binding of igg to b cell via hla molecules

Binding of IgG to B cell via HLA molecules D. Chia, P. I. Terasaki, H. Chan, M. Ozawa. Binding of IgG to B cell via HLA molecules. Tissue Antigens 1991: 37: 49-55.

Abstract: Binding of immunoglobulins to major histocompatibility complex (MHC) molecules was demonstrated by two different assays: the binding of IgG to B cells by flow cytometry, and purified MHC antigens with an Elisa assay. Fc fragment from immune-complex binds to the Fc receptor on B lymphocytes. Here, Fab was also shown to bind to B cells. This binding was inhibited by specific human all0 anti-HLA Class I and I1 sera directed at the polymorphic sites. Thus, in addition to the Fc receptor, MHC can also serve as a binding site for IgG. In an Elisa assay using purified antigens, IgG was shown to bind to HLA Class I and I1 molecules. Other proteins such as transferrin, human serum albumin, gelatin, etc., did not bind to the MHC proteins. Immunoglob- ulins bound to MHC molecules by sites on the Fab fragment independent of the hypervariable region. This was demonstrated by the retention of antibody activity even after binding of antibody (anti-lactoferrin) to MHC. The relative avidity between Fab and HLA Class I and I1 was 4-8 x lo5 M-'.

The immunoglobulin superfamily has been grow- ing rapidly, as more protein sequences suggest a common evolutionary origin for diverse molecules (1). These cell surface molecules with immunoglobulin-like domains appear to mediate cell adhesion by their homophiiic or heterophilic interactions (2-4). This occurred between CD2 and LFA-3 (5), CD4 and MHC Class I1 (6), and CD8 and MHC Class I(7). More recently, human CD4 was shown to bind immunoglobulins, through the Fab portion of the IgG molecule (8).

The binding of IgG with B cells through the Fc receptors is well established (9). In this study, we demonstrate that immunoglobulin could also bind to B cells through the MHC molecules by a site on the Fab fragment other than the hypervariable region.

Material and Methods Flow cytometry

Lymphocytes were separated into T and B cells by adherence of B cells on nylon wool (10). The cells were treated with the various biotinylated reagents, and then developed by strepavidin-FITC (Sigma Chemical Co., St. Louis, MO). Leu 4, and Leu 16 were used as markers for T and B cells, respectively

This work was supported in part by a grant from .the National Institute of Diabetes, Digestive and Kidney Diseases DK 02375- 30.

David Chia, Paul 1. Terasaki, Henry Cbm and Miyuki Ozawa UCLA Tissue Typing Laboratory, Department of Surgery, UCLA School of Medicine, Los Angeles, California, USA

Key words: IgG - HLA - binding - B cell

Received 14 May, revised, accepted for publi cation 20 November 1990

(Becton Dickinson, Mountain View, CA). The stained cells were then analyzed by FACSCAN (Becton Dickinson). The inhibition assays were performed by first incubating the B cells with specific monoclonal antibodies, or specific all0 anti- HLA Class I and I1 sera, followed by IgG-biotin. The inhibition was determined as median channel shift of control minus median channel shift of inhibitors/median channel shift of control x 100%.

Micro Elisa assay

The amount of human IgG bound to various antigens was determined by a micro-Elisa assay, as described previously (1 1). Protein A, anti-human IgG specific for Fc or Fab, HLA Class I and 11, B2 microglobulin, transferrin, gelatin, BSA, and HSA at 20 pg/ml in phosphate-buffered saline were adsorbed on non-wettable Terasaki trays (Robbins Scientific Co., Sunnyvale, CA) overnight at 4°C and then at 37°C for 2 h, and blocked by 0.5% HSA'for 1 h at 37°C. The HSA blocking had no influence on the Elisa result. Phosphate-buffered saline with 0.1% NP40 was used as diluent in all experiments. IgG conjugated with peroxidase was added at different concentrations and incubated at 37" C for 1 h. The substrate used for the Elisa tests was OPD (0-phenylenediamine), and reactions were stopped by 2.5 M H2S04. The absorbance at 488 nm was measured with a microplate reader TR-200 (Dynatech Lab., Alexandria, VA).

