Report of the second international soluble HLA (sHLA) workshop

Special Issue on the Second I n t e r n a t i o n a l Soluble H L A ( s H L A ) W o r k s h o p

Report of the Second International Soluble HLA (sHLA) Workshop F. Carl Grumet, Roland Buelow, Hans Grosse-Wilde, Britta Kubens, Marvin Garovoy, and Philippe Pouletty

O R G A N I Z I N G COMMITTEE: Marvin Garovoy, F. Carl Grumet, and Philippe Pouletty. SCIENTIFIC COMMITTEE: Francoise Amesland, Roland Buelow, William Burlingham, Dominique Charron, Frans Claas, Rene Duquesnoy, Soldano Ferrone, Frank Gelder, Hans Grosse-Wilde, and Jorge Kalil. STUDY P A R T I C I P A N T S (laboratory number): F. Amesland (1), R. Fauchet (2), E. Ivaskova (3), J. Tkaczuk (4), P. Mackintosh (5), J. Flament (6), W. Burlingham (7), L. DeVito (7), P. M. Edmonds (8), F. Claas (9), R. Kerman (10), B. Colombe (11), M. Garovoy (11), C. Russo (12), S. Hashemi (13), E. Yang (13), F. Baroni (14), W. Mantovani (14), R. Mancini (14), C. Curuso (15), H. Grosse-Wilde (16), U. Westhoff (16), B.

ABBREVIATIONS BOLA bovine class I antigen CYNAP cytotoxicity negative, absorption positive ELISA enzyme-linked immunoassay FACS fluorescence-activated cell sorter CREG cross-reactive group IEP isoelectric point

Kubens (16), F. Gelder (17), S. Ferrone (18), R. Holdsworth (19), J. Kalil (20), M. Hagihara (21), K. Tsuji (21), D. Xian-hui (22), V. Lazda (23), C. Navarrete (24), F. Puppo (25), A. Menicucci (26), R. Baricordi (26), P. Robbins (27), W. Green (28), E. Carbone (29), S. A. Alharbi (30), R. E. Mahmaoud (31), A. Nocera (32), S. Barocci (32), S. D. Borelli (33), R. K. Charlton (34), A. Ferreira (35), W. He (36), M. S. Leffel (37), L. M. Villar (38), C. D. Qing (39), R. Duquesnoy (40), F. C. Grumet (41), R. Buelow (42), P. Pouletty (42), S. NehlsenCannarella (43), J. Neumann (44), B. K. Shenton (45), E. A. Torres (46), V. Taneja (47), L. Huilin (48), P. Terasaki (49), D. Chia (49), and G. Wang (49). Human Immunology 40, I 5 3 - 1 6 5 (1994)

LCL mAb RaHC sHLA 1D-IEF

lymphoblastoid cell line monoclonal antibody rabbit-anti-human class 1 heavy chain soluble HLA antigen one-dimensional isoelectric focusing

INTRODUCTION The complementary physiologic roles of proteins expressed in membrane-bound or secreted forms as a result of regulated alternative splicing mechanisms are well established for immunoglobulins. More recently, soluble HLA antigens (sHLA) have also received attention as an

From the Department of Pathology (F.C.G.), Stanford University, Stanford," SangStat Medical Corporation (R. B., P.P. ), Menlo Park; and the Immunogenetics and Transplantation Laboratory ( M. G. ), UCS F Medical Center, University of California-San Francisco, San Francisco, California, USA; and the Institute of Immunology (H.G.-W., B.K.), Essen University Hospital, Essen, Germany. Address reprint requests to Dr. F. C. Grumet, Stanford University Blood Center, 800 Welch Road, Palo Alto, CA 94304, USA. Received (U) April I1, 1994; acceptedApril I4, 1994.

Human Immunology40, 153-165 (1994) © AmericanSocietyfor Histocompatibilityand lmmunogenetics, 1994

important analogous component of the H L A prote family. To enhance exchanges and c o m m u n i c a t i l among investigators of this topic, the First Internatior Soluble HLA (sHLA) Workshop [1] allowed a validati, of enzyme-linked immunoassays (ELISAs) to quantit~ sHLA antigens by 15 laboratories. Furthermore, a fi~ sHLA class I antigen international standard (t.sB7) defined for interlaboratory standardization. For seve HLA alleles, various publications have demonstrat that alloantigenic reactivity is identical for both t membrane-bound and the naturally occurring solut form of that specificity [2-7]. To determine whether tl held true for most known HLA allele specificities, and provide a forum focused on investigation of sHLA, t 1 0198-8859/94/$7

154

Second International Soluble HLA Workshop was held in Phoenix, Arizona, in October 1993, as a satellite workshop of the 19th Annual American Society for Histocompatibility and Immunogenetics (ASHI) Meeting. In the wet part of the workshop, performed during the 6 months preceding the meeting, sHLA class I antigens in the supernatants of 45 phenotyped lymphoblastoid cell lines (LCLs) expressing a total of 37 alleles were studied. A total of 49 laboratories participated in the study, using several sHLA-typing techniques: inhibition of lymphocytotoxicity, inhibition of flow cytometry, sHLA ELISA, and isoelectric focusing (IEF). The wet workshop demonstrated detectability of a very high number of class I sHLA allele speciflcities. With currently available reagents, many sHLA antigens could be recognized in non-cell-dependent techniques, (e.g., ELISA and IEF), suggesting that sHLA-containing fluids such as plasma or LCL culture supernatants may constitute a potential source of antigen for HLA typing or detection of anti-HLA antibodies. The presentations and manuscripts submitted for the workshop open meeting raised and reviewed a number of interesting possible functions for class I sHLA. These antigenically reactive molecules are known to occur naturally in normal body fluids, with several different sizes of heavy (or) chains: a 44- to 45-kD species of shed, intact membrane antigens; --39-kD secreted molecules lacking the transmembrane domain because of alternate splicing; and species at 34-35 kD and 36-37 kD resulting from proteolytic digestion of the 39- and/or 44- to 45-kD species [8-11]. Kubens et al. [12], Westhoffet al. [13], Drouet et al. [14], and Hagihara et al. [15] examined the different patterns and genetic determinants of this sHLA expression; specific biological functions, however, were not assigned to any particular species. An immunomodulatory function, self-tolerance, has previously been suggested for sHLA [ 16], and additional immune regulatory function was suggested by Saririan et al. [ 17], who reported correlation of prevaccination plasma sHLA levels with response to influenza vaccine. Whether the elevated plasma sHLA levels associated with gastric carcinoma [18] and pulmonary tuberculosis [19] also represent immunomodulatory functions or just catabolic reactions to disease is not known. No new information was available regarding another previously proposed physiologic function for sHLA, that of olfactory stimuli for individuality recognition [20, 21]. With respect to possible significance for transplantation, a role for sHLA had been implied earlier because of the very high plasma sHLA levels observed following placement of liver allografts, an organ that is relatively well tolerated [22]. Additional prior reports had shown that sHLA can specifically downregulate cytotoxic lymphocytes [23] and that complexes of allogeneic sHLA

