Proc. Natl. Acad. Sci. USA Vol. 76, No. 11, pp. 5834-5838, November 1979
Immunology
Dissociation and exchange of the f32-microglobulin subunit of HLA-A and HLA-B antigens (subunit association equilibrium/radioimmunoassay/cell surface probe)
FRANTOIS HYAFIL* AND JACK L. STROMINGER Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138
Contributed by Jack Leonard Strominger, August 9, 1979
ABSTRACT Human histocompatibility antigens HLA-A, HLA-B, and HLA-C are a complex of two noncovalently associated subunits: a heavy chain glycoprotein (a) carrying the genetic polymorphism and an invariant light chain, globulin (mm). Upon incubation of papain-solubilized HLA with radiolabeled urinary 02m, the latter is incorporated into HLA, where it substitutes for the preexisting f2m that has dissociated from the complex. The association-dissociation equilibrium that governs this #2m exchange reaction was investigated and found to be characterized by a long lifetime of the complex (half-life of 80 min at 370C) and a relatively low Kd (4 nM). The fi2m exchange was used as the basis of a radioimmunoassay for HLA antigens with radiolabeled ft2m as a unique label for all HLA specificities. In a similar fashion, radiolabeled #2m can be incororated into HLA at the cell surface. Although the process is slower and less extensive than in solution, it can be used as a means to tag cells with specific probes for HLA antigens. The HLA-A, HLA-B, and HLA-C regions of the human major histocompatibility complex (H2-K and H2-D in the mouse) produce a highly polymorphic group of antigens present at the surface of most cells and involved in a variety of immunological phenomena (1-5). These antigens are composed of a glycoprotein of molecular weight 44,000 (a), which is noncovalently associated with 132-microglobulin (/2m), a protein of molecular weight 12,000 (6). Genes in the same region have been found to control the induction of effector T lymphocytes in allogeneic systems and the killing of target cells by those lymphocytes (2) and to restrict the interaction of cytotoxic T lymphocytes with modified syngeneic cells (7). That the HLA and H2 antigens are the actual targets of the effector lymphocytes emerges from antiserum blocking studies (7) and more convincingly from the biochemical analysis of a mutant H2-K molecule (8) and fromr the capacity of liposome-carried purified HLA to elicit a specific cytotoxic response (9). At least in one case (ankylosis spondilytis), it is not unlikely that a disease susceptibility statistically associated with an HLA allele (HLA-B27) is determined by the HLA gene itself rather than by a closely associated gene (see discussion in ref. 3). Indeed, mice differing only in the structure of the H2-K antigen have been recently shown to have different susceptibilities to autoimmune thyroiditis (10). Possibly relevant is the fact that HLA and H2 may be used as cell surface receptors by viruses (11) and bacteria (12). The rationale in the experiments presented here is to take advantage of the structure of the HLA-A, -B, and -C antigens to introduce at the surface of cells specific noninterfering probes of the function of these antigens. The heavy glycoprotein chain is polymorphic and spans the plasma membrane. In contrast, /32m is nonpolymorphic and outside the plasma membrane. Furthermore, it is readily available in large quantities from the
urine of patients with kidney dysfunctions. Investigation of conditions in which exogeneous urinary f2m might replace the /2m carried by HLA antigens at the surface of cells was, therefore, carried out in a model system using papain-solubilized (pap) HLA-A and -B antigens [which lack the membrane-spanning hydrophobic region and the COOH-terminal hydrophilic region (13)]. The exchange of urinary f32m with /2m present in HLA molecules is demonstrated and shown to be a consequence of an association-dissociation equilibrium between the subunits of the HLA-A and -B antigens. The thermodynamics of this equilibrium allow the development of a radioimmunoassay (RIA) for HLA-A and -B antigens in which radiolabeled.2m is used to label all the specificities. A similar process occurs at the surface of cultured lymphoblasts, where radiolabeled /32m can be incorporated into HLA-B antigens by exchange upon incubation at 37°C. Thus many probes (radioactive, spectroscopic, chemical) carried by /32m could potentially be introduced into HLA antigens and might be useful in unraveling the intracellular and extracellular interactions in which those molecules are involved.
72-micro-
MATERIALS AND METHODS Cells and Sera. The lymphoblastoid cell lines used in these experiments were: JY (HLA-A2; B7 homozygote), LB (HLAA28; B40; Cw3 homozygote), Laz 007 (HLA-A3,A?; B5,Bw39), and Daudi (no detectable HLA). The volunteer donors from which blood was obtained were typed by D. Fitzpatrick and B. Canaday in the laboratory of E. Yunis. Tissue typing sera were obtained from E. Yunis, B. Amos, and the Transplantation Branch of the National Institute of Allergy and Infectious Diseases. 02m and HLA. Human urinary /2m (14) (a gift of R. Robb) was iodinated by the chloramine-T method (15) or with the Bolton-Hunter reagent (16) to specific activities of 103 or 2-5 X 104 cpm/ng, respectively. HLA-A2pap was purified as described (17). Reductive Methylation of HLA. A solution of HLApap (2 mg of a mixture of HLA-A2 and B7) in 0.2 M Na2B407, pH 9.0/0.5 M NaCl, to which approximately 1 mg of NaB3H4 (New England Nuclear, 276 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels) had been added was made 1 M in formaldehyde by five successive additions at 5-min intervals. After 1 hr on ice and dialysis, [3H]HLA was repurified on DE 52 DEAE-cellulose by a linear gradient of Tris/PO4 (17). [3H]HLA migrated as a single peak with a specific activity of 5 cpm/ng, representing an incorporation of about eight CH3 groups per molecule. Preparation of Internally Labeled HLA. JY cells (107) were Abbreviations: 132m, 132-microglobulin; a chain, HLA heavy chain; HLA papain-solubilized HLA; Lch, Lens culinaris hemagglutinin; NaDodSO4, sodium dodecyl sulfate; RIA, radioimmunoassay. * Present address: Unite de Genetique Cellulaire, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, Cedex 15 France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5834
Proc. Natl. Acad. Sci. USA 76 (1979)
Immunology: Hyafil and Strominger cultured for 24 hr with 5 mCi of L-[4,5-3H(N)]lysine (New England Nuclear, 60 Ci/mmol). Labeled cells were resuspended in buffer A (10 mM Tris-HCl, pH 7.3/0.15 M NaCI/3 mM NaN3) containing bovine serum albumin at 6 mg/ml and digested with 20 units of papain (Worthington) activated with 0.4 mg of cysteine. After 30 min at 37°C, the cells were pelleted and [3H]HLA was purified from the supernatant by: (i) DE 52 chromatography to get rid of papain, (ii) affinity chromatography on agarose coupled with Lens culinaris hemagglutinin (Lch), (iii) gel filtration on Sephadex G-150 of the methyl a-mannoside eluate from the Lch-agarose. About 1 ,g of pure HLApap containing 5 X 105 cpm was obtained as judged by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis (18) and fluorography. Crude Papain Extracts from Platelets and Peripheral Blood Lymphocytes. Cells were isolated from freshly drawn blood (19, 20) and digested with papain as above but for the
B 2010
F
10(0
F
2 c) -0 .? T 4
I I I
^ C
I
C
1.
