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Immunogenic and Antigenic Epitopes of Immunoglobulins Binding of Human Monoclonal Anti-D Antibodies to FcRI on the Monocyte-Like U937 Cell Line

M.R. Walkera, B.M. Kumpelb, K. Thompsonc, J.M. Woofd, D.R. Burtond, R. JefferiP aDepartment of Immunology, Medical School, Birmingham; bUK Transplant Service, South Western Regional Transfusion Centre, Bristol; CMRC Centre, Cambridge; dDepartment of Biochemistry, University of Sheffield, UK

Abstract. Seventeen human monoclonal IgG1- or IgG3 anti-D-secreting clones have been examined for their ability to sensitise O+ red cells for Fc-receptor-mediated rosette formation with U937 cells. IgG3 but not IgGl anti-D antibodies were able to mediate stable rosette formation with unstimulated U937 cells via interaction with the FcRI receptor. Decreasing FcRI density by incubating U937 cells with di-butyryl CAMP almost completely abolished rosette formation, whilst increasing FcRI density by incubating U937 cells with interferon-y increased the percentage of cells forming rosettes with IgG3- and IgGl-sensitised red cells. These data suggest that rosette formation between IgG anti-D-sensitised red cells and FcRI-expressing cells is dependent upon the density of IgG3 on the red cell surface, the density of FcRI on the effector cell, multiple FcRI/IgG interactions are required for stable rosette formation and that more FcRI/IgG 1 than FcRI/IgG3 interactions are required.

Introduction

Hyperimmune anti-D immunoglobulin is widely used in immuno-prophylaxis for haemolytic disease of the new-born (HDN). Sera having a high anti-D titre may be obtained from sensitised mothers or immunised male volunteers, although the anti-D titre may be unrelated to biological potency evaluated by in vitro assay [Barclay et al., 1985; Parinaud et al., 19851. A consistent finding in studies to further characterise the anti-D response has been a restriction to the IgGl and IgG3 subclasses [Ur- baniak and Greiss, 1980; Litwin, 1973; Michaelson and Kornstad, 19871. Litwin [ 19731 reported that in individ- uals heterozygous for Glm(a) and Glm(f) there was a predominance of anti-D antibody of the Glm(a) allotype, however, Parinaud et al. [ 19851 reported that the level of Glm(f) antibody correlated with the severity of HDN.

The mechanism(s) of red cell destruction in HDN has not been definitively demonstrated but the studies of Urbaniak and Greiss [ 19801 suggested that the ability of anti-D antibody to sensitize red blood cells for antibody- dependent cellular cytotoxicity (ADCC) correlates with the severity of HDN and that severe HDN is character- ised by a predominant IgG3 subclass response. However some high-titre anti-D sera, particularly those produced

in male volunteers, have been shown to be unable to sensitise red cells for ADCC, with both the IgGl and IgG3 antibody fractions being inactive [Barcley et al., 19851. In the study of Parinaud et al. [ 19851 it was concluded that the severity of HDN was correlated with the level of IgGl subclass anti-D antibody.

These inconsistent findings suggest that the anti-D response is complex and that functional heterogeneity may be- evident within both IgGl and IgG3 subclass antibodies. The availability of human monoclonal anti-D antibodies of each IgG subclass and of each of the major allotypes offers the potential to determine the functional profile of individual monoclonal antibodies. The func- tional activities may be related to the role of anti-D antibody in mediating HDN or successful prophylaxis in D- women although each may proceed by quite distinct mechanisms. In this report we have characterised twelve monoclonal anti-D antibodies and studied their interac- tion with the FcRI receptor on U937 cells both before and following stimulation with interferon-? (IFN-y) and di- butyryl cyclic AMP (Bt2cAMP) which modulate FcRI and FcRII expression [Dougherty et al., 19871 as a model for human monocytes. These studies may be particularly relevant to red cell destruction by monocyte-mediated ADCC and/or phagocytosis.

Human Monoclonal Anti-D Antibody Interaction with FcRI 223

Materials and Methods

Human Monoclonal Anti-Rhesus D Antibodies Human monoclonal anti-D antibodies (hMcAb) were produced

by stable EBV-transformed cell lines [Doyle et al., 1985; Kumpel et a]., 19871 or by heterohybridomas established by fusing EBV-trans- formed lines with the mouse X63-Ag8.653 plasmacytoma cell line [Thompson et al., 19861. The origin, subclass, light-chain type and heavy-chain allotype of the hMcAb studied are presented in table I. In most experiments culture supernate containing hMcAb was used to sensitise human RlRz cells. In other experiments hMcAb purified by immuno-affinity chromatography using a mouse monoclonal anti- human kappa-chain antibody (clone 72/2) immobilised onto gluta- raldehyde-activated silicate (Boehringer-Mannheim) was used.