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Chia et al.

The binding of HLA Class I and I1 to various antigens was shown in another way. After coating various antigens as described above, 5 pl of HLA Class I (1 mg/ml) was added for 2 h at 37"C, followed by monoclonal antibody against Class I, W6-32 peroxidase (2.5 pg/ml) for 1 h at 37°C. 5 pl of HLA Class I1 (0.25 mg/ml) was added for 2 h at 37"C, followed by mouse monoclonal anti- DR peroxidase (0.17 pg/ml) for 1 h at 37" c. The amount of peroxidase bound was determined as above.

To test for the retention of antibody activity after binding, HLA Class I or 11, or HSA, coated trays were first incubated for 1 h at 37°C with 5 pl of affinity-purified rabbit anti-lactoferrin (Accu- rate Chemical, Westbury, NY) at concentrations of 0, 0.5, and 1 .O mg/ml. Then lactoferrin conjugated to biotin at different concentrations was added, and streptavidin-peroxidase (Calbiochem Corp., La Jolla, CA) was added to develop the reaction.

Purification of Class I and Class II substances

HLA Class I was isolated by affinity chromatography from pooled platelets (12). Briefly, detergent (0.5% Nonidet P40, NP40) extract from platelets was passed through a mouse IgG column then a mouse monoclonal antibody (1 3) W6-32 coupled sepharose CL-4B column, and eluted by 0.05 M diethylamine at pH 11.5. HLA Class I1 was obtained by passing detergent extract of pooled human spleen cells through the same two columns as noted above. In addition, we used a column with an anti-human Ia monoclonal antibody (14) coupled to sepharose. The purity of both Class I and I1 was determined by micro-Elisa assay using W6- 32 peroxidase and anti-Ia peroxidase. The antibodies reacted with their respective antigens only. Furthermore, it was noted that HLA Class I and I1 had molecular weights of 45 K, 12 K, and 33 K, 29 K respectively on SDS-PAGE as the major bands.

Purification of human IgG fragments

IgG, Fab,, Fab, and Fc were obtained from Orga- non Teknika-Capple (West Chester, PA). IgG from an individual was purified from serum using a Pro- tein A affinity column (Bio-rad, Richmond, CA) by HPLC. Fab fragments were prepared as described previously (1 5). Pooled human IgG (Orga- non Tekknika-Capple) was mixed with immobil- ized papain (Pierce, Rockford, IL) for 4 h. The papain was then removed, and the supernatant passed through a Protein A column to remove IgG and Fc. IgG, Fab2, Fab, Fc, and HSA were biotinylated (16) by reacting 10 mg of protein with

1 mg of Biotinyl-e-amino-caproic acid N-hydroxys- uccinimide ester (Calbiochem Corp.) in PBS.

Scatchard plot determination

The concentration of bound and free Fab labelled with biotin which bound to HLA Class I and I1 was determined. The standard was Fab-biotin bound to anti-Fab antibody-coated tray developed by strepavidin-peroxidase (Calbiochem Corp.). The bound Fab was determined by comparing the absorbance HLA-coated tray with trays coated with various amounts of Fab-biotin. At the lower concentrations of Fab used in the experiments, all the Fab-biotin bound to the tray. K, was calculated as a negative value of the slope when bound/free was plotted against bound.

Results Immunoglobulin binding to B and T cells

The binding of immunoglobulin to HLA present on T and B cells was examined by flow cytometry. IgG, Fab, Fc, and HSA labelled with biotin, were reacted to the cells. B cells could bind Fab, IgG, and Fc, but not HSA (Fig la). Fc receptors on B lymphocytes could account for the binding of IgG and Fc. However, the binding of Fab to the cells was unexpected. T cells showed reactions similar to B cells, but at lower levels (Fig. lb).