E 3 20C
-
FIG. 1. Radiolabeled urinary 32m can be incorporated into HLA. (A) Gel filtration profile of 1251-132m (1.6 x 106 cpm/1.6 gg) incubated with HLA-A2pap (16 ,g) for 3 hr at 25°C in buffer A and run on a Sephadex G-75 column (1.2 X 35 cm) equilibrated in buffer A
containing lysozyme at 1 mg/ ml. Fraction size is 0.75 ml: Arrows indicate the elution positions of blue dextran (VO),
1 OC
addition of 10% citric acid/citrate/dextrose in the case of platelets. The supernatants were treated with 0.8 mg of iodoacetic acid and used immediately or stored at -20'C until needed. RESULTS #2m Exchanges with the Small Subunit of HLApap. Radiolabeled urinary f2m (labeled with '25I by the chloramine-T method) migrates as a single peak on Sephadex G-75. When it is incubated with HLA-A2pap, part of the radioactivity elutes early from the column at the same position as HLA-A2pap (Fig. 1A). To show that in this heavier material /2m was associated with HLA-A2pap, immune complex formation was assessed by gel filtration on Sephadex G-150 (Fig. 1 B, C, and D). Whereas 90% of the radioactivity associated with HLA-A2 could be complexed by an HLA-A2 antiserum (Fig. 1B), normal human serum (Fig. 1C), or alloantisera of other specificities (not shown) failed to bind any labeled material. As a control, it was also shown that the HLA-A2 alloantiserum used above does not have any anti-f2m activity (Fig. 1D). Transfer of radioactive material between f2m and the a chain was ruled out by NaDodSO4/polyacrylamide gel electrophoresis and gel filtration in 6 M guanidine hydrochloride, which showed that all the radioactivity incorporated into HLA was associated with the polypeptide of 12,000 daltons. It was important to demonstrate that there is actual exchange between the /2m originally present on HLA-A and -B antigens and the added urinary 12m. HLA-A2,1B7pap was thus labeled by reductive methylation (21) in the presence of NaB3H4, a modification that does not alter the antigenic properties of HLA-A and -B antigens (22). When reductively methylated HLApap was incubated with increasing amounts of urinary /2m and then separated from /2m by gel filtration, an increasing amount of radioactivity eluted with free 32m (Fig. 2A). Furthermore, analysis by NaDodSO4/polyacrylamide gel elec-
0 I 0 x
E
E 0
a
3C 2C
E E 0
C
E C.)
A
4C
)c
C)r D 500
_5W-0) wc) -T 4 4 4 4
400 300 200 100
0I I
20
30 40 Fraction
50
of Davis [an HLA-A2 antiserum (B, D)l or normal human serum (C). After 24 hr at 4°C, the samples were run on a Sephadex G-150 column (1.0 X 60 cm). Fractions (1 ml) were assayed for radioactivity and absorbance at 280 nm. Arrows indicate the eluition position of major serum proteins. In B, C, and D, the peak at tube 20 is the immune complex of HLAA2, that at tube 35 is HLA-A2 itself, and that at tube 46 is free f32m. Alb, albumin.
Of
1C
a I 0 10
[3H]HLApap, and 1251-f2m in-
cubated in the absence of HLA. Fractions 19-23 were pooled and a 0.05-ml aliquot (B, C) of this pool or 20 ng of 1251-fl2m (D) was incubated with 0.2 ml
5835
a
a
I
100"150 200 250
50
A2M, ,ug
B a a
chain
/)2m 13
b
__X
M
c
d
_
e
f
-
h
g
i
bd%
U
_P
FIG. 2. Exchange between the small subunit of radiolabeled HLApap and urinary f2m. Reductively methylated [:H]HLAPaP (120,000 cpm/24 ,ug) was incubated for 5 hr at 370C with increasing amounts of unlabeled f,3m and analyzed on a Sephadex G-75 column equilibrated in 0.1 M NH4HCO:i to separate HLApap from free f32m. Fractions were assayed for radioactivity (A), and the material mi,grating at the position of HLAj)aP was pooled, freeze-dried, run on NaDodSO4/polyacrylamide gel electrophoresis (23), stained with Coomassie blue, and fluorographed (B).
5836
Immunology: Hyafil and Strominger
Proc. Natl. Acad. Sci. USA 76 (1979)
trophoresis of the HLA-A and -B antigens revealed a constant ratio of the intensities of the heavy chain and O2m bands after Coomassie blue staining (not shown) but a progressive loss of the [3H]32m band as visualized by fluorography (Fig. 2B). Thus, at neutral pH and isotonic salt concentration, all of the [3H]32m in 13H]HLApap was replaced by urinary O2m. The two HLA specificities, HLA-A2 and HLA-B7, exchanged with '25I-02m at the same rates in an experiment in which separated HLA-A2pap and HLA-B7pap were used. Thermodynamics of the Interaction Between the Subunits of HLApap. The simplest explanation of the above findings is an association-dissociation equilibrium between the a chain of HLA-A and -B antigens and /32m (designated in this section as 3): aO/
k~ff (. a
kon
+
/3,
Kd
=
koff/kon0
[1]
of appearance of labeled free displayed an exponential behavior and yielded a value for koff of 1.4 X 10-4 s-1 at 370C. In another set of experiments, the dissociation was measured directly in the absence of added /2m. In these conditions [a] = 1/] and, after 5 hr at 370C, it can be shown that the system has reached equilibrium so that [/], the free /32m concentration, can be expressed by Eq. 2, in which HLAt1t is the initial concentration of HLA: [2] [/3] = Kd[(l + 4 HLAtot/Kd)1/2 - 1]/2. This effect was monitored by using either internally labeled [3H]HLApap (Fig. 3C) or a tracer amount of '25I-/2m (Fig. 3D). Experimental results were in good agreement with Eq. 2, indicating a Kd of about 4 nM at 370C. Parameters obtained by using 125I-/2m (labeled with the Bolton-Hunter reagent) as a tracer were very similar to those obtained by using reductively methylated or [3H]lysine internally labeled HLApap, thus suggesting that labeling /2m with 125I by using the Bolton-Hunter reagent does not modify its interaction with the chain. In contrast, some experiments seem to indicate that /32m iodinated on its tyrosines has a weaker affinity for the chain. Whatever technique was used to radioiodinate /2m (chloramine-T, lactoperoxidase/H202, lactoperoxidase/glucose oxidase/glucose, or Bolton-Hunter reagent), only about 40% of the label could be incorporated into HLApap (see Fig. 3D), and the residual material could not be incorporated when incubated again with HLA. Although this could be due to radioiodination damage, it can be explained better by the heterogeneity of the batch of /2m used in these experiments (unpublished data). RIA for HLA Antigens. At 370C, the half-life of the ao/ complex is 80 min. As shown in Fig. 3A, it is increased by about two orders of magnitude at 0C, so that the complex is stable for most practical applications even at concentrations much lower than its Kd. An immediate consequence is that HLA-A and -B antigens can be labeled at 370C with exogeneous /2m and then assayed in the cold to prevent dissociation of the complex. On this basis, a direct RIA for HLA was devised with the following steps: (i) removal of HLA-A and -B antigens (and other proteins) from cells by treatment with papain, (ii) incubation for 4 hr at 370C with '25I-/2m, (iii) gel filtration on Sephadex G-75 (or affinity chromatography on Lch-agarose) a
E
x
En QL C
E
u
U.