F.C. Grumet et al.

plus antibody could induce prolongation of allograft survival [24]. In new animal models, Wang et al. [25] have now shown soluble class I alloantigen alone can prolong rat heart allografts, while Grumet et al. [26] have demonstrated that sHLA alone can block specific alloantibody induction in mice. At a more immediate and practical level, sHLA appears to hold promise as a noninvasive monitor for allograft rejection. Donor-specific sHLA was used by Claas et al. [27] to identify heart graft rejection episodes, and by DeVito et al. [28] to identify kidney or kidneypancreas rejection epidoses; while total sHLA were used by Puppo et al. [29] to identify liver allograft rejection, and by Shroeder et al. [30] to follow renal graft rejection. Overall, class I sHLA molecules clearly deserve continued study to clarify and extend their roles as physiologic immunomodulators, as specific immunosuppressive therapeutic agents, and as noninvasive monitors of allograft rejection. In addition, it is likely that novel and nonimmunologic functions (e.g., olfactory stimuli) are likely to be further defined as well. Hopefully, future sHLA workshops will be able to encourage and facilitate study of this very interesting class of molecules. WET WORKSHOP REPORT Production of sHLA Although normal donor serum contains class I sHLA antigen, the ready availability of many stable lymphoblastoid B cell lines (LCLs) homozygous for HLA-A and/ or -B, as well as their relative safety regarding transportation of known infectious material led us to select the LCL fluids rather than sera as the source for the test sHLA distributed in the workshop. The Epstein-Barr virus (EBV)-transformed human LCLs that had been generated from peripheral blood lymphocytes previously phenotyped by microlymphocytotoxicity were from SangStat Medical Corporation (Menlo Park, CA, USA). To increase the production of sHLA, cells were stimulated with mitogens as described [5]. After removal of the cells by centrifugation, the supernatant was concentrated by ultrafiltration. The sHLA concentration was determined using the sHLA-STAT class I ELISA kit (SangStat). The final sHLA concentration (200-3000 ng/ ml) was very similar to that observed in the serum of normal individuals [6, 7]. Table 1 lists the phenotypes of the LCLs and the sHLA concentration of each fluid. Typing of sHLA by Cytotoxicity Inhibition Inhibition of cytotoxicity is a relatively simple modification of standard typing techniques that was easily set up in most HLA-typing labs to test for class I sHLA antigens. The objectives of this phase of the workshop, therefore, were (a) to determine whether cytotoxicity in-

Second sHLA Workshop

TABLE 1

15

Phenotypes of LCLs and the s H L A c o n c e n t r a t i o n of each fluid Panel A

Code name U-100 U-200 U-300 U-500 U-700 U-800 U-900 U- 1000 U- 1200 U-1300 U- 1400 U- 1500 U- 1600 S-2100 C-2200 G-2300 G-2700 G-3200 G-3500 C-3700 S-4300 C-4400 S-4700 S-5700 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 U100 U200 U100 diluted 1:3 U100 diluted 1:9 U100 diluted 1:27

Comments

Phenotype

No antigen control Plasma sHLA Plasma sHLA Replicate S-5800 Replicate U- 1600

A24 B7 A1 B52 A30 B18 A23 B 14(65) A32 B38 A29 B61 A1 B41 A33 B44 A2 B35 A32 B44 A24 B60 A31 B60 A31 B62 A l l A32 B39 B62 A3 A23 B50 B55 A l l A33 B51 B54 A24 B57 A26 B39 B44 A31 A32 B13 B51 A25 A28 B51 A24 B27 B44 A2 A25 Bl8 B45 A1 A2 B39 B58 A1 A2 B8 B27 None Not available Not available B7 A31 B62 A24 B7 A1 B52 A24 B7 A24 B7 A24 B7

sHLA concentration (ng/ml) 2044.8 1008.0 1918.3 995.5 1072.8 428.3 951.0 785.8 780.5 927.8 1144.8 611.0 984.8 1364.8 1268.5 1019.8 910.8 991.3 451.8 797.5 2242.5 991.3 959.5 844.0 0.0 1629.5 1409.3 2456.8 1086.5 1869.5 1060.0 584.7 190.7 45.8

Panel B

Code n a m e U-400 U-1700 U-1800 G-2400 G-2500 G-2600 G-2900 G-3400 G-3600 B-3900 B-4000 B-4100 B-4200 S-4500 S-4600

Comments

Phenotype A26 B38 A3 B57 A24 A26 B51 B61 A3 A31 B27 B35 A2 B41 A2 B54 B62 A2 A28 B8 B50 A1 B61 A3 A l l B18 B52 A2 B8 B41 A1 A30 B8 A2 B52 A2 A26 B49 B57 A2 A24 B27 A l l A25 B7

sHLA concentration (ng/ml) 397.7 438.5 601.3 1541.3 742.0 528.8 214.8 811.3 1491,8 600,5 542.3 614.3 544.8 1907.8 199.5 (Continued)

156

F.C. Grumet et al.