The binding site of IgG on B cell was probed by inhibition experiments using antibodies to HLA. B cells were first treated with monoclonal antibodies (W6-32, and anti-Ia), or specific all0 anti-HLA Class I and I1 sera, followed by incubation with IgG-biotin. The amount of IgG bound was measured by flow cytometry. As shown in Fig. 2, pre- treatment with W6-32 antibody and not with anti- Ia, inhibited the reaction of IgG to B cells. Only the specific all0 anti-HLA Class I and I1 sera for that particular cell could inhibit the binding of IgG, while the non-specific sera anti-B7 and anti- B8 in Fig. 2a and 2b, respectively, did no. The binding of all0 anti-HLA antibodies to B cells resulted in an inhibition of IgG binding, but binding to the cell did not always result in an inhibition, as in the case of anti-Ia.

Binding of IgG and Fab to HLA

Since the above experiments were performed on B- cell surface, it would be important to demonstrate the same results with purified MHC antigens. MHC antigens were first purified by affinity chromatography, and then used in Elisa assay to study the binding with immunoglobulins. The binding of peroxidase-conjugated IgG to Terasaki

50

IgG binding to HLA

11

Relative Fluorescence Intensity Figure 1. Binding of IgG and its fragments to B and T lymphocytes by flow cytometry. Biotinylated IgG, Fab, Fc and HSA were incubated with B cells (A), and T cells (B), then strepavidin-FITC was used to develop the reactions.

trays precoated with different proteins is shown in was evident at concentrations of 4 pg/ml. About Fig. 3. Three broad levels of binding were noted. 100- to 1000-fold higher concentrations were As expected, the highest degree of binding was the necessary to achieve equivalent binding for HLA conventional antigen-antibody reaction with anti- Class I1 and I molecules. Transferrin, gelatin, BSA IgG. Similarly, Fc-specific binding with protein A and HSA had the lowest binding activity to IgG,

100 90 80 70

$! 30 20 10 0

A1 ,A26,B8,B38,DR4,DRwl8 .......................................................................... 100 90 80 70 60 50 40 30 20 10 0

A1 ,A2,67,645,DR4,DRwl5 ............................................................................................

Figure 2. Inhibition of binding of IgG by anti-MHC antibodies. Binding of IgG to splenic B cells as shown by flow cytometry was inhibited by specific HLA antibodies directed to the B cells. The HLA types of cells A and B are given. W6-32 can be seen to completely inhibit binding of IgG, whereas the monoclonal Ia antibody was not inhibitory. Allogeneic antibodies were inhibitory if the cell possessed the antigen, but were inactive if the cell did not have the antigen to which it was directed. After addition of the antibodies IgG biotin (2 mg/ml), streptavidin-FITC was added. The quantity of IgG bound was determined by flow cytometry.

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Chia et al.

2.2 2.0

E 00 1.6

a 1.2

0.8

2 0.4 0.0

C

00 Tr

a, 0 c

.c,

z v)

..................................................... 3

.....................................................

I I I I I I

0.0 0.2 0.4 0.6 0.8 1 .o Ig G-Peroxidase Conc. (mg/ml)

Protein A anti-I G(Fab), anti-lgG(Fc) HLA &ass II

HLA Class I

Transferrin Gelatin, BSA,HSA

Figure 3. Binding of human IgG to antigens. The reagents listed on the right side of the figure were first coated on plastic trays then various concentrations of IgG-peroxidase were added. OPD was added and the Elisa reaction read as absorbance at 488 nm. The values given are the mean of two to five experimetns.

at another 10-fold lower concentration. Thus, a clear selective binding of IgG to MHC was shown between the extremes of antigen-antibody reaction and non-binding to albumin.