C
Ea 4
.4
4
i X
EEL
~+
80B 70r 60;
30-
50 -\
20-
E 40E c~ 30 CL
D
C
A series of experiments aimed at testing the validity of this model and at measuring its parameters was carried out. The dissociation of the ao/ complex is the rate-limiting step when radiolabeled is displaced from ao3 by an excess of free unlabeled d. Such experiments, using either reductively methylated [3H]HLA (Fig. 3A), or HLApap labeled in its d subunit by exchange with 125I-02m (Fig. 3B), showed that the time course
a
4
-J
0 T 0 C
E
CL
I
~
~
20
I
40 Time, ks
60
I
-9 -8 -7 -6 log [HLA pap
FIG. 3. Kinetics (A, B) and thermodynamics (C, D) of the association-dissociation reaction between the subunits of HLApap. (A) Reductively methylated HLApap (240,000 cpm/48 ug) was incubated at 00C (ti), or at 370C (0, 0) with 0 jig (0) or 100 ,g (0, A) of I32m. (B) 1251-HLA was prepared by exchange of 1251-/32m labeled by the Bolton-Hunter reagent and gel filtration as in Fig. 1A. Samples (1.2 mg) were incubated at 370C with 10 mg of unlabeled f2m. (C) [3H]Lysine-labeled HLApap (20,000 cpm/40 ng) or (D) 1251-32m (20,000 cpm labeled with the Bolton-Hunter reagent) was incubated at 370C with various amounts (expressed in M) of unlabeled HLApap. At various times (5 hr for C and D), samples were analyzed as in Fig. 1A. The experimental results were fitted to the theoretical equations by using a three-parameter iterative regression computer program. The best-fit values with their standard deviations are: (A) kff = (1.44 i 0.16) X 10-4 S-1, (B) kzff = (1.34 i 0.49) X 10-4 S-1, (C) Kd = (3.38 ± 0.60) nM, and (D) Kd = (5.31 + 1.93) nM. Theoretical curves using these parameters are drawn as continuous lines.
to remove excess '25I-/2m and lower the background in subsequent immunoprecipitation, (iv) incubation overnight at 4VC with alloantiserum, and (v) immunoprecipitation with cells of Staphylococcus aureus strain Cowan I (24). By using pure HLA-A2pap, subnanogram quantities of antigen could be specifically labeled and immunoprecipitated by this technique. Furthermore, both specificities in all the mixtures tested could be detected, namely: A2 and B7, A28 and B40, or B40 and Cw3. To screen a variety of specificities, crude papain extracts from human cells of known types were assayed by this method. A good correlation between the typing patterns obtained in classical cytotoxicity tests and the RIA was obtained (Table 1). Some of the false negative results can be explained by a lower representation of some antigens [HLA-B12 on platelets (25)1. Other discrepancies could be due to a peculiar behavior of some antigens in the association-dissociation equilibrium or more likely to slight differences between the HLA serology defined by microlymphocytotoxicity and by the RIA described here. fl2m Incorporates into HLA at the Cell Surface. To determine whether a similar exchange process would also occur on the plasma membrane of living cells, the following experiments were performed in the presence of puromycin to prevent the turnover of HLA-A and -B antigens at the cell surface. Under those conditions, there is no significant degradation of HLA-A and -B antigens in 24 hr, whereas other cell surface markers, such as 1g, are shed in a few hours (measured with internally labeled cells). 125I-/2m labeled by the Bolton-Hunter reagent binds specifically to the lymphoblastoid cell line JY, but there is no binding to Daudi cells, which are known to lack HLA-A and
Immunology: Hyafil and Strominger
Antiserum
Specificity
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 1. RIA for HLA-A and HLA-B antigens* Donor,- HLA type, and sourcet BF FH GK A2; B5,7 A3,10; B5,? A2,9; B7,15 platelets platelets platelets G-75 Lch G-75 G-75 Lch
5837
HP A2,3; B7,12 PBLs Platelets G-75 G-75
232 82 362 360 Davis A2 308 86 260 ND A3 400 19 22 77 Canaday 238 119 ND ND ND ND 280 ND Fe125 A9 39 24 15 ND 12 Keyis 310 164 A10 22 ND B5 76 57 16 20 31 Filting 28 B7 190 Jackson 377 46 30 410 206 380 B12 11 30 210 SLA 35 57 27 50 Gommol ND 82 25 B15 56 25 217 5 * Results are expressed as cpm recovered in the S. aureus pellet after three washes. Expected positive results are underlined. ND, not done. The last line in each column heading indicates how excess 32m was removed from the antigen, gel filtration on Sephadex G-75 or affinity chromatography on Lch-agarose. PBLs, peripheral blood lymphocytes.
-B antigens at their surface (26, 27) (Fig. 4). A nonspecific binding (not inhibited by unlabeled 132m) was also observed for both cell types at high cell concentrations. As in the case for HLApap, only a portion of the '2-132m could be exchanged, but in this case it amounted to only about 4% of the total radioactivity. The residual 96% could not be exchanged in further attempts. The cell-bound radioactivity was displaced in the presence of unlabeled /2m with a half-time of about 12 hr at 370C (and at least ten times longer at 0°C). To show that cellbound radioactivity was incorporated into HLA-A and -B antigens, cells were incubated with alloantisera and lysed with detergent, and the supernatants were immunoprecipitated by addition of cells of S. aureus strain Cowan I. Exogeneously added 1251-I2m incorporated preferentially into HLA-B7 (or -B40) at the surface of cells rather than into HLA-A2 (or -A28) (Table 2). In these experiments the labeled cells were mixed
2000
with unlabeled cells of a different HLA type before detergent solubilization. The absence of radioactivity in the HLA-A and -B antigens of these cells shows that the association of /32m with HLA-B and to a lesser extent with HLA-A antigens occurs at the surface of cells and not during the immunoprecipitation. Is There Exchange at the Cell Surface? In order to answer this question, JY cells were labeled with [3H]lysine and then incubated at 37°C in the presence of puromycin with or without the addition of urinary 12m at 50 ug/ml. At various times, samples were harvested and surface HLA-A and -B antigens were purified by (i) papain digestion of whole cells terminated by iodoacetic acid, (ii) affinity chromatography on Lch-agarose with elution by methyl a-mannoside, and (iii) affinity chromatography on rabbit anti-32m IgG coupled to Sepharose with elution by glycineFHCl, pH 2.2 (28). The relative radioactivity of the chain and the !2m were assessed by gel filtration under denaturing conditions (10% acetic acid). The fraction of radioactivity in the 32m peak did not change significantly in the absence of /2m (55.4%, 59.3%, and 55.6% after 3, 11, and 25 hr, respectively) but decreased with /2m in the medium (62.8%, 55.3%, and 46.4% at 3, 11, and 25 hr, respectively). The decrease was not large and amounted to a maximum of 25% exchange during the 25-hr incubation. One possible interpretation is that the observed binding of exogeneous f2m is on HLA molecules lacking /2m at the cell a
E
Table 2. Incorporation of 1251-f32m into HIA-A and HLA-B antigens in living lymphoblast cells Labeled cells Antiserum JY (A2; B7) Specificity LB (A28; B40)
~0 c
0, E
Davis Jackson White
Canaday
0
3000
750
188
Cells X 10 -3
47 per
12
well
FI(;. 4. Binding of radiolabeled /32m to cells. 1251-/32m (30,000 cpm labeled with the Bolton-Hunter reagent) was incubated with JY (O, 0) or I)audi (A, *) cells in 0.I ml of RPMI 1640 medium containing l)ovine serum albumin at 6 mg/ml and puromycin at 50 Mg/ml in the presence (0, ) or the absence (*, A) of unlabeled 132m at 50 Pg/mI. Alter 4 hr at 37°C, the cells were washed three times in buffer A, resuspended in 1 M NaOH, and counted.