TABLE 1

(Continued) Panel B

Code name B-4800 B-5000 B-5300 S-5500 C-5600 S-5800 Sample 1 Sample 2 Sample 3 Sample 4 Sample 6 U 100 U200 U100 diluted 1:3 U100 diluted 1:9 U100 diluted 1:27

Comments

No antigen control Plasma sHLA Plasma sHLA Replicate S-5800 Replicate U-400

Phenotype A25 A30 B13 B55 A29 A32 B35 B45 A3 A33 B7 B41 A1 B8 B58 A24 A28(69) B44 B49 B7 None Not available Not available B7 A26 B38 A24 B7 A1 B52 A24 B7 A24 B7 A24 B7

hibition could be used by many labs to identify accurately a broad range of class I sHLA antigens, (b) to determine relative sensitivity and specificity for this technique, and (c) to provide a set of multicenter data to compare this technique with other sHLA antigen identification methods. The common technique used by the participating labs is summarized as follows: Typing antisera were loaded into a Terasaki typing tray at 1 ~l/well. Then, 1 ~I of sHLA-containing fluid or saline (or balanced salt solution) control was then added to each well and incubated for 2 hours at 20°-22°C. Next, 1 gtl of target peripheral blood lymphocyte suspension (at 2000/1~1) was next added to selected wells and incubated for 30 minutes at 20°-22°C. Wells were selected by expectation of a positive reaction between the target cells and the antiserum in the well (e.g., an A1 + cell added to wells with antiA1, an A2 + cell to wells with anti-A2, etc.). Cells were washed (in the wells) three times by soft-drop addition of balanced salt solution, followed by flicking or gentle blotting. Then, 5 ~1 of rabbit serum (complement) were added and, after 90 minutes of incubation at 22°C, each was read and scored for cytotoxicity in the laboratory's routine manner. The sHLA fluids were provided in two panels, A and B (Table 1); half of the labs received panel A and half received panel B. Panels o f s H L A fluids were sent to 45 requesting labs, of whom 26 returned their test results. Because the samples were not sent out completely blinded, several investigators tested only for the presence of the expected A and/or B alleles, and therefore false ( + ) and/or ( - ) results could not be assessed for those labs. Based on the

sHLA concentration (ng/ml) 358.0 340.8 705.8 1094.8 1040.3 6861.6 0.0 1629.5 1409.3 2456.8 397.7 1869.5 1060.0 584.7 190.7 45.8

results submitted, particularly evidence of measurable false ( + ) and ( - ) rates (since none of us is perfect) or a definitive statement that testing for most HLA antigens was performed, the data from 16 labs were selected for the combined analysis. Test results were considered "specific ( + ) " if the expected A- or B-locus antigens were identified as being present, and "cross-reactive ( + ) " if closely cross-reactive group (CREG) (e.g., A23 for A24, and B55 for B56) sHLA antigens were identified. To assess test reproducibility among samples, several fluids were blind duplicates of panel fluids containing known antigens. Figure 1 shows the overall results of that assessment. As seen in the top panel, the percent of labs identifying the correct antigens in each known sample differed somewhat from the percent of labs identifying the same antigens in the cognate hidden duplicate. For example, 83% of labs detected the B7 in fluid S-5800, while 59% also detected B7 in the cognate hidden duplicate fluid 4; and 62% detected A26 in fluid U-400, while 33% detected that same antigen in the duplicate, fluid 6. The reporting of extra reactions, as shown in the bottom panel, appeared somewhat more consistent for several pairs (e.g., U-1600 and 5, U-400 and 6) than for others (e.g., S-5800 and 4). In general, the reproducibility data showed there was substantial room for improvement and standardization of cytotoxicity inhibition methodology. Sensitivity of the common technique was initially assessed by testing fixed dilutions of a culture fluid from the panel's A24,B7 double-homozygous LCL (Table 2). Detection of the expected antigens was best at the highest concentration of test fluid and worsened with dilu-

Second sHLA Workshop

15

GROUPED BY FLUID

TABLE 2

Controls and replicates 100

Antigens identified in dilutions of A24,B7 fluid Number of labs identifying

gO

b 0 (.)

Number of labs reporting

A24

B7

A-locus extras

Undiluted 1:3 1:9 1:27

12 10 10 10

12 8 7 4

11 9 8 4

2 1 0 0

a Diluted

control

B-loc extra

40

O0

1 52

1 52

3162

3162

2638

Specific antigen only

[]

2.638 24 7

24 7

0 7

in negative

culture

medium.

Specific + cross-reactive

io 40

20

1

580o

FLUID # --->

i A Locus []S

4 4 l 0

0 7

8O

m

Dilutiona of A24,B7 fluid

Locus

FIGURE 1 Analysis of sets of duplicates of individual fluids. (Top) Detection of component antigens within each fluid: The horizontal axis shows the antigens present in each fluid, and the vertical axis indicates the percent of laboratories detecting each antigen. (Bottom) Reporting of extra reactions: the horizontal axis shows the A- and B-locus extras for each fluid, and the vertical axis indicates the percent of laboratories reporting such extras. The fluids with known duplicates are A1,B52 vs U-200; and A24,B7 vs U-100. The pairs with known and blind (respectively) duplicates are S-5800 vs 4, U-1600 vs 5, and U-400 vs 6. Fluid 1 is negative culture fluid control. tion. In contrast, reporting of extra antigens was most problematic at the highest concentration of test fluid, indicating that higher sensitivity carried the anticipated cost of lower specificity. Sensitivity and specificity were further addressed by examining the cumulative data of sHLA antigens identified in all fluids, and comparing the numbers of expected, observed, and extra antigens reported (Table 3). Using cross-reactive ( + ) rather than specific ( + ) increased overall sensitivity slightly (55%, up from 49%), with a concomitant one-third reduction of false ( + ) resuits. Because cytotoxicity inhibition depends upon sHLA binding of alloantibody (which thus becomes unavailable to effect cytotoxicity on the target cell) in what functionally parallels C Y N A P (cytotoxicity negative, ab-