The converse of the above experiment, the binding of Class I molecule to various proteins coated on the tray, is shown in Fig. 4. For Class I antigens (Fig. 4a), presence was detected by the monoclonal

A

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

B

0 012 Ol4 0:s 0:s IlO 1I2 1I4 Absorbance at 488 nm

Figure 4 . Binding of HLA Class I and I1 molecules to various antigens. The proteins listed were first coated on plastic trays. For data shown on the left hand side (4a), HLA Class I was added followed by monoclonal antibody against Class I, W6-32 peroxidase. For data shown on the right hand side, (4b), HLA Class I1 was added, followed by mouse monoclonal anti-Ia peroxidase. The results shown are the mean of quadruplicates.

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IgG binding to HLA

antibody W6-32-coupled peroxidase. Fab, and IgG bound HLA Class I strongly, but Fc, IgM, BSA, HSA and transferrin did not. The reactivity of Class I1 molecules was similar to Class I (Fig. 4b). The binding of Class I to Class I1 could be of interest.

Binding of HLA to autologous and heterologous IgG

Since the IgG used for these experiments were de- rived from pooled human serum, the possibility existed that anti-HLA antibodies from serum donors immunized by pregnancies was present in the IgG preparations. The antibodies would then be expected to react to HLA substances. IgG was therefore prepared from the serum of a male donor whose spleen was used to isolate the HLA antigens. The serum did not show any anti-HLA activity. As is evident in Fig. 5, autologous IgG adhered to HLA Class I and Class I1 to about the same degree as allogeneic IgG from 2 other donors.

Binding of antigen to HLA-immunoglobulin complex

The ability of the antibody to bind specific antigen after its reaction with HLA was examined. Follow- ing the binding of an affinity-purified anti-lactoferrin antibody to HLA, the level of lactoferrin antigen-binding capacity was measured (Fig. 6). It is apparent that, even after binding to Class I and I1 substances, anti-lactoferrin antibody retained its activity against lactoferrin. Thus, the binding site of antibody with MHC was not the primary antigen binding site of the antibody.

0 1 2 3 4 5

Table 1. The association constant between Fab and HLA Class I and II

K~ M-I x 10-5 Experiment No. HLA Class I HLA Class I1

1 2 3 4 Mean f S.D

4.8 8.1 4.6 9.3 6.2 9.0 2.5

4.5 f 1.5 -

8.8 f 0.66

Two more experiments were performed to estab- lish the independence of HLA Class I and I1 binding with antibody and the specific antigen-antibody reaction (data not shown). After coating trays with anti-lactoferrin, adherence of Class I and I1 re- mained constant despite the prior addition of in- creasing concentrations of lactoferrin from 1 to 80 ng/ml. In the converse experiments, the prior reaction of Class I and Class I1 to anti-lactoferrin did not influence the binding of lactoferrin to its antibody. Thus, there was no cross-interference of MHC binding and antigen binding to antibody.

Scatchard analysis of HLA with Fab

To further quantitate the binding reaction between HLA and Fab, the relative avidity was measured by the Scatchard plot method (17). The association constants of the interactions between Fab and HLA Class I and I1 were 4.5+ 1.5 x lo5 M-' and 8.8 k0.6 x lo5 M-' respectively (Table 1).

1 .............................................................................

...........................................................

........................................

................... .....

...........................................

0 1 2 3 4 5 Conc. (mg/ml)

Figure 5. Binding of autologous or allogeneic IgG to HLA Class I and 11. HLA Class I and I1 isolated from the spleen cells of one person were first coated on the tray. Then, IgG isolated from the serum of the same individual (autologous) and from 2 other people (allogeneic) was added at different concentrations. Anti-human Fab coupled to peroxidase was then added, and the Elisa reaction was carried out. The results are the mean of triplicates.

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Chia et al.

E C 03 03 d

1.6 Ab Ag Conc.

1.2

0.8

0.4

0.0

H I A A l m s Class II 0.5 mg

0 0 m g

HSA ~ l m g 0.5 mg

o o m g

0 250 500 750 1000 Antigen Conc. (ng/ml)

Figure 6. Antigen binding to antibody previously complexed with HLA Class I or I1 molecules. HLA Class I- or 11-, or HSA- coated trays, were first incubated with affinity purified rabbit anti-lactoferin. Then lactofemn conjugated to biotin at different concentrations was added, and finally streptavidin coupled peroxidase was added to develop the reaction. The results are the mean of triplicates.