A2,A28 B7 B40 A3 B5
7 69
4
-
56 1
1
9 Dols Holzer 3 2 Bw39,B8,B114 Cells (5 X 105) in 1 ml of' RPMI 1640 medium containing gelatin at 1 mg/ml and puromycin at 50 ig/ml were incubated with 20 Mig of' '251-32m, labeled with the Bolton-Hunter reagent. After 4 hr at 370C, the cells were mixed with 5 X 10" unlabeled Laz 007 cells (HLA-A,3,A?; B5,Bw39), washed, and resuspended in buffer A. Aliquots were incubated 2 hr at 370C with various antisera, after which the cells were lysed by addition of Nonidet P-40 to a 0.5%/i, final concentration. After 12 hr at 40C, the samples were centrifuged 30 min at 20,000 X g and the supernatants (containing approximately 90%(, of the radioactivity) were immunoprecipitated (24). Results are expressed as percent of the detergent-soluble radioactivity recovered after immunoprecipitation.
5838
Immunology: Hyafil and Strominger
surface. However, the existence of such molecules is very unlikely because both the expression of HLA-A and -B antigens at the cell surface (26, 27) and the binding of exogeneous 12m (Fig. 4) strongly depend on the expression of /2m by the cells. We rather favor the hypothesis that only a subpopulation of HLA molecules can participate in the exchange process during the time course of our experiments (e.g., only HLA-B7 or a fraction thereof). The other molecules might either not dissociate at all or, at least, have a much longer lifetime of the complex. The longer incubation periods needed to resolve these questions could not be employed because cell viability in the presence of puromycin begins to fall after 24 hr. DISCUSSION The crux of this investigation is the determination of the thermodynamic parameters of the association equilibrium between the a chain of HLA and 12m. The knowledge of these parameters potentially provides a nondenaturing technique for the preparation of isolated a chain and the investigation of its serological and functional properties. It is noteworthy that HLApap did not turn out to be a very good model system for the situation occurring at the cell surface. The lifetime of the complex is longer at the cell surface than with HLApap and probably depends on the specificity of the a chain. It is thus tempting to speculate that the af2m complex might be stabilized by interaction of 32m with cell surface lipids or other surface components. The choice of HLApp as a model system led to the serendipitous discovery of a simple labeling method for HLA-A and -B specificities by exchange with radiolabeled /2m. RIAs based on this method allow the quantitative detection of most HLA specificities, eliminating the need for live cells in tissue typing. Moreover, indirect RIAs can be set up, overcoming the need for pure HLA and the difficult problems of degradation of HLA during radioiodination. Such assays for HLA may prove valuable in biomedical research-e.g., for investigating the relationship between some tumor antigens and HLA (29). It is quite possible that human /2m can also be incorporated into H2-K and -D at the surface of mouse cells. The binding of human 32m to murine cells has been reported and correlated with the H2 antigen density of the cells under study (30). Preliminary experiments confirm a significant binding of human (32m to murine lymphocytes, in contrast to a total absence of binding to embryonal carcinoma cells, which lack H2 antigens or analogs thereof (31). The same authors failed to demonstrate any binding of /2m to human peripheral blood lymphocytes. This may be due to a short incubation time and the use of peripheral blood lymphocytes instead of Epstein-Barr virustransformed lymphoblasts. In the latter case, the expression of HLA antigens is dramatically enhanced (32) so that HLA is the main molecular species found in association with f2m (28). In other types of cells, other minor histocompatibility antigens such as TLa, Qa (33), or HY (34) have been reported in association with /2m and could also be detected and probed by the methods described here. Finally, the specific introduction via f2m of haptenic groups such as the Bolton-Hunter reagent or trinitrophenyl on HLA molecules at the cell surface would constitute a molecularly defined example of "altered self." Study of the biological effects of such modifications could be very rewarding. We thank Dr. Abe Fuks for many stimulating discussions and advice during the course of this work, Dr. Doron Lancet for assistance with the manuscript, and Dr. Philippe Dessen for computer analysis. This work was supported by a research grant from the National Institutes of Health (Al 10736) and a fellowship of the Delegation Generale a la Recherche Scientifique et Technique to F.H.
Proc. Natl. Acad. Sci. USA 76 (1979) 1. Munro, A. & Bright, S. (1976) Nature (London) 264, 145-152. 2. Bach, F. H., Bach, M. L. & Sondel, P. (1976) Nature (London) 259,223-281. 3. Bach, F. H. & Van Rood, J. J. (1976) N. Engl. J. Med. 295, 806-813, 872-878, 927-936. 4. Snell, G. D., Dausset, J. & Nathenson, S. (1976) Histocompatibility (Academic, New York). 5. Munro, A. & Waldmann, H. (1978) Br. Med. Bull. 34, 253258. 6. Barnstable, C. J., Jones, E. A. & Crumpton, M. J. (1978) Br. Med. Bull. 34, 241-246. 7. Shearer, G. M. & Schmitt-Verhulst, A. M. (1977) Adv. Immunol. 25,55-91. 8. Brown, J. L. & Nathenson, S. G. (1977) J. Immunol. 118, 98102. 9. Engelhard, V. H., Strominger, J. L., Mescher, M. & Burakoff, S. J. (1978) Proc. Natl. Acad. Sci. USA 75,5688-5691. 10. Maron, R. & Cohen, I. R. (1979) Nature (London) 279, 715716. 11. Helenius, A., Morein, B., Fries, E., Simons, K., Robinson, R., Schirrmacher, V., Terhorst, C. & Strominger, J. L. (1978) Proc. Natl. Acad. Sci. USA 75,3846-3850. 12. Klareskog, L., Banck, G., Forgren, A. & Peterson, P. A. (1978) Proc. Natl. Acad. Sci. USA 75,6197-6201. 13. Robb, R. J., Terhorst, C. & Strominger, J. L. (1978) J. Biol. Chem. 253,5319-5324. 14. Cejka, J. & Kithier, K. (1972) Arch. Biochem. Biophys. 153, 854-857. 15. Hunter, W. M. (1973) in Handbook of Experimental Immunology, ed., Weir, D. M. (Blackwell, Oxford), Vol. 1, pp. 17.117.36. 16. Bolton, A. E. & Hunter, W. M. (1973) Biochem. J. 133, 529539. 17. Parham, P., Alpert, B. N., Orr, H. T. & Strominger, J. L. (1977) J. Biol. Chem. 252,7555-7567. 18. Laemmli, U. K. (1970) Nature (London) 227,680-685. 