sorption positive) binding, it was not surprising th when specific ( + ) were used, one-third of the extra a tigens called were specificities highly cross-reactive wi the valid or true ( + ) antigens. The distribution of ext reactions showed many more false ( + ) antigens report, at the B than at the A locus. O f a total of 420 testin (i.e., the number of fluids times the number of la testing each fluid), 222 showed no extras, and only ' showed three or more extras. To determine whether there was a discernible patte of accuracy of antigen determination within each flui test results were first analyzed by fluid. Sample data shown in Figs. 2 and 3. As seen in Fig. 2, many fluk particularly double homozygotes, showed similar degrc of either good (e.g., fluid A24,B7) or poor (e.g., fluid recognition for both A- and B-locus antigens. Still ott fluids showed discordant rates of detection for their co~ ponent antigens (e.g., most of the fluids shown in F 3). The detectability of antigens did not correlate w the concentration of total sHLA in each fluid; corre tions for each specificity were not determinable becal concentrations of each antigen within the fluids were i known. In the bottom half of Figs. 2 and 3, the f quency of false ( + ) or extra reactions is depicted. though by visual inspection there appeared to be a tre toward fewer extras in fluids with higher rates of cal correct ( + ) s , this might have been a secondary effect labs tending to avoid calling triplets. This trend was TABLE 3

Cumulative data for all fluids Number of extr~ antigens identific

sHLA antigens identified Specific Specific and cross-reactive

Exp (+ )

Obs (+ )

A locus

1 lo(

1231

609

249

3

1231

674

146

2:

158

F.C. Grumet et al.

GROUPED BY FLUID

GROUPED BY FLUID

(Listed by anligcn)

(Listed by antigen)

100

lO0

80

~a

60

O

40

20

O 0

1 152

60

t

7 . 3162

2638

/E

' 24 7

24 7

1 52

J

3018

i

2638

2314

© cj

4O

9.0

3238

29

I

1 61

Specific antigen only

[]

I

Specific + cross-reactive

33 41

2 44

I

F

35

32 44

r

Specific antigen only gO

24 31 31 60 60 62

~gens I

[]

I

24 57'

I

2°IH

r~

J

i

!o

40

11

,

39 32

62

0/ i

80

i°

51 26 61

Specific + cross-reactive

'11

I

40

/

B

0

c

l

10£ l.S

3

5

24.7

[ 1200

300

5O0

L

[]

A Locus []B

1800

70O

800

Flu d:

FLUID # --. >

20

Locus

FIGURE 2 Analysis of individual fluids. (Top) Detection of component antigens: the horizontal axis shows the component antigens present in each fluid, and the vertical axis indicates the percent of laboratories detecting each antigen. (Bottom) Reporting of extra reactions: the horizontal axis shows the Aand B-locus extras for each fluid, and the vertical axis indicates the percent of laboratories reporting such extras. supported by the regression analysis of Fig. 4 (see below). No other trends were evident. Correlations between specificity and sensitivity at each locus were also sought statistically to determine whether greater sensitivity correlated with higher false ( + ) rates (Fig. 4). No correlation (r < 0.3) between percent of valid ( + ) and percent of false ( + ) for each fluid was seen at either locus, indicating that extra reactions were not necessarily a price to be paid for greater sensitivity. Finally, results were analyzed to determine whether some antigens were more reliably detected than others. Figures 5 and 6 show representative samples of this analysis. The trend appeared to show that specificities most reliably identifiable in cytotoxicity testing and for which good typing sera are readily available were generally detectable by most laboratories using cytotoxicity inhibition (Fig. 5). Those antigens that are less well identified by cytotoxicity and that have fewer good sera available

:

[700

'2~0O

Fluids F L U I D # --->

FIGURE 3 Fig. 2.

~

A Locus [ ] B

Locus

Analysis of individual fluids. See the legend for

were less consistently detectable by cytotoxicity inhibition (Fig. 6). It is likely, therefore, that the quality of the test sera used substantially affected sHLA determination in this phase of the workshop. T y p i n g o f sHLA by ELISA In contrast to typing of sHLA by inhibition of lymphocytotoxicity, only five laboratories undertook the attempt to type sHLA by ELISA. This probably reflects the difficulty in setting up a new assay that is not used in most HLA laboratories. Four laboratories developed a sandwich ELISA, using several different monoclonal antibodies to capture the sHLA on the ELISA plate. One laboratory (no. 43) developed a competition assay: plates were coated with human lymphocytes that were subsequently fixed with glutaraldehyde. The binding of allelespecific monoclonal antibodies (mAbs) to the lymphocytes was inhibited by sHLA molecules reactive with a particular mAb, while nonreactive sHLA molecules were much less inhibited [31]. The mAbs that were used for the sHLA typing by ELISA are listed in Table 4. When the culture superna-

Second sHLA Workshop

15

GROUPED BY ANTIGEN

A LOCUS VA[ I]) VS EXTRA POSITIV[;~%

(Listed by antigen) 100

80

:2

~q <

,10 I ml

•

•

< .A

60

~o

40

•

20 •

•

• •

•

Ii

I

•

i

•

0 1 ---> F1LUID # . - - >

i

20

12(I % LABS WITH VALID POSITIVES

2

--->

Specific antigen o n l y

B LOCUS

23

24 23

--->

[]

S p e c i f i c + cross-reactive

FIGURE 5 Analysis by antigen: the horizontal axis sho each antigen of different fluids, clustered by specificity; a the vertical axis shows the percent of laboratories identifyi that antigen in each fluid.