Discussion

Currently, the major interest in MHC molecules focuses on the peptide that is found in the groove (18). In the studies described here, attention is . drawn to a separate, somewhat unexpected observation: MHC interaction with immunoglobulin. Evidence is provided that IgG adheres to MHC at sites other than the groove, as monoclonal antibody W6-32 would block the IgG adherence, but its binding site was far away from the groove (19). The IgG does not bind through a specific reaction of the hypervariable region, but rather by other sites on the Fab portion of the molecule.

Binding of IgG to MHC was demonstrated both by binding to B lymphocytes and by binding to purified Class I and Class I1 molecules, as detected by Elisa reactions. The binding to B cells was much stronger than to T cells. Clearly, this binding was through the Fab which was distinct from the Fc binding previously recognized (9). It has often been mentioned that B cells have a high background when tested by flow cytometry (20). The binding of the 2nd anti-IgG antibody resulted in “non- specific” background which can now be explained by binding to MHC. Specific antibodies to the HLA antigens present on the B cells were able to block the adherence of IgG. For example, anti-B8 antibodies blocked IgG adherence to cells with B8, but did not block IgG adherence to cells that did not have B8. Similar reactions were noted with anti-B7 antibodies.

Though a strictly quantitative relationship be-

tween the number of surface molecules of each specificity with the degree of inhibition could not be shown in these experiments, some measure of additivity was suggested by the increased inhibition of IgG binding by a combination of antibodies. The ability of anti-B8 to block 80-90% of the IgG binding, when it should bind to less than one- quarter of the MHC molecules, was puzzling. While the steric blockage produced by antibodies reacting to certain epitopes may have a greater effect on the IgG binding than certain other epitopes, it would be difficult to reconcile the above observation. One possible explanation could be that the close proximity of all the MHC molecules would react with one of the molecules, sterically blocking the binding of IgG to most of the MHC molecules.

A major reason why the Elisa reaction was not readily applicable to HLA typing was because of the non-specific binding of the 2nd anti-immunoglobulin antibodies. We show here that, with the Elisa test, using purified MHC products, mouse IgG readily binds to MHC “non-specifically”. This binding, generally considered to be “stickiness” of antibodies, is actually “specific” to a certain site on the cell in that it can be blocked by antibodies to MHC.

The IgG binding is through a site on the Fab that is distinct from the hypervariable site, since antibody activity is retained after binding to MHC. This finding leads us to suggest that the biologic significance of the adherence phenomenon we de- scribe might be that MHC serves to transport, or

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IgG binding to HLA

temporarily fix antibodies. The cell which has the greatest quantity of MHC on its surface, the B lymphocyte, may be the one that transports the greatest quantitites of immunoglobulin. The rela- tively low avidity of the adherence may favor the ready disassociation of the antibody once it inter- acts with its antigen. The association constant between MHC and IgG of 4-8 x M-’ is weak compared to specific antigen binding, but is never- theless strong in that it is resistant to repeated washings and is very reproducible in repeated experiments.

The additional findings of adherence of Class I and Class I1 molecules to each other as well as adherence of free B2 microglobulins to IgG are experimental, unexplained observations. The interaction between Class I and I1 could be due to a nonspecific binding of their hydrophobic domains. However, others have also observed the binding of MHC Class I to Class I1 molecules (21). The recent demonstration that IgG adheres to CD4 molecules (also through the Fab fragment) (8), with an avidity similar to that reported here for IgG and MHC, suggests that cell surface structures might function as a transport mechanism for antibodies, perhaps into targeted sites specifically sought out be cells.