19. Colombani, J. & Colombani, M. (1976) in Manual of Clinical Immunology, eds. Rose, N. R. & Friedman, H. (American Association for Microbiology, Washington, DC), pp. 805-810. 20. Amos, D. B. & Pool, P. (1976) in Manual of Clinical Immunology, eds. Rose, N. R. & Friedman, H. (American Association for Microbiology, Washington, DC), pp. 797-804. 21. Rice, R. H. & Means, G. E. (1971) J. Biol. Chem. 246, 831832. 22. Parham, P., Terhorst, C., Herrman, H., Humphreys, R. E., Waterfield, M. D. & Strominger, J. L. (1975) Proc. NatI. Acad. Sci. USA 72, 1594-1598. 23. Ziegler, A., Harrison, S. C. & Leberman, C. (1974) Virology 59, 509-515. 24. Kessler, S. W. (1975) J. Immunol. 115, 1617-1624. 25. Dausset, J., Colombani, J., Legrand, L. & Fellous, M. (1970) in Histocompatibility Testing, ed. Terasaki, P. (Munksgaard, Copenhagen), 53-75. 26. Fellous, M., Kamoun, M., Wiels, J., Dausset, J., Clements, G. & Klein, G. (1977) Immunogenetics 5,423-436 27. Arce-Gomez, B., Jones, E. A., Barnstable, C. J., Solomon, E. & Bodmer, W. F. (1978) Tissue Antigens 11, 96-112. 28. Robb, R., Mann, D. L. & Strominger, J. L. (1976) J. Biol. Chem. 251,5427-5428. 29. Thomson, D. M. P., Tataryn, D. N., O'Connor, R., Rauch, J., Friedlander, P., Gold, P. & Shuster, J. (1979) Cancer Res. 39, 604-611. 30. Anderson, C. L., Kubo, R. T. & Grey, H. M. (1975) J. Immunol. 114,997-1000. 31. Morello, D., Gachelin, G., Dubois, P., Tanigaki, N., Pressman, D. & Jacob, F. (1978) Transplantation 26, 119-125. 32. McCune, J., Humphreys, R. E., Yocum, R. R. & Strominger, J. L. (1975) Proc. Nati. Acad. Sci. USA 72,3206-3209. 33. Viretta, E. S. & Capra, J. D. (1978) Adv. Immunol. 26, 147193. 34. Fellous, M., Gunther, E., Kemler, R., Wiels, J., Berger, R., Guenet, J. L., Jakob, H. & Jacob, F. (1978) J. Exp. Med. 148,58-70.
Immunology
Dissociation and exchange of the f32-microglobulin subunit of HLA-A and HLA-B antigens (subunit association equilibrium/radioimmunoassay/cell surface probe)
FRANTOIS HYAFIL* AND JACK L. STROMINGER Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138
Contributed by Jack Leonard Strominger, August 9, 1979
ABSTRACT Human histocompatibility antigens HLA-A, HLA-B, and HLA-C are a complex of two noncovalently associated subunits: a heavy chain glycoprotein (a) carrying the genetic polymorphism and an invariant light chain, globulin (mm). Upon incubation of papain-solubilized HLA with radiolabeled urinary 02m, the latter is incorporated into HLA, where it substitutes for the preexisting f2m that has dissociated from the complex. The association-dissociation equilibrium that governs this #2m exchange reaction was investigated and found to be characterized by a long lifetime of the complex (half-life of 80 min at 370C) and a relatively low Kd (4 nM). The fi2m exchange was used as the basis of a radioimmunoassay for HLA antigens with radiolabeled ft2m as a unique label for all HLA specificities. In a similar fashion, radiolabeled #2m can be incororated into HLA at the cell surface. Although the process is slower and less extensive than in solution, it can be used as a means to tag cells with specific probes for HLA antigens. The HLA-A, HLA-B, and HLA-C regions of the human major histocompatibility complex (H2-K and H2-D in the mouse) produce a highly polymorphic group of antigens present at the surface of most cells and involved in a variety of immunological phenomena (1-5). These antigens are composed of a glycoprotein of molecular weight 44,000 (a), which is noncovalently associated with 132-microglobulin (/2m), a protein of molecular weight 12,000 (6). Genes in the same region have been found to control the induction of effector T lymphocytes in allogeneic systems and the killing of target cells by those lymphocytes (2) and to restrict the interaction of cytotoxic T lymphocytes with modified syngeneic cells (7). That the HLA and H2 antigens are the actual targets of the effector lymphocytes emerges from antiserum blocking studies (7) and more convincingly from the biochemical analysis of a mutant H2-K molecule (8) and fromr the capacity of liposome-carried purified HLA to elicit a specific cytotoxic response (9). At least in one case (ankylosis spondilytis), it is not unlikely that a disease susceptibility statistically associated with an HLA allele (HLA-B27) is determined by the HLA gene itself rather than by a closely associated gene (see discussion in ref. 3). Indeed, mice differing only in the structure of the H2-K antigen have been recently shown to have different susceptibilities to autoimmune thyroiditis (10). Possibly relevant is the fact that HLA and H2 may be used as cell surface receptors by viruses (11) and bacteria (12). The rationale in the experiments presented here is to take advantage of the structure of the HLA-A, -B, and -C antigens to introduce at the surface of cells specific noninterfering probes of the function of these antigens. The heavy glycoprotein chain is polymorphic and spans the plasma membrane. In contrast, /32m is nonpolymorphic and outside the plasma membrane. Furthermore, it is readily available in large quantities from the
urine of patients with kidney dysfunctions. Investigation of conditions in which exogeneous urinary f2m might replace the /2m carried by HLA antigens at the surface of cells was, therefore, carried out in a model system using papain-solubilized (pap) HLA-A and -B antigens [which lack the membrane-spanning hydrophobic region and the COOH-terminal hydrophilic region (13)]. The exchange of urinary f32m with /2m present in HLA molecules is demonstrated and shown to be a consequence of an association-dissociation equilibrium between the subunits of the HLA-A and -B antigens. The thermodynamics of this equilibrium allow the development of a radioimmunoassay (RIA) for HLA-A and -B antigens in which radiolabeled.2m is used to label all the specificities. A similar process occurs at the surface of cultured lymphoblasts, where radiolabeled /32m can be incorporated into HLA-B antigens by exchange upon incubation at 37°C. Thus many probes (radioactive, spectroscopic, chemical) carried by /32m could potentially be introduced into HLA antigens and might be useful in unraveling the intracellular and extracellular interactions in which those molecules are involved.