VAI .[D \,'F, }!X'l IG\ POSITIVI~

so

at

•

3 --->11-->

Antigens

M•

% LABS WITH VALID POSITIV]~

FIGURE 4 Correlation between valid and extra ( + ) reaction rates for A-locus (top) and B-locus (bottom) antigens for test fluids. Each solid square represents one fluid. The horizontal axis shows the percent of laboratories that identified the valid (specific and cross-reactive) antigens for that fluid, and the vertical axis shows the percent of labs reporting extra reactions belonging to the same locus. For analysis at each locus, only fluids derived from cell lines with homozygous antigen expression at that locus were used. rants of 57 different LCLs were typed by sandwich ELISA using the highly specific mAbs 1-12, the correlation between typing of microlymphocytotoxicity and ELISA was high (Table 4). mAbs 1,3, and 6 were also used in the cellular inhibition ELISA with similar results to the sandwich ELISA. However, the correlation between microlymphocytotoxicity and typing by ELISA was low (r ~< 0.67) when less specific mAbs (nos. 13-17) were used (Table 4). Due to the varying reactivities of these mAbs with different HLA antigens, it was not possible to define a cutoff value that allowed reliable differentiation between positive and negative reactions. This extensive analysis of sHLA antigens by ELISA demonstrated that mAbs react with sHLA identically to membrane-bound HLA antigens. In addition, it confirmed previous results that typing by ELISA correlates with HLA class I typing by microlymphocytotoxicity [2,

3]. HLA typing by ELISA may offer several advanta~ over conventional serologic methods. ELISAs provide c jective reading and automated analysis of results, a therefore should be more readily standardized and repl ducible. In contrast to microlymphocytotoxicity, no l~ cell preparations are required, large batch testing can performed, and less time is required. The major curr~ limitation of the ELISA is the unavailability of mAbs many alleles. In the future, different ELISA formats rr allow the use of alloantisera instead of mAbs. Biochemical C o m p o n e n t Based on the still rare reports that sHLA class I mc cules can be identified as allotypic variants by ol dimensional isoelectric focusing followed by Wesu blot (1D-IEF), a biochemical component was introdu, FIGURE 6

See the legend for Fig.

5.

GROUPED BY ANTIGEN (I.istcd by anligcn) 100

80

~<60 ,.,/ © (J

40

20

0 25--->26---> # --->

28-->292930-->31

IFLUlfi

S p e c i f i c antigen o n l y

. . . .

>

32 - - - >

Antigens []

Specific + cross-reactive

33 - - >

F.C. Grumet et al.

160

TABLE 4

Monoclonal antibodies

No.

Antibody

Isotype

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

anti-A2 IgM anti-A2/24 IgM anti-A3 IgM anti-A 11 IgM anti-A29 IgM anti-B8 IgM anti-B8/14 IgM anti-B 13 IgM anti-B 17 IgM anti-B27 IgG anti-B27/44 IgM MA2.1 MB40 CRll-351 HO2 SY1

IgM IgM IgM IgM IgM IgM IgM IgM IgM lgG lgM lgG IgG IgG IgG IgG

17

KS4

IgG

18 19

TP25 w6/32

IgG IgG

Reactivity by microlymphocytotoxicity A2 A2/24 A3 All A29 B8 B8/14 B13 B57/58 B27 B27/44 A2/B 17 B7/40/60 A2/28

A2/B17 B13 B7/27/w42/ w54/w55/w56/

r Cytotoxicity 1.00 0.89 1.00 0.97 1.00 0.92 0.95 1.00 0.77 0.71 0.42 -------

w67/w73 A/B/C A/B/C

Reactivity by ELISA

Used by lab no.

r

TP

TN

FP

FN

A2 A2/24 A3 All A29 B8 B8/14 B13 B17 B27 B44 A2/B 17 B7/60(/40fl A 1/2/3/11/24/(28) a --B7/27

42/43 42 42/43 42 42 42/43 42 42 42 42 42 42 7/42 7/12/18/42 18 18

0.89 0.96 1.00 1.00 0.86 0.84 0.93 0.86 1.00 0.86 0.72 0.92 0.67 0.53 0.46 <0

17 20 12 4 3 6 7 3 7 3 5 18 7 50 8 0

42 35 51 53 53 55 49 53 50 53 47 31 86 46 8 16

1 1 0 0 1 1 1 1 0 0 0 2 3 42 6 6

2 0 0 0 0 1 0 0 0 1 4 0 3 0 1 1

(w42/w54/w55/

7/18/42/43

0.34

17

67

26

8

w56/w67/w73) a A/B/C a A/B/C °

7/42 7/12

a Not tested. r is the correlation coefficient. TP, TN, FP, and FN are true positive, true negative, false positive, and false negative, respectively.

to type the specimens of panel A. Two workshop participants (Essen and Ferrara) submitted their results for joint analysis. Detergent-solubilized membrane HLA class I molecules from immunogenetically defined European Collection for Biological Research (ECBR) cells lines [32] served as reference antigens. To detect HLA class I molecules after focusing, a polyclonal rabbit antiserum toward denaturated human class I heavy chains (RaHC) provided by Dr. H. L. Ploegh (MIT, Cambridge, MA, USA) was used [33]. The second antibody was an alkaline-phosphatase-conjugated goat-anti-rabbit immunoglobulin G (IgG) (Dianova GmBH, Hamburg, Germany). For immunoadsorption of sHLA class I, ascites of mAbs w6/32 was used [34]. The immunoadsorption steps using paramagnetic beads (Dynal Deutschland, Hamburg, Germany) were accomplished as described [35], with minor modifications. In short, the beads precoated with rabbit-antimouse IgG were incubated with anti-HLA class I mAb w6/32 ascites overnight and, after appropriate washings, the immunobeads were mixed with 0.5 ml of the respective panel-A specimens. Captured sHLA class I molecules were desialyzed by neuraminidase treatment [36] and thereafter suspended in sample buffer. 1D-IEF was done under denaturating conditions [37], and the separated proteins were electrophoretically transferred at 412