In this report, we wish to draw attention to the adhesion of IgG to purified MHC as well as MHC on B cells. Although the potential biologic significance is unclear, the finding is of immediate im- portance in studies of the MHC molecules using antibodies: many reactions using secondary antibodies are “non-specific” in the sense that the antibodies are not reacting through their hypervariable site, but rather through a common Fab site. Experi- ments on the anti-idiotypic reactions of antibodies to HLA (22) can also be expected to be complicated by this phenomenon.

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2. Williams, AF. Surface molecules and cell interactions. J Theoret Biol 1982: 98 221-34.

3. Anderson P, Morimoto C, Breitmeyer JB, Schlossman SF. Regulatory interactions between members of the immunoglobulin superfamily. Immunof Today 1988: 9: 199-203.

4. HunkapilIer T, Hood L. Diversity of the immunoglobulin gene superfamdy. Adv Immunol 1989: 44: 1-63.

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used by human cytotoxic T-cell clones. Nature 1986: 323 2624. Doyle C, Strominger JL. Interaction between CD4 and class I1 MHC molecules mediates cell adhesion. Nature 1987: 330 256-9. Salter RD, Norment AM, Chen BP et al. Polymorphism in the a3 domain of HLA-A molecules affects binding to CD8. Nature 1989: 338: 345-7. Lenert P, Kroon D, Spiegelberg H, Golub ES, Zanetti M. Human CD4 binds immunoglobulins. Science 1990: 248: 1639-43. Dickler HB. Lymphocyte receptors for immunoglobulin. Adv Immunol 1976: 24: 167-214. Danilovs JA, Ayoub G, Terasaki PI. B lymphocyte isolation by thrombin-nylon wool. In: Terasaki PI, ed. Histocornpat- ibility Testing 1980, Los Angeles: UCLA Tissue Typing Laboratory, 1980: 287-8. Katz DV, Chia D, Hermes M, Galton J, Hirota M, Teraski PI. Detection of monoclonal antibodies for tumor diagnosis with the use of inhibition of micro-enzyme-linked immuno- sorbent assay. J National Cancer Inst 1985: 14: 335-9. Cook DJ, Scornik JC. Purified HLA antigens to probe human alloantibody specificity. Human Immunol 1985: 14: 234-44. Parham P, Barnstable CJ, Bodmer WF. Use of a monoclonal antibody (W6/32) in structural studies of HLA- A,B,C antigens. J Immunoll979: ’123 342-9. Iwaki Y, Terasaki PI, Kinukawa T, Billing R, Thai TH, Root T. Crossreactivity of Ia antigens between human and canine by a mouse monoclonal antibody (CIA). Transplan- tation 1983: 36: 189-91. Porter RR. The hydrolysis of rabbit r-globulin and antibodies with crystalline papain. Biochem J 1959: 73: 119-27.

16. Hnatowich Dj, Virzi F, Rusckowski M. Investigations of avidin and biotin for imaging applications. J Nucl Med 1987: 28: 1294-302.

17. Chia D, Nasu H, Yamagata J, Barnett EV. Avidity indices of anti-IgG antibodies in diseases. Arth Rheum 1981: 24: 80ML

18. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strom- inger JL, Wiley DC. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987: 329: 506-12.

19. Kahn-Perles B, Boyer C, Arnold B, Sanderson Ar, Ferrier P, Lemonnier FA. Acquisition of HLA class I W6/32 defined antigenic determinant by heavy chains from different spe- cies following association with bovine b,-microglobulin. J Immunoll987: 138: 2190-6.

20. Garovoy MR, Rheinschmidt MA, Bigos M, et al. Flow cytometry analysis: a high technology crossmatch technique facilitating transplantation. Transplant Proc 1983: 15: 1939-44.

21. Szzo”l1o”si J, Damjanovich S, Balks M, et al. Physical association between MHC class I and Class I1 molecules detected on the cell surface by flow cytometric energy trans- fer. J Immunot 1989 143: 208-13.

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Address: David Chia, Ph.D. 950 Veteran Ave. Los Angeles Calif. 90024 USA

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binding of igg to b cell via hla molecules

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