72-micro-
MATERIALS AND METHODS Cells and Sera. The lymphoblastoid cell lines used in these experiments were: JY (HLA-A2; B7 homozygote), LB (HLAA28; B40; Cw3 homozygote), Laz 007 (HLA-A3,A?; B5,Bw39), and Daudi (no detectable HLA). The volunteer donors from which blood was obtained were typed by D. Fitzpatrick and B. Canaday in the laboratory of E. Yunis. Tissue typing sera were obtained from E. Yunis, B. Amos, and the Transplantation Branch of the National Institute of Allergy and Infectious Diseases. 02m and HLA. Human urinary /2m (14) (a gift of R. Robb) was iodinated by the chloramine-T method (15) or with the Bolton-Hunter reagent (16) to specific activities of 103 or 2-5 X 104 cpm/ng, respectively. HLA-A2pap was purified as described (17). Reductive Methylation of HLA. A solution of HLApap (2 mg of a mixture of HLA-A2 and B7) in 0.2 M Na2B407, pH 9.0/0.5 M NaCl, to which approximately 1 mg of NaB3H4 (New England Nuclear, 276 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels) had been added was made 1 M in formaldehyde by five successive additions at 5-min intervals. After 1 hr on ice and dialysis, [3H]HLA was repurified on DE 52 DEAE-cellulose by a linear gradient of Tris/PO4 (17). [3H]HLA migrated as a single peak with a specific activity of 5 cpm/ng, representing an incorporation of about eight CH3 groups per molecule. Preparation of Internally Labeled HLA. JY cells (107) were Abbreviations: 132m, 132-microglobulin; a chain, HLA heavy chain; HLA papain-solubilized HLA; Lch, Lens culinaris hemagglutinin; NaDodSO4, sodium dodecyl sulfate; RIA, radioimmunoassay. * Present address: Unite de Genetique Cellulaire, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, Cedex 15 France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5834
Proc. Natl. Acad. Sci. USA 76 (1979)
Immunology: Hyafil and Strominger cultured for 24 hr with 5 mCi of L-[4,5-3H(N)]lysine (New England Nuclear, 60 Ci/mmol). Labeled cells were resuspended in buffer A (10 mM Tris-HCl, pH 7.3/0.15 M NaCI/3 mM NaN3) containing bovine serum albumin at 6 mg/ml and digested with 20 units of papain (Worthington) activated with 0.4 mg of cysteine. After 30 min at 37°C, the cells were pelleted and [3H]HLA was purified from the supernatant by: (i) DE 52 chromatography to get rid of papain, (ii) affinity chromatography on agarose coupled with Lens culinaris hemagglutinin (Lch), (iii) gel filtration on Sephadex G-150 of the methyl a-mannoside eluate from the Lch-agarose. About 1 ,g of pure HLApap containing 5 X 105 cpm was obtained as judged by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis (18) and fluorography. Crude Papain Extracts from Platelets and Peripheral Blood Lymphocytes. Cells were isolated from freshly drawn blood (19, 20) and digested with papain as above but for the
B 2010
F
10(0
F
2 c) -0 .? T 4
I I I
^ C
I
C
1.
E 3 20C
-
FIG. 1. Radiolabeled urinary 32m can be incorporated into HLA. (A) Gel filtration profile of 1251-132m (1.6 x 106 cpm/1.6 gg) incubated with HLA-A2pap (16 ,g) for 3 hr at 25°C in buffer A and run on a Sephadex G-75 column (1.2 X 35 cm) equilibrated in buffer A
containing lysozyme at 1 mg/ ml. Fraction size is 0.75 ml: Arrows indicate the elution positions of blue dextran (VO),
1 OC
addition of 10% citric acid/citrate/dextrose in the case of platelets. The supernatants were treated with 0.8 mg of iodoacetic acid and used immediately or stored at -20'C until needed. RESULTS #2m Exchanges with the Small Subunit of HLApap. Radiolabeled urinary f2m (labeled with '25I by the chloramine-T method) migrates as a single peak on Sephadex G-75. When it is incubated with HLA-A2pap, part of the radioactivity elutes early from the column at the same position as HLA-A2pap (Fig. 1A). To show that in this heavier material /2m was associated with HLA-A2pap, immune complex formation was assessed by gel filtration on Sephadex G-150 (Fig. 1 B, C, and D). Whereas 90% of the radioactivity associated with HLA-A2 could be complexed by an HLA-A2 antiserum (Fig. 1B), normal human serum (Fig. 1C), or alloantisera of other specificities (not shown) failed to bind any labeled material. As a control, it was also shown that the HLA-A2 alloantiserum used above does not have any anti-f2m activity (Fig. 1D). Transfer of radioactive material between f2m and the a chain was ruled out by NaDodSO4/polyacrylamide gel electrophoresis and gel filtration in 6 M guanidine hydrochloride, which showed that all the radioactivity incorporated into HLA was associated with the polypeptide of 12,000 daltons. It was important to demonstrate that there is actual exchange between the /2m originally present on HLA-A and -B antigens and the added urinary 12m. HLA-A2,1B7pap was thus labeled by reductive methylation (21) in the presence of NaB3H4, a modification that does not alter the antigenic properties of HLA-A and -B antigens (22). When reductively methylated HLApap was incubated with increasing amounts of urinary /2m and then separated from /2m by gel filtration, an increasing amount of radioactivity eluted with free 32m (Fig. 2A). Furthermore, analysis by NaDodSO4/polyacrylamide gel elec-
0 I 0 x
E
E 0
a
3C 2C
E E 0
C
E C.)
A
4C
)c
C)r D 500
_5W-0) wc) -T 4 4 4 4
400 300 200 100
0I I
20
30 40 Fraction
50
of Davis [an HLA-A2 antiserum (B, D)l or normal human serum (C). After 24 hr at 4°C, the samples were run on a Sephadex G-150 column (1.0 X 60 cm). Fractions (1 ml) were assayed for radioactivity and absorbance at 280 nm. Arrows indicate the eluition position of major serum proteins. In B, C, and D, the peak at tube 20 is the immune complex of HLAA2, that at tube 35 is HLA-A2 itself, and that at tube 46 is free f32m. Alb, albumin.
Of
1C
a I 0 10
[3H]HLApap, and 1251-f2m in-
cubated in the absence of HLA. Fractions 19-23 were pooled and a 0.05-ml aliquot (B, C) of this pool or 20 ng of 1251-fl2m (D) was incubated with 0.2 ml
5835
a
a
I
100"150 200 250
50
A2M, ,ug
B a a
chain
/)2m 13
b
__X
M
c
d
_
e
f
-
h
g
i
bd%
U
_P
FIG. 2. Exchange between the small subunit of radiolabeled HLApap and urinary f2m. Reductively methylated [:H]HLAPaP (120,000 cpm/24 ,ug) was incubated for 5 hr at 370C with increasing amounts of unlabeled f,3m and analyzed on a Sephadex G-75 column equilibrated in 0.1 M NH4HCO:i to separate HLApap from free f32m. Fractions were assayed for radioactivity (A), and the material mi,grating at the position of HLAj)aP was pooled, freeze-dried, run on NaDodSO4/polyacrylamide gel electrophoresis (23), stained with Coomassie blue, and fluorographed (B).