mA for 2.5 hours onto Immobilon + membranes/(Millipore, Neu-Isenburg, Germany) according to the method described by Towbin et al. [37]. HLA class-Ispecific bands were visualized by incubation with RaHC followed by the second antibody by using the staining substrate bromocloro-indolylphosphate and 4-nitroblueterazolium chloride [38] (Essen lab) or the luminol system (Ferrara lab). Prior to 1D-IEF, all specimens analyzed were tested for sHLA class I concentrations by an ELISA as previously described [39]. Because the Ferrara data unequivocally identified sHLA (very weak HLA-A24.1, -A31, -B7.2, and -B44.1) in only a total of four specimens (fluids A24/B7, 100, 1300, and 1600), the following results are based on the data from Essen. Here samples 1400 and 2700 were reported as technical failures and the three dilutions of sample A24,B7 were not tested. The original 1D-IEF banding pattern of samples 1 through 1200, and 1300 through 5700, are seen in Fig. 7A and B, respectively. Figure 8 represents the schematic drawings of the relevant bands and the corresponding antigen assignments based on comparative analysis with 1D-IEF banding patterns of membrane-extracted HLA class I antigens from the reference cell lines. For the sake of simplicity, those patterns are not shown. It can be seen from the figures that, for most specimens of panel A, alignment of the sHLA antigens with bands of similar isoelecrric points

Second sHLA Workshop

1(

2 ~¸~i~¸¸ i

1.52

4

120(

A

FIGURE 7 1D-IEF gels. The number of each fluid sample is shown at the bottom of each lane: (A) fluids 1 through U-1200, and (B) fluids U-1300 through S-5700.

B

(IEPs) of the membrane extracts of reference cells was possible. Antigen specificities were tentatively assigned on the basis of the matching IEP of bands from the reference membrane extracts. There was no obvious correlation between the intensity of sHLA class-I-specific bands and measured sHLA class I concentration (ranging from 0.39 through 5.0 ~g/ml) in the specimens. Table 5 summarizes the biochemical typing results in

comparison with the given HLA class I types of the, lines. Discrepancies and unexpected results are indica by bold italic. The most notable are as follows: In negative control specimen (cell culture medium sup[ mented with 10% fetal calf serum), a faint band • observed at the position typical for HLA-B44.2. Sinc is known that antibodies, like mAb HC-10, rai against human HLA antigens cross-react also with

162

F . C . Grumet et al,

~,2.2

\29.,~

~18"

A33

1361

]35.2 \24.1 344.1

B14

800

300

A

A2.2

F I G U R E 8 Schematic drawing of bands observed in Fig. 7. The number of the fluid is shown at the bottom of each lane. Antigen assignments to each band have been made on the basis of IEP similarities to bands seen with membrane extracted class I antigens from reference cell lines.

A2.2

A2,2

1000

1200

Second sHLA Workshop

TABLE 5

1(

Summary of the biochemical typing results HLA type

Sample no.

sHLA p.g/ml

1 1.52 2 3 4 5 24.7 100 200

0.80 1.60 1.00 0.60 2.40 0.50 5.00 0.69 1.94 1.47 1.71 2.21 1.39 0.56 1.57 0.39 1.00 0.55 0.76 1.22 0.56 0.40 0.66 1.10 2.35 0.49 1.07 2.91 1.46 1.20 0.68

3OO 500 70O 800 900 1000 1200 1300 1400 1500 1600 2100 2200 2300 2700 3200 3500 3700 4300 4400 4700 5700

1D-IEF (Essen)

A

B

-1

A

-52

1 3w

31 24 24 1 30 23 32 29 1 33 2 32 24 31 31 11 3 II 24 26 31 25 24 2 1 1

7 62 7 7 52 18 i4 38 61 41 44 35 44 60 60 62 32 23 33

32 28 25 2 2

24.1 24.1 24.1

I 24.1 23 32 29.2

Undefined band 24,1

44. l 14 44.1

35.: (37,

Technical failure

62 55

51 57 39 13 51 27 18

54

8

44.2 w 52 27.1 52/44. A3/B7, A24. l/A26.2/B38.2/B35.3 (B47) 7* 61 7.2 7.2 52 30.0 18.1 14 38. l 61

1 33 2w 32

39 50

39

B

31 31 11 3.2 w 33

32 w 23

60 62 62 55 w 51.1

(44.

Technical failure 44 51

44 45 58 27

26 24.1 2w 24.1 2 w 1 1

25 25 2 28

39.2 51.1 51.2 18 17

44.

45 (54.

w, weak; (x), tentative banding position; x/y, cobanding; and *, untypical banding position.

