Tissue Antigens (1986) 28, 170-175

Antibody dependent cellular cytotoxicity mediated by HLA-class I1 specific monoclonal antibodies FRITS KONING, MARRIE KARDOL and HANS BRUNING

Department of Immunohaematology and Bloodbank, University Hospital, Leiden, The Netherlands

The ability of mouse monoclonal antibodies (MoAbs) directed against HLA-class I1 molecules to mediate in Antibody Dependent Cellular Cytotoxicity (ADCC) was investigated. The results indicate that both MoAbs to monomorphic and polymor- phic HLA-DR and DQ determinants are able to mediate ADCC in an antigen spe- cific manner. However, not all antibodies mediate ADCC to a similar extent. Fur- thermore, antibodies were identified that appeared to mediate ADCC in an HLA-DR haplotype dependent fashion. These results indicate that the inhibition of HLA-class I1 specific proliferative responses by anti-class I1 MoAbs may be influen- ced by ADCC directed against class I1 positive stimulator cells.

Received for publication 12 March, accepted 12 May 1986

Monoclonal antibodies have in the last few years been very useful for the study of the HLA-class I1 gene products. They have facili- tated the biochemical characterization of the molecules encoded in the HLA-DR, DQ and DP region (Kaufman et al. 1984, Giles & Ca- pra 1985), are likely to become important for tissue typing since they reveal additional poly- morphism in the class I1 region which is not detected by alloantisera (Koning et a1 1985. Schreuder et al. 1986), and finally they have been important for the identification of gene products carrying allogeneic stimulatory de- terminants or epitopes which restrict antigen presentation to antigen primed T-cells (Otten- hoff et al. 1985, Horibe et al. 1984). In the lat- ter functional assays, monoclonal antibodies are usually present throughout the culture

time. Inhibition of 3H-thymidine uptake by a given antibody forms the basis for assigning either the allogeneic stimulatory determinant or restriction epitope to the HLA-class I1 gene products recognised by the monoclonal anti- body. A factor which might influence the out- come of such experiments is Antibody De- pendent Cellular Cytotoxicity (ADCC) medi- ated by the monoclonal antibodies resulting in the kill of allogeneic stimulator cells or anti- gen presenting cells. We therefore tested the capability of a panel of anti-HLA-class I1 monoclonal antibodies to mediate ADCC against HLA-class I1 positive target cells. The results indicate that most of these antibodies can induce efficient lysis of HLA-class I1 posi- tive target cells in an antigen specific manner. The implications of this are discussed.

ADCC MEDIATED BY ANTI-CLASS 11 MOABS 171

Material and methods

Monoclonal antibodies

All monoclonal antibodies used (Table 1) have been described previously and were used in the form of diluted ascites. Two antibody

concentrations were tested with final ascites dilution in the testplates being 300 and 600. Earlier experiments had shown that these concentrations saturate the binding sites on the target cells.

Table I . Characteristics of the antibodies used and their capacity to mediate in antibody dependent cellular cytotoxicity. Percent specific lysis was calculated as described in Material and methods.

% specific lysis” Target cells (HLA-DR) 111 212 313 414 515 w6lw6 717

Moabs &-(sub) Specificityb class Monomorphic Reference

Control B8.11.2 LD1.l 7.5.10.1 PdVS.2 1c2 XAlM IVA12

TU22 SPV-L3.8

7.3.19.1

MCS7 PD3 IB6 HIE3 1 6 2 3

I-LR2

SFR3-DR5

IIB3 IVD12

-

G2b G2 G2a G1 G2a ? GI

G2a G2a

G2b G2b ? A G3 GI G3 G2b

G2a G1

- DR D R class I1 class I1 class 11 class I1 class I1

DQ DQ

Polymorphic DRw52-like DRw52-like DRw52-like DR DR DR DR3 + w6 DRS

DQwl-like DQw3

- -

++ ++ + ++++ ++++ ++ ++ ++ + ++ ++ + ++ + ++

-

+ -

- -

+ ++++ ++++ + ++ ++ + - - -

- -

+ -

- +++ + ++++ ++ +++ ++ +++ - +++

+++ ++++ ++++ ++++ +++ ++ ++++ -

- +++ + ++++ ++ +++ ++ ++ ++ ++

- ++++ ++++ ++++ + - - + ++

- ++ + ++++ + ++ + + ++ ++

+ + ++++ ++ +++ + ++++ -

- +

-

+++ ++++ ++ +++ ++ +++ + +++

-

++ +++ ++++ +++ +++ ++ +++ -

+ -

-

++ + +++ + ++ ++ ++ + ++

-

+++ + + + -

Rebai (1983) Koning (1986) Koning (1984) Koning (1985) Koning (1986) Koning (1986) Giles (1983)

Wernet (1983) Spits (1984)

Koning (1984) Nadler (1981) Tanigaki (1983) Koning (1986) Koning (1986) Hurley (1982) Johnson (1982) Radka (1984)

Koning (1986) Giles (1983)

a _ = 0-20% lysis. + = 20-40% lysis. ++ = 40-60% lysis. +++ = 60-80% lysis. + + + + = 80- 100% lysis.