5836
Immunology: Hyafil and Strominger
Proc. Natl. Acad. Sci. USA 76 (1979)
trophoresis of the HLA-A and -B antigens revealed a constant ratio of the intensities of the heavy chain and O2m bands after Coomassie blue staining (not shown) but a progressive loss of the [3H]32m band as visualized by fluorography (Fig. 2B). Thus, at neutral pH and isotonic salt concentration, all of the [3H]32m in 13H]HLApap was replaced by urinary O2m. The two HLA specificities, HLA-A2 and HLA-B7, exchanged with '25I-02m at the same rates in an experiment in which separated HLA-A2pap and HLA-B7pap were used. Thermodynamics of the Interaction Between the Subunits of HLApap. The simplest explanation of the above findings is an association-dissociation equilibrium between the a chain of HLA-A and -B antigens and /32m (designated in this section as 3): aO/
k~ff (. a
kon
+
/3,
Kd
=
koff/kon0
[1]
of appearance of labeled free displayed an exponential behavior and yielded a value for koff of 1.4 X 10-4 s-1 at 370C. In another set of experiments, the dissociation was measured directly in the absence of added /2m. In these conditions [a] = 1/] and, after 5 hr at 370C, it can be shown that the system has reached equilibrium so that [/], the free /32m concentration, can be expressed by Eq. 2, in which HLAt1t is the initial concentration of HLA: [2] [/3] = Kd[(l + 4 HLAtot/Kd)1/2 - 1]/2. This effect was monitored by using either internally labeled [3H]HLApap (Fig. 3C) or a tracer amount of '25I-/2m (Fig. 3D). Experimental results were in good agreement with Eq. 2, indicating a Kd of about 4 nM at 370C. Parameters obtained by using 125I-/2m (labeled with the Bolton-Hunter reagent) as a tracer were very similar to those obtained by using reductively methylated or [3H]lysine internally labeled HLApap, thus suggesting that labeling /2m with 125I by using the Bolton-Hunter reagent does not modify its interaction with the chain. In contrast, some experiments seem to indicate that /32m iodinated on its tyrosines has a weaker affinity for the chain. Whatever technique was used to radioiodinate /2m (chloramine-T, lactoperoxidase/H202, lactoperoxidase/glucose oxidase/glucose, or Bolton-Hunter reagent), only about 40% of the label could be incorporated into HLApap (see Fig. 3D), and the residual material could not be incorporated when incubated again with HLA. Although this could be due to radioiodination damage, it can be explained better by the heterogeneity of the batch of /2m used in these experiments (unpublished data). RIA for HLA Antigens. At 370C, the half-life of the ao/ complex is 80 min. As shown in Fig. 3A, it is increased by about two orders of magnitude at 0C, so that the complex is stable for most practical applications even at concentrations much lower than its Kd. An immediate consequence is that HLA-A and -B antigens can be labeled at 370C with exogeneous /2m and then assayed in the cold to prevent dissociation of the complex. On this basis, a direct RIA for HLA was devised with the following steps: (i) removal of HLA-A and -B antigens (and other proteins) from cells by treatment with papain, (ii) incubation for 4 hr at 370C with '25I-/2m, (iii) gel filtration on Sephadex G-75 (or affinity chromatography on Lch-agarose) a
E
x
En QL C
E
u
U.
C
Ea 4
.4
4
i X
EEL
~+
80B 70r 60;
30-
50 -\
20-
E 40E c~ 30 CL
D
C
A series of experiments aimed at testing the validity of this model and at measuring its parameters was carried out. The dissociation of the ao/ complex is the rate-limiting step when radiolabeled is displaced from ao3 by an excess of free unlabeled d. Such experiments, using either reductively methylated [3H]HLA (Fig. 3A), or HLApap labeled in its d subunit by exchange with 125I-02m (Fig. 3B), showed that the time course
a
4
-J
0 T 0 C
E
CL
I
~
~
20
I
40 Time, ks
60
I
-9 -8 -7 -6 log [HLA pap
FIG. 3. Kinetics (A, B) and thermodynamics (C, D) of the association-dissociation reaction between the subunits of HLApap. (A) Reductively methylated HLApap (240,000 cpm/48 ug) was incubated at 00C (ti), or at 370C (0, 0) with 0 jig (0) or 100 ,g (0, A) of I32m. (B) 1251-HLA was prepared by exchange of 1251-/32m labeled by the Bolton-Hunter reagent and gel filtration as in Fig. 1A. Samples (1.2 mg) were incubated at 370C with 10 mg of unlabeled f2m. (C) [3H]Lysine-labeled HLApap (20,000 cpm/40 ng) or (D) 1251-32m (20,000 cpm labeled with the Bolton-Hunter reagent) was incubated at 370C with various amounts (expressed in M) of unlabeled HLApap. At various times (5 hr for C and D), samples were analyzed as in Fig. 1A. The experimental results were fitted to the theoretical equations by using a three-parameter iterative regression computer program. The best-fit values with their standard deviations are: (A) kff = (1.44 i 0.16) X 10-4 S-1, (B) kzff = (1.34 i 0.49) X 10-4 S-1, (C) Kd = (3.38 ± 0.60) nM, and (D) Kd = (5.31 + 1.93) nM. Theoretical curves using these parameters are drawn as continuous lines.
to remove excess '25I-/2m and lower the background in subsequent immunoprecipitation, (iv) incubation overnight at 4VC with alloantiserum, and (v) immunoprecipitation with cells of Staphylococcus aureus strain Cowan I (24). By using pure HLA-A2pap, subnanogram quantities of antigen could be specifically labeled and immunoprecipitated by this technique. Furthermore, both specificities in all the mixtures tested could be detected, namely: A2 and B7, A28 and B40, or B40 and Cw3. To screen a variety of specificities, crude papain extracts from human cells of known types were assayed by this method. A good correlation between the typing patterns obtained in classical cytotoxicity tests and the RIA was obtained (Table 1). Some of the false negative results can be explained by a lower representation of some antigens [HLA-B12 on platelets (25)1. Other discrepancies could be due to a peculiar behavior of some antigens in the association-dissociation equilibrium or more likely to slight differences between the HLA serology defined by microlymphocytotoxicity and by the RIA described here. fl2m Incorporates into HLA at the Cell Surface. To determine whether a similar exchange process would also occur on the plasma membrane of living cells, the following experiments were performed in the presence of puromycin to prevent the turnover of HLA-A and -B antigens at the cell surface. Under those conditions, there is no significant degradation of HLA-A and -B antigens in 24 hr, whereas other cell surface markers, such as 1g, are shed in a few hours (measured with internally labeled cells). 125I-/2m labeled by the Bolton-Hunter reagent binds specifically to the lymphoblastoid cell line JY, but there is no binding to Daudi cells, which are known to lack HLA-A and
Immunology: Hyafil and Strominger
Antiserum
Specificity
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 1. RIA for HLA-A and HLA-B antigens* Donor,- HLA type, and sourcet BF FH GK A2; B5,7 A3,10; B5,? A2,9; B7,15 platelets platelets platelets G-75 Lch G-75 G-75 Lch
5837
HP A2,3; B7,12 PBLs Platelets G-75 G-75
232 82 362 360 Davis A2 308 86 260 ND A3 400 19 22 77 Canaday 238 119 ND ND ND ND 280 ND Fe125 A9 39 24 15 ND 12 Keyis 310 164 A10 22 ND B5 76 57 16 20 31 Filting 28 B7 190 Jackson 377 46 30 410 206 380 B12 11 30 210 SLA 35 57 27 50 Gommol ND 82 25 B15 56 25 217 5 * Results are expressed as cpm recovered in the S. aureus pellet after three washes. Expected positive results are underlined. ND, not done. The last line in each column heading indicates how excess 32m was removed from the antigen, gel filtration on Sephadex G-75 or affinity chromatography on Lch-agarose. PBLs, peripheral blood lymphocytes.
