily accounts for the cross-reactivity seen be tveen B15 and B17.

Anti-Z antibody did not react with any member of the BS-CREG (other than B15) by direct cytotoxicity, absorption, immunoprecipitation, or fluoresceinated

qLA-BI 5 Has Two Public Antigens 343

Protein A binding, which in our hands is the most sensitive assay for publk antigens. In contrast, ouJr previous study on the cross-reactivity of the BS- CREG demonstrated that B15 was only one of several antigens that bore antigen Y [7]. Thus, B5, BIS, B18, and Bw35, but not B17 or Bw21, bore tiffs determinant. The distinct reaction patterns of anti-Y and anti-Z indicate that Y and Z are two distinct antigenic determinants. Thus, immunochemicaily, the B15 molecule has been demonstrated to bear at least three allodeterminants: B15, Y, and Z.

Serologically, the antigenic composition of the B13 molecule is even more complex. Two sets of alternative aliodeterminants (Bw4 or Bw6 and B~62 (f0rmerl~ B15.1) or Bw63 (formerly B15.2)) have been associated with BI5 by serological techniques, and in fact have defined the "splits" of this antigen [5]. Since Bw62 and Bw63 are not present on B17 molecules, and Bw4 or Bw6 are present on all HLA-B molecules [5, 23], it is highly improbable that they are identical to Y or Z. Thus, there are at least five distinct allodeterminants defined by alloantibodies present on the single HLA glycoprotein chain pos- sessing the determinant B 15. Th!s antigenic complexity begins to rival that long known in the murine H-2 system [24].

In addition, other antigenic determinants on HLA molecules have been defined using murine monoclonal xenoantibodies. A framework determinant W6/32 present on all HLA-A, -B, and -C molecules can be detected by a monoclonal xenoantibody [25]. More recently, monochmal xenoantibodies have been used to detect an allodeterminant, MB40.1, which is present on some, but not all HLA-A and -B molecul,.~s including HLA-B15 [26], and an allodeterminant 38.95, which is shared by ]-ILA-A2 and HLA-B17 [27].

While the definition of new antigenic determinants may lend insight into the evolution of molecules determined by the major histocompatibility compiex [25, 26], consideration must also be given to the biological importance of the various determinants. Thus, distinction should be made between those determinants which have biological roles, and those that are merely serological curiosities. The private determinants (e.g., B15) on the HLA molecule have been shown to be target antigens of cell-mediated cytolysis [28]. Recently we have demonstrated that alloantibody defined public armgens (e.g., Y) can also act as target antigens in this assay [29]. These results suggest that these determinants may play a significant role in immunologic processes, such as HLA restrictions on cytotoxic T tymphocytes directed against viruses or minor histocompatibility antigens [30-35].

Similarly, we have suggested that the findings that public antigen X is more common than HLA-B27 among patients with spond,Aoarthropathy [36], and that public antigen Y is more common than HLA-B5 among patients with Behcet's disease (Luehrman LK, Schwartz BD: Unpublished data) indicate that public antigens may play a role in disease predisposition. Whether the antigens defined by monoclonal antibodies can serve in similar roles is a question that has yet to be resolved.

ACKNOWLEDOMENT This work was supported in part by NIH Grant AI-15353, by Grant IM-131 from the American Cancer Society, ~.nd by grants from the Kroc Foundation and the American National Red Cross. The authors wish to thank Dr. Susan E. Cullen for fruitful discussions and helpful suggestions, and Lorraine Bourisaw and Judy Craig for expert preparation of the manuscript.

344 Benjamin D. Schwartz et al.

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HLA serological cross-reactivity: HLA-B15 has two public antigens