DR: reacts with DR only DQ: reacts with DQ only Class 11: reacts with DR and other locus products

Results are shown for the 20:l effector:target cell ratio in the presence of 300 X diluted antibody samples only. The 10:l effector:target cell ratio gave similar results but weaker reactions.

172 KONING ET AL

TARGET CELLS

I)R I

DR2

I)R3

DR4

DRS

DRw6

DR7

% SPECIFIC LYSlS 0 50 0 50 0 50 0 50 0 50 0 50 0 50

16-23 I-LRZ 7.3.19.1 SPV- L3 B8.11.2

MONOCLONAL ANTIBODIES

I PdV5.2 3 7.5.10.1

Figure I . Antibody dependent cellular cytotoxicity mediated by the monoclonal antibodies indicated. Re- sults are shown for the 20:l effector:target cell ratio in the presence of 300 x diluted antibody samples. % specific lysis was calculated as described in Material und rnerhods.

Antibody dependent cellular cytotoxicity

ADCC was carried out using a non-radioac- tive assay as has been described previously (Bruning et al. 1979) with some modifica- tions. Freshly isolated PBL were used as ef- fector cells and tested in 20:l and 1 O : l effec- tor: target cell ratios against 2,500 carboxyflu- orescein (C-F) labelled target cells. Labelling of the target cells took place as follows: twice washed target cells were resuspended in 1 ml PBS containing 50 pg carboxyfluorescein di- acetate (C-FDA) and incubated at 37°C for 25 min. After one wash, the cells were resuspen- ded in RPMI 1640 supplemented with 10% FCS. EBV-transformed lymphoblastoid B- cell lines, homozygous for the HLA-antigens, were used as target cells. The assays were set up as follows: 10 p1 effector cells, 10 pl target cells and 10 p1 antibody solution were mixed in Terasaki type trays with a well volume of 40 pl (Greiner, cat. nr. 726180). A s negative con- trols, heat treated effector cells (15 min, 46°C)

and antibody solution containing irrelevant antibody were used. As positive control a rab- bit heteroantiserum against B-cell lines was used.

After spinning the trays for 2 min at 150g, the relative number of C-F labelled cells was determined, as an arbitrary fluorescence value per well, using an automated inverted epi-illuminated fluorescence microscope equipped with filters for C-F. All values were expressed as per tray normalised percentages. Next, the trays were incubated at 37°C for 3 h in a humidified 5% CO, incubator. Subse- quently, 10 pl ink-suspension (Leitz ink, 1:300 in isotonic 5% EDTA, p H = 7) was added to each well. The ink quenches the released me- dium C-F but not the C-F in the unlysed sedi- mented target cells. After a 10 min equili- bration, the amount C-F remaining in the tar- get cells was determined with the automated microscope. Percent lysis was calculated as follows : the latter assay values are corrected for well to well variability by dividing them by

ADCC MEDIATED BY ANTI-CLASS 11 MOABS 173

the preincubation values and used in the fol- lowing formula: (1 - A/B) X 100%. A is the average corrected value of effector + target cells with anti-class I1 MoAb solution. B is the average corrected value of effector + target cells with control antibody solution. The method used has been shown to give results which are comparable to those obtained with the "CR-release assay (Bruning et al. 1979, unpublished results).

Results

In two experiments the capability of the monoclonal antibodies to mediate in ADCC was investigated. In both experiments, a panel of 7 HLA-homozygous B-cell lines was used as target cells. The panel contained cell lines of the D R M , DR2/2, DR3/3, DR4/4, DR5/5, DRw6/DRw6 and DR717 haplotype. Since both experiments gave comparable re- sults, those obtained in the first experiment are shown only. Table 1 gives a summary of the results indicating which antibodies medi- ated ADCC. Figure 1 illustrates this in more detail for a selected number of antibodies. Both antibodies to