-B antigens at their surface (26, 27) (Fig. 4). A nonspecific binding (not inhibited by unlabeled 132m) was also observed for both cell types at high cell concentrations. As in the case for HLApap, only a portion of the '2-132m could be exchanged, but in this case it amounted to only about 4% of the total radioactivity. The residual 96% could not be exchanged in further attempts. The cell-bound radioactivity was displaced in the presence of unlabeled /2m with a half-time of about 12 hr at 370C (and at least ten times longer at 0°C). To show that cellbound radioactivity was incorporated into HLA-A and -B antigens, cells were incubated with alloantisera and lysed with detergent, and the supernatants were immunoprecipitated by addition of cells of S. aureus strain Cowan I. Exogeneously added 1251-I2m incorporated preferentially into HLA-B7 (or -B40) at the surface of cells rather than into HLA-A2 (or -A28) (Table 2). In these experiments the labeled cells were mixed
2000
with unlabeled cells of a different HLA type before detergent solubilization. The absence of radioactivity in the HLA-A and -B antigens of these cells shows that the association of /32m with HLA-B and to a lesser extent with HLA-A antigens occurs at the surface of cells and not during the immunoprecipitation. Is There Exchange at the Cell Surface? In order to answer this question, JY cells were labeled with [3H]lysine and then incubated at 37°C in the presence of puromycin with or without the addition of urinary 12m at 50 ug/ml. At various times, samples were harvested and surface HLA-A and -B antigens were purified by (i) papain digestion of whole cells terminated by iodoacetic acid, (ii) affinity chromatography on Lch-agarose with elution by methyl a-mannoside, and (iii) affinity chromatography on rabbit anti-32m IgG coupled to Sepharose with elution by glycineFHCl, pH 2.2 (28). The relative radioactivity of the chain and the !2m were assessed by gel filtration under denaturing conditions (10% acetic acid). The fraction of radioactivity in the 32m peak did not change significantly in the absence of /2m (55.4%, 59.3%, and 55.6% after 3, 11, and 25 hr, respectively) but decreased with /2m in the medium (62.8%, 55.3%, and 46.4% at 3, 11, and 25 hr, respectively). The decrease was not large and amounted to a maximum of 25% exchange during the 25-hr incubation. One possible interpretation is that the observed binding of exogeneous f2m is on HLA molecules lacking /2m at the cell a
E
Table 2. Incorporation of 1251-f32m into HIA-A and HLA-B antigens in living lymphoblast cells Labeled cells Antiserum JY (A2; B7) Specificity LB (A28; B40)
~0 c
0, E
Davis Jackson White
Canaday
0
3000
750
188
Cells X 10 -3
47 per
12
well
FI(;. 4. Binding of radiolabeled /32m to cells. 1251-/32m (30,000 cpm labeled with the Bolton-Hunter reagent) was incubated with JY (O, 0) or I)audi (A, *) cells in 0.I ml of RPMI 1640 medium containing l)ovine serum albumin at 6 mg/ml and puromycin at 50 Mg/ml in the presence (0, ) or the absence (*, A) of unlabeled 132m at 50 Pg/mI. Alter 4 hr at 37°C, the cells were washed three times in buffer A, resuspended in 1 M NaOH, and counted.
A2,A28 B7 B40 A3 B5
7 69
4
-
56 1
1
9 Dols Holzer 3 2 Bw39,B8,B114 Cells (5 X 105) in 1 ml of' RPMI 1640 medium containing gelatin at 1 mg/ml and puromycin at 50 ig/ml were incubated with 20 Mig of' '251-32m, labeled with the Bolton-Hunter reagent. After 4 hr at 370C, the cells were mixed with 5 X 10" unlabeled Laz 007 cells (HLA-A,3,A?; B5,Bw39), washed, and resuspended in buffer A. Aliquots were incubated 2 hr at 370C with various antisera, after which the cells were lysed by addition of Nonidet P-40 to a 0.5%/i, final concentration. After 12 hr at 40C, the samples were centrifuged 30 min at 20,000 X g and the supernatants (containing approximately 90%(, of the radioactivity) were immunoprecipitated (24). Results are expressed as percent of the detergent-soluble radioactivity recovered after immunoprecipitation.
5838
Immunology: Hyafil and Strominger
surface. However, the existence of such molecules is very unlikely because both the expression of HLA-A and -B antigens at the cell surface (26, 27) and the binding of exogeneous 12m (Fig. 4) strongly depend on the expression of /2m by the cells. We rather favor the hypothesis that only a subpopulation of HLA molecules can participate in the exchange process during the time course of our experiments (e.g., only HLA-B7 or a fraction thereof). The other molecules might either not dissociate at all or, at least, have a much longer lifetime of the complex. The longer incubation periods needed to resolve these questions could not be employed because cell viability in the presence of puromycin begins to fall after 24 hr. DISCUSSION The crux of this investigation is the determination of the thermodynamic parameters of the association equilibrium between the a chain of HLA and 12m. The knowledge of these parameters potentially provides a nondenaturing technique for the preparation of isolated a chain and the investigation of its serological and functional properties. It is noteworthy that HLApap did not turn out to be a very good model system for the situation occurring at the cell surface. The lifetime of the complex is longer at the cell surface than with HLApap and probably depends on the specificity of the a chain. It is thus tempting to speculate that the af2m complex might be stabilized by interaction of 32m with cell surface lipids or other surface components. The choice of HLApp as a model system led to the serendipitous discovery of a simple labeling method for HLA-A and -B specificities by exchange with radiolabeled /2m. RIAs based on this method allow the quantitative detection of most HLA specificities, eliminating the need for live cells in tissue typing. Moreover, indirect RIAs can be set up, overcoming the need for pure HLA and the difficult problems of degradation of HLA during radioiodination. Such assays for HLA may prove valuable in biomedical research-e.g., for investigating the relationship between some tumor antigens and HLA (29). It is quite possible that human /2m can also be incorporated into H2-K and -D at the surface of mouse cells. The binding of human 32m to murine cells has been reported and correlated with the H2 antigen density of the cells under study (30). Preliminary experiments confirm a significant binding of human (32m to murine lymphocytes, in contrast to a total absence of binding to embryonal carcinoma cells, which lack H2 antigens or analogs thereof (31). The same authors failed to demonstrate any binding of /2m to human peripheral blood lymphocytes. This may be due to a short incubation time and the use of peripheral blood lymphocytes instead of Epstein-Barr virustransformed lymphoblasts. In the latter case, the expression of HLA antigens is dramatically enhanced (32) so that HLA is the main molecular species found in association with f2m (28). In other types of cells, other minor histocompatibility antigens such as TLa, Qa (33), or HY (34) have been reported in association with /2m and could also be detected and probed by the methods described here. Finally, the specific introduction via f2m of haptenic groups such as the Bolton-Hunter reagent or trinitrophenyl on HLA molecules at the cell surface would constitute a molecularly defined example of "altered self." Study of the biological effects of such modifications could be very rewarding. We thank Dr. Abe Fuks for many stimulating discussions and advice during the course of this work, Dr. Doron Lancet for assistance with the manuscript, and Dr. Philippe Dessen for computer analysis. This work was supported by a research grant from the National Institutes of Health (Al 10736) and a fellowship of the Delegation Generale a la Recherche Scientifique et Technique to F.H.
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