Erythrocyte Sensitisation Human RIR2 erythrocytes (RBC) were obtained from Dr. D.

MacDonald (Blood Transfusion Service, Vincent Drive, Birming- ham, UK), washed five times in sterile phosphate-buffered saline (PBS) and packed by centrifugation. One volume of packed RBC was incubated with five volumes of hMcAb supernatant (or purified hMcAb appropriately diluted in PBS) for 1 h at 37°C followed by an overnight incubation at 4°C. Sensitised RBC were then washed three times with PBS and resuspended in PBS to 1% for rosetting studies or 0.2% for haemagglutination analyses. In some experiments, RBC were sensitised with a mixture of IgG3 and IgGl achieved by mixing different proportions of IgGl or IgG3 hMcAb containing superna- tants (containing 5-10 pgml IgG).

Table 1. Source and generation of human monoclonal anti-rhe- sus D antibodies

Clone Subclass Allotype Source Method of produc- tion

PhT 1A3 IgGld Glm(f) Bristol PhT 1A3 IgG3~l G3m(g) Bristol EBV transforma- JoFe 2B6 IgGld Glm(f) Bristol tion of lympho- JoFe 2B6 IgGld ND Bristol cytes FC3 IgGllc Glm(az) Bristol

REG-A IgGlK Glm(z) Cambridge EBV transforma- FOG-1 IgGllc Glm(z) Cambridge tion and subse- FOG-B IgGl Glm(z) Cambridge quent fusion with PAG-1 IgGl Glm(z) Cambridge X63.Ag8-653 cells

CB6 IgG3~l G3m(g) Bristol CB6 I g G l d Glm(az) Bristol EBV transforma- JoFe6F12 I g G h G3m(g) Bristol tion of lympho- JoFe6DlO IgG3lc G3m(g) Bristol cytes JoFe6El 1 g G 3 ~ G3m(g) Bristol

GAD-2 IgG3 G3m(b) Cambridge EBV transforma- FOG-3 IgG3lc G3m(b) Cambridge tion and subse- FOG-A IgG3r G3m(b) Cambridge quent fusion with

X63.Ag8-653 cells ~~

ND = Not determined. These clones 'switched' in their expression of heavy chain.

U937 Cell Line The human monocyte-like U937 cell line was maintained in

RPMI containing 10% FCS at 37°C in a 5% C02 humidified incuba- tor. U937 cells (at ( 0 . 5 ~ lo6 ml-I) were stimulated with recombinant IFN-y (Janssen Life Sciences) by incubation for 72 h in medium containing 100 or 1,000 unitdm1 IFN-y or with 1 mM Bt2cAMP (Sigma) for 48 h, as described previously [Kay et al., 19831. For use in the assays, stimulated and unstimulated cells were washed four times in sterile PBS and resuspended to the desired concentration in PBS.

Rosef ring Assays The ability of anti-RhD-hMcAb-sensitised RBC to form rosettes

with U937 cells was assessed using the protocol described by Ander- son et al. [1986]. Essentially, 25p1 of a 1% suspension of sensitised RBC and 25 p1 of U937 cells at 1 O6 cells/ml in PBS were incubated in each well of V-shaped microtitre trays (Titertek) containing 50 pl of PBS for 1 5 min at room temperature (RT). Plates were centrifuged for I min at 50 g and further incubated for 45 mins at RT. One drop of acridine orange was added, the pellets gently resuspended and ali- quots transferred to a haemocytometer. The number of U937 cells forming rosettes per 100 cells was determined under UV illumination for each of 4 replicate wells; rosettes formed between U937 cells and greater than 5 RBC being scored as a positive result. The ratio of RBC:U937 cells in each well was calculated to be of the order of 1OO: l .

Inhibition of rosette formation was performed as above except that the 50 p1 of PBS was replaced by 50 p1 inhibitor, either as culture supernatant or dilutions of purified anti-RhD hMcAb in PBS.

Haemagglutination Assay Haemagglutination analyses were performed in U-shaped micro-

titre trays (Titertek) as previously described [Lowe et al., 19821. Ascitic fluid containing murine McAb specific for epitopes on human IgG [Lowe et al., 1982; Nik Jaafar et al., 19831 were doubly diluted from an external dilution of 1/10 in HEPES buffer containing 2% fetal calf serum. One drop (30 pl) of a 0.2% suspension of sensitised RBC were added to each well and the agglutination pattern assessed after 1-2 h.