vine class I antigens (BOLAs) [40], this might be the first and inadvertent detection of soluble BOLA class I molecules. The two plasma samples with unknown HLA types of the donors could not be definitely typed by 1D-IEF due to cobanding of certain HLA-A and -B products [38] as seen in fluid 3 for A3 and B7. In fluid 4, the supernatant of T.sB7 transfectant, a band with an 1DIEF position more acidic than the known IEP of both HLA-B7.1 and -B7.2 was found. This new IEP might be explained by the missing transmembrane and cytoplasmic parts of the molecule. The 1D-IEF result in fluid 5 (blind replicate of U- 1600) with bands at IEP for A24.1 and B61 contrasts with the given HLA type of the LCL and with the specificities assigned for the bands in its replicate, U- 1600. In total, the 1D-IEF analysis of sHLA class I allotypes compared with the given antigen assignments revealed a concordance rate of 84.4% (27 of 32) for the HLA-A locus and of 67.6% (23 of 34) for the HLA-B locus. The

discrepancies consisted of three additional antlge[ eight antigens missing, and six antigens different fro those predicted by LCL phenotyping. There was no ch pattern of detectability dependent upon either locus specific alleles. Although in most cases the EIP allo~ antigen assignment of a band, additional work rema to be done to calibrate and ascertain the IEP of shed actively secreted class I sHLA compared with their me brane-extracted counterparts. This is not an unexpecl finding since the former molecules have excised polypc tide streches or truncated carboxyl-terminal ends wl" compared with the canonical membrane-bound specit ity, resulting in a different IEP. Typing of sHLA by Flow Cytometry Only one laboratory attempted to perform typing us~ the fluorescence-activated cell sorter (FACS). The rest obtained are reported in detail in the article by Nehls

164

Cannarella et al. in this issue and therefore are not reiterated here.

Summary of the Joint Results This workshop represented the first attempt to type a common set of many sHLA class I antigens by numerous laboratories utilizing several techniques. The "gold reference standard" was the microlymphocytotoxicity phenotype of the LCLs generating the tissue culture fluid containing the sHLA. Microlymphocytotoxicity inhibition was used by the largest number of participants by far, presumably because of its relatively familiar methodology; its use of only small amount of reagents, including readily available alloantisera; and its high throughput. This technique appeared to be of a similar order of sensitivity to the other techniques, but more extensive direct comparisons still should be made. Specificity and sensitivity would probably be improved by the availability of better alloantisera or allospecific mAbs. ELISA was the next most common method but was tested with substantially fewer antibodies and only by a small number of laboratories. Nevertheless, when good mAbs were used, this method showed high r values for specific antigen identification. If additional monoclonal reagents can be obtained, ELISA has the promising technical potential of enabling good throughput with machine-readable test results. For the third method, 1D-IEF, most of the data came from only a single laboratory and no quantitative estimate of sensitivity was obtained. Antigen identification was based upon reference bands of membrane-extracted class I molecules, but because the secreted molecules may have different IEPs, restandardization will be necessary for this technique to be employed by many laboratories. The final technique employed was inhibition of antibody as detected by FACS. Only one participant tested this method. Those data suggested equivalent or greater sensitivity but required substantially greater amounts of reagents compared with cytotoxicity inhibition. As with cytotoxicity inhibition and ELISA, quality of test results appeared to depend upon availability of good allospecific antibodies.

REFERENCES 1. Pouletty P, Ferrone S, Amesland F, Cohen N, Westhoff U, Charron D, Shimizu RM, Grosse-Wilde H: Summary report from the first international workshop on soluble HLA antigens, Paris, August 1992. Tissue Antigens 42: 45, 1993. 2. Russo C, Fotino M, Carbonara A, Ferrone S: A double determinant immunoassay for HLA class I typing using serum as an antigen source. Hum Immunol 19:69, 1987. 3. Sakaguchi K, Ono R, Tsujisaki M, Richiardi P, Carbonara

F. C Grumet et al.

A, Park MS, Tonal R, Terasaki PI, Ferrone S: Anti-HLAB7, B27, Bw42, Bw54, Bw55, Bw56, Bw57, Bw73 monoclonal antibodies: specificity, idiotypes, and application for a double determinant immunoassay. Hum Immunol 21:193, 1988. 4. Pouletty P, Chang C, Atwood E, Kalil J, Ferrone S, Shimuzu R, Howson W, Mazahari R, Del Villano B, Grumet FC: Typing of serum soluble HLA-B27 by ELISA. Tissue Antigens 42: 14, 1993. 5. Villar LM, Roy G, Lazaro I, Alvarez-Carmeno JC, Brieva JA, Del la Sen ML, Leoro G, Bootello A, GonzalesPorque P: Detection of soluble class I molecules (non HLA-A or HLA-B) in serum, spleen membranes and lymphocytes in culture. Eur J Immunol 19:1835, 1989. 6. Russo C, Pellegrino MA, Ferrone S: A double-determinant immunoassay with monoclonal antibodies to the HLA-A,B,C complex. Transplant Proc 15:66, 1983. 7. Shimuzu B, Sra K, Ferrone S, Pouletty P: sHLA-STAT TM class I, an ELISA for quantitation of HLA antigens in serum. Hum Immunol 32(Suppl 1):289, 1992. 8. Krangel MS: Secretion of HLA-A and -B antigens via an alternative RNA splicing pathway. J Exp Med 163: 1173, 196. 9. Krangel MS: Two forms of HLA class I molecules in human plasma. Hum Immunol 30: 155, 1987. 10. Dobbe LMS, Stam NJ, Neef]esJJ, Giphart MJ: Biochemical complexity of serum HLA class I molecules. Immunogenetics 27:203, 1988. 11. HagaJA, She J-X, Kao K-J: Biochemical characterization of 39-kDa class I histocompatibility antigen in plasma. J Biol Chem 266:3695, 1991. 12. Kubens B, P/iBler M, Grosse-Wilde H: Segregation study of the soluble 39-kD HLA class I heavy chain. Hum Immunol 40:247, 1994 [in this issue]. 13. WesthoffU, Stiewe T, Gross-Wilde H: Distribution pattern of soluble HLA class I heavy chains after Western blot using MAB HC-10 and a polyclonal antiserum towards COOH-terminus [abst 35]. Hum Immunol 37: 132, 1993. 14. Drouet M, Aussel L, Fauchet R: Plasmatic HLA-B molecules: quantification and molecular definition [abst 8]. Hum Immunol 37: 123, 1993. 15. Hagihara M, Shimura T, Takebe K, Tsuji K: Do HLAA24, A2 and A l l allospecificities influence the production of serum soluble HLA antigens [abst 10]? Hum Immunol 37:124, 1993. 16. King DW, Reed E, Suciu-Foca N: Complexes of soluble HLA antigens and anti-HLA autoantibodies in human sera: possible role in maintenance of self-tolerance. Immunol Res 8:249, 1989. 17. Saririan K, Contini P, Indiveri F, Gross P, Russo C: Serum HLA class I levels in elderly humans: utilization in following the response to influenza vaccine. Hum Immunol 40:202, 1994 [in this issue].