Results

Epitope expression was investigated in a haemaggluti- nation assay employing anti-D-hMcAb-sensitised RBC and a panel of 28 murine monoclonal antibodies each specific for a defined gamma-chain epitope [Lowe et al., 1982; Nik Jaafar et al., 19831. This study confirmed the subclass and allotype of the anti-D hMcAb (table I) and indicated that the IgG antibody molecules secreted had a normal structure and conformation.

In direct rosetting with U937 cells, IgG3 subclass anti- D hMcAb of both G3m(g) and G3m(b) allotype were able

224 Walker/Kurnpel/Thompson/Woof/Burton/Jefferis

to sensitise RBC for rosette formation whilst IgG1 sub- class anti-D hMcAb of either Glm(f) or Glm(z) allotype did not (fig. 1). No association between rosette formation and light-chain type was evident. A similar result was obtained in an indirect procedure in which U937 cells were sensitised with the hMcAb pre-bound to the FcR.

Fig. 1. Percentage of U937 cells forming rosettes with red cells sensitised by IgG1 or IgG3 anti-D hMcAb.

Following washing, U937 cells exposed to IgG3 anti-D hMcAb rosetted with unsensitised R IR:! cells whilst U937 cells exposed to IgGl anti-D hMcAb did not.

The ability of anti-D hMcAb to inhibit rosette forma- tion between U937 cells and IgG3 anti-D hMcAb sensi- tised RBC was not, however, restricted to those hMcAb belonging to the IgG3 subclass (table 11). The degree of inhibition varied between individual anti-d hMcAb used as inhibitor and for RBC sensitisation; however, there was no correlation with subclass or allotype of the inhib- itor. No inhibition of rosette formation was evidenced when supernatant ofthe anti-FcRII McAb IV3 (a kind gift from Dr. C.L. Anderson), which under identical condi- tions has been demonstrated to block ligand binding by FcRII [Anderson et al., 19861, was used as the inhibi- tor.

Three anti-D-hMcAb-producing clones were observed to ‘switch’ the heavy-chain isotype expressed following continuous culture for several months. Thus clones JoFe 2B6 and PhT lA3 ‘switched‘ from IgGl to IgG3 expres- sion and clone CB6 from IgG3 to IgG1. R I R ~ cells sensi- tised with each isotype of these anti-D hMcAb were eval- uated for rosette formation with unstimulated U937 cells. Rosette formation was obtained with the IgG3 hMcAb only (table 111). The supernates from each hMcAb pair were mixed in varying proportions and used to sensitise RBC thereby varying the proportion of IgG 1 and IgG3 on the RBC surface. Haemagglutination of these sensitised

Table 11. Inhibition of rosette formation between IgG3 anti-RhD hMcAb and U937 cells IgGl and IgG3 anti-RhD hMcAb

Inhibitor Percent inhibition of rosetting using RBC sensitised with

PhT 1A3 JoFe 6F12 CB6 FOG-3 FOG-A JoFe 6D10 JoFe 6EI GAD-? IgG3 IgG3 IgG3 IgG3 IgG3 IgG3 IgG3 IgG3

PhT IA3 JoFe6F 1 2 CB6 FOG-3 FOG-A JoFe6D 10 JoFe 6EI JoFe 286 PAC- 1 FOG-B FC3 FOG- I REG-A GAD-2

IgG3 IgG3 IgG3 IgG3 IgG3 IgG 3 IgG3 IgG 1 IgG 1 IgG 1 IgG 1 IgG 1 IgG 1 IgG3

89 100 49

100 84 97 92

100 86

100 100

9 67 73

95 89 48 99 97 87 44 97 65 79 99 11 94 5 5

53 67 35 91 63 53 40 95 85 99

100 31 60 64

53 51 31 89 81 67 43

100 56 95

100 13 71 57

68 48

7 98 79 43 46 98 93 98 98 25 81 44

69 78 31 94 97 70 37 87 36 76 97 I I 28 31

5 5 71 26 94 80 67 42 68 42 71 86 11 63 24

74 47

0 42 74 68

I00 74 98

I00 56 99 44

7 7 --

~~

Inhibitors as culture supernates containing 5-1 0 &ml IgG.

775 Human Monoclonal Anti-D Antibody Interaction with FcRI --

cells with murine anti-IgG subclass monoclonal antibod- ies demonstrated that both IgGl and IgG3 antibody bound. Using a constant ratio of sensitised RBC to unstimulated U937 cells, the percentage of U937 cells forming rosettes increased linearly with the increasing proportion of IgG3 hMcAb culture supernatant used for sensitisation (fig. 2a).