Second sHLA Workshop

18. Shimura T, Hagihara M, Yamamoto K, Takebe K, Munkhbat B, Ogoshi K, Mitomi T, Nagamachi Y, Tsuji K: Quantification of serum-soluble HLA class I antigens in patients with gastric cancer. Hum Immunol 40: 183, 1994 [in this issue]. 19. Inostroza J, Munoz P, Espinoza R, Millaqueo L, Diaz P, Leiva L, Sorensen R: Quantitation of soluble HLA class I heterodimers and ~2-microglobulin in patients with active pulmonary tuberculosis. Hum Immunol 40:000, 1994 [in this issue]. 20. Zavazava N, Westphal E, Mueller-Ruchholtz W: Characterization of soluble HLA molecules in sweat and quantitative HLA differences in serum of healthy individuals. J Immunogenet 17:387, 1991. 2I. Singh PB, Brown RE, Roser B: Class I transplantation antigens in solution in body fluids and in the urine: individuality signals of the environment. J Exp Med 168: 195, 1988. 22. Pollard SG, Davies HffS, Calne RY: Preoperative appearance of serum class I antigen during liver transplantation. Transplantation 49:659, 1990. 23. Zavazava N, Hausmann R, Mueller-Ruchholtz W: Inhibition of anti-HLA-B7 alloreactive CTL by affinitypurified soluble HLA. Transplantation 51:838, 1991. 24. Sumimoto R, Kamada N: Specific suppression ofallograft rejection of soluble class I antigen and complexes with monoclonal antibody. Transplantation 50:678, 1990. 25. Wang J, Geissler E, Knechtle SJ: In vivo transfer ofgene encoding soluble MHC class I prolongs heart transplant survival [abst 23]. Hum Immunol 37:121, 1993. 26. Grumet FC, Krishnaswamy S, See-Tho K, Filvaroff E, Hiraki DD: Soluble form of an HLA-B7 class I antigen specifically suppresses humoral alloimmunization. Hum Immunol 40:228, 1994 [in this issue]. 27. Claas FHJ, Jankowska-Gan E, DeVito LD, Jutte N, Balk A, Weimar W, Burlingham WJ: Monitoring of heart transplant rejection using a donor-specific soluble HLA class I ELISA [abst 3]. Hum Immunol 37:121, 1993. 28. DeVito-Haynes LN, Jankowska-Gan E, Sollinger HW, Knechtle SJ, Burlingham WJ: Monitoring of kidney and simultaneous pancreas-kidney transplantation rejection by release of donor-specific, soluble HLA class I. Hum Immunol 40:191, 1994 [in this issue]. 29. Puppo F, Pellicci R, Brenci S, Nocera A, Morelli N, Dardano G, Bertocchi M, Antonucci A, Ghio M, Scudeletti M, Barocci S, Valente U, Indiveri F: HLA class-I-

16

soluble antigen serum levels in liver transplantation: predictor marker of acute rejection. Hum Immunol 4( 166, 1994 [in this issue]. 30. Schroeder T, Pouletty P, Shimizu R, Benson N, Micha A, Hariharan S, Alexander W, First MR: Soluble class HLA antigens in serum following renal transplantatic [abst 21]. Hum Immunol 37:130, 1993. 31. Nehlsen-Cannarella SL, Buckert L, Fagoaga O, Folz Grinde S, Hisey C, Schmitt R, Zappia J: Assessment, three methods of evaluating soluble class I HLA molecul, in cell culture supernatants and serum samples from tt Second International Workshop on sHLA. Hum Imml nol 40:2 10, I994 [in this issue]. 32. Grosse-Wilde H, Ferrara GB: European Collection f, Biomedical Research. Eur J Immunogenet 19:180, 1991 33. Neefjes JJ, Doxiadis I, Stam NJ, Beckers CJ, Ploegh HI An analysis of class I antigens of man and other species [ one-dimensional IEP and immunoblotting. Immunog netics 23:164, 1986. 34. Parham P, Barnstable CJ, Bodmer WF: Use of monocl nal antibody (w6/32) in structural studies of HLA-A,B, antigens. J Immunol i23:324, 1979. 35. Grosse-Wilde H, Doxiadis I: Allotyping for HLA clas~, using plasma as antigen source. J Immunogenet 16:14 1989. 36. Neefjes JJ, Breur-Vriesendorp B, van Seventer G~ Ivanyi P, Ploegh HL: An improved biochemical methq for the analysis of HLA-class I antigens: definition of nc HLA-class I subtypes. Hum Immunol 16: 169, 1986. 37. Towbin H, Staehelin T, Gordon J: Electrophoretic trar fer of proteins from acrylamide gels to nitrocellulc sheets: procedure and some applications. Proc Natl Ac Sci USA 76:4350, 1979. 38. Frenz G, Doxiadis I, Voegeler U, Grosse-Wilde H: HI class I biochemistry: definition and frequency determir tion of subtypes by one-dimensional isoelectric focusi and immunoblotting. Vox Sang 56:190, 1989. 39. Doxiadis I, Westhoff U, Grosse-Wilde H: Quantificati of soluble HLA class I gene products by an enzyme link immunosorbent assay. Blut 59:449, 1989. 40. Viuff B, Ostergard H, Aasted dimensional isoelectric focusing bovine major histocompatibility molecules and correlation with Genet 22:147, 1991.

B, Kristensen B: Or and immunoblotting complex (BOLA) clas class I serology. An