IgG 1 -anti-D-hMcAb-sensitised RBC gave 5-20 and 35-50% of rosettes with U937 cells stimulated with 100 and 1,000 unitdm1 IFN-)I, respectively. However, the addition of 20 or 10% IgG3 to the sensitising supernatant gave plateau values (fig. 2b). The ability of IgG1- or IgG3- anti-D-hMcAb-sensitised RBC to form rosettes with U937 cells was almost completely abolished if U937 cells were pre-incubated with 1 m M Bt2cAMP (fig. 2c).

Discussion

The monoclonal anti-D antibodies under study were secreted by stable cell lines obtained by EBV transforma-

tion or similar cell lines stabilised by fusion to the X63- Ag8.653 plasmacytoma cell line with the generation of heterohybrids. To establish that the secreted products were structurally normal, RBC were sensitised with the anti-D antibody and human IgG epitope display was detected by agglutination using a panel of mouse mono- clonal antibodies to human IgG [Nik Jaafar et al., 1983;

Table 111. Rosette formation using RBC sensitised with 'switched' anti-RhD hMcAb

Clone Subclass Rosetk formation (U937)

CB6 CB6 PhT 1A3 PhT 1A3 JoFe 2B6 JoFe 2B6

IgG I IgG3 IgG 1 IgG3 IgG 1 IgG3

4 + 4 67 k 7

2 + 2 70 2 8 2 k 2

73 * 3

Fig. 2. Effect ofU937 FcRI density upon rosette formation with RBC sensitised by culture supernatants mixed to give different proportions of IgGl:IgG3 anti-D hMcAb. a Unstimulated U937 cells and red cells sensitised by anti-D hMcAb CB6 (o), Jofe 2B6 (D) and PhTlA3 (0).

b U937 cells stimulated with 100 unitdml (o=CB6; +=PhTIA3) or 1,000 u n i t s h l (.=CB6; .=PhTlA3) IFN-1. c BtzcAMP-stimulated U937 cells (o=CB6; o=PhTlA3).

226 Walker/Kurnpel/Thornpson/Woof/Burton/Jefferi s

Lowe et al., 1982; Bird et al., 19841. The results obtained demonstrated normal epitope display in each of the CH1, C H ~ and C H ~ domains, the hinge region of IgG3 and also allowed the subclass and allotype of the target antibody to be determined (table I). These data show that the anti-D antibodies effectively sensitise the RBC and may be expected to be the functional homologues of anti-D anti- bodies of the same subclass and allotype present in the blood of sensitised mothers or immunised male volun- teers.

RBC sensitised with each hMcAb were evaluated for their ability to form rosettes with U937 cells. This is demonstrated to be mediated via interaction with FcRI since down-regulation of FcRI and up-regulation of FcRII on U937 by Bt2cAMP almost completely abolished rosette formation (fig. 2c). Rosette formation could not be inhibited by pretreatment of the U937 cells with cul- ture supernatant of the anti-FcRII mouse monoclonal antibody IV3 [Anderson et al., 19861 but was inhibited by monomer IgGl or IgG3 anti-D hMcAb (table 11). Since culture supernatants containing 5-10 pg/ml were used as inhibitors it may be that the differences in the inhibitory capacity of some hMcAbs reflect differences in the IgG concentration. Indeed, using purified hMcAb sat 0.1 mg/ml produced 100% inhibition of rosetting by RBC sensitised with the autologous hMcAb. No rosette forma- tion was observed if the FcRI-/FcRII+ Daudi or K562 cell lines were used as effector cells (data not shown). Further- more, Dougherty et al. [1987] have shown that rosette formation can be completely inhibited by an anti-FcRI antibody.

All IgG3 hMcAb tested formed a high percentage of rosettes with U937 cells (>70%) whilst IgGl hMcAb failed to sensitise for rosette formation (fig. I ) . Further compelling evidence for the difference between IgGl and lgG3 hMcAb in this system was provided by hMcAb clones CB6, PhT 1 A3 and JoFe 2B6 that ‘switched’ iso- type in culture and were available as the IgGl and IgG3 isotypes secreted at equivalent concentrations in the supernates [B. Bradley; pers. commun.]. Since only the IgG3 hMcAb molecules were effective in promoting rosette formation with unstimulated U937 cells (table III), this property appears to be independent of the epi- tope specificity or affinity of the antibody for the D- antigen. Indeed, the panel of hMcAb used contained antibodies of both IgG1 and IgG3 isotypes recognising the same or different epitopes on the D antigen [N.C. Hughes-Jones; pers. commun.]. The lack of rosette for- mation with IgGl anti-D hMcAb is at first sight surpris- ing since it is generally accepted that IgGl and IgG3 have

similar affinity for FcRI [Burton, 19851. Furthermore, in this system it was shown that IgGl could inhibit rosette formation between IgG3 sensitised RBC and U937 cells (table 11). The explanation for the ineffectiveness of IgG 1 is probably associated with a requirement for multiple interactions between FcRI on the U937 cell and the hMcAb anti-D on the RBC for stable rosette formation. Evidence for a requirement for multiple interactions was obtained using U937 cells cultured in the presence of IFN-y which increases FcRI expression [Anderson et al., 19861. Cells cultured in the presence of 100 or 1,000 units/ml IFN-y gave 5-15 and 35-50% rosettes with IgGl-sensitized RBC, respectively (fig. 2b). In contrast down-regulation of FcRI expression by Bt2cAM P pre- vented rosette formation by both IgG 1 and IgG3 hMcAb (fig. 2c). This demonstrates that multiple FcRI/hMcAb interactions are necessary and that FcRWhMcAb inter- actions (if possible) alone cannot promote rosette forma- tion with anti-D-hMcAb-sensitised RBC. Since stable rosette formation is dependent upon close approach of target and effector cell which generates charge repulsion, the number of IgG3/FcRI interations necessary to over- come the charge repulsion generated may be less than those required for IgGl by virtue of the extended hinge of IgG3 (fig. 3) [Gregory et al., 19871. Indeed, it has been demonstrated that using IFN-y-stimulated monocytes 100 molecules of IgG3 anti-D per RBC mediate rosette formation, whereas 10,000 molecules of TgGl are required per RBC [Weiner et al., 19871. Similar results have been demonstrated for human monocytes and lym- phocytes by Zupanska et al. [ 19861.

Within a polyclonal anti-D response both IgGl and IgG3 antibodies are normally produced but the propor- tions of each varies between individuals.

It would appear therefore that the clearance mecha- nism(s) activated would also vary according to which isotype predominated. Suggestive evidence for this pro- posal was obtained when RBC were sensitised with mix- tures of IgGl and IgG3 hMcAb (fig. 2a). An essentially linear response was obtained providing further evidence of the necessity for multiple FcRI/IgG interactions. Thus it may be assumed that stable rosette formation requires two or more IgG3 molecules to be in an appropriate proximity to each other to allow multiple binding with FcRI molecules. The frequency of IgG3 molecules thus favourably displayed will increase with its proportion of the total antibody-sensitising the RBC. This requirement may be similarly achieved by increasing the RcRI density which allows widely distributed 1gG3 molecules to bind to a sufficiently large number of FcRI molecules. This is

Human Monoclonal Anti-D Antibody Interaction with FcRI 227

Fig. 3. Schematic repre- sentation of IgG1 (a) and IgG3 (b) molecules binding to antigen and the monocyte FcRI receptor.

illustrated using the IgG1 and IgG3 variants of PhTlA3; with an IgGl:IgG3 ratio of 4:1, 30%, 75% and >90°/o rosettes were obtained using unstimulated, 100 or 1,000 units/ml IFN-y-stimulated U937 cells, respectively.

These studies suggest that several parameters may be critical to the formation of a stable interaction between an antibody-coated target cell and an effector cell. Some parameters have been defined thus; (1) the density of antibody on the target cell, (2) the density of FcRI on the effector cell, (3) the availability of the interaction site for FcRI on the IgG molecule - the extended hinge region of IgG3 favouring interaction to occur at low antibody and FcRI densities, (4) the charge on the surface of the inter- acting cells. These are events preceding activation of the effector cell which may have further requirements to enable activation of the elimination mechanism(s).

Acknowledgements

The authors would like to thank Dr. C. L. Anderson for the kind gift of McAb IV.3, and Peter Richardson and Julie Campbell for assistence with establishing the rosette assays. This work was sup- ported by a grant from the Wellcome Trust. DRB is a Fellow of the Lister Institute for Preventitive Medicine.

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Addendum

The suggestion that clones CB6 and PhTIA3 ‘switched’ expres- sion of heavy chain isotype cannot be substantiated since subsequent karyotype analysis revealed that cross-contamination of the culture had occurred in the laboratory of origin.

Received: December 11, 1987 Revised manuscript received: April 8, 1988 Accepted April 9, 1988

M.R. Walker, PhD Department of Clinical Chemistry Wolfson Research Laboratories Queen Elizabeth Medical Centre Edgbaston, Birmingham B 15 2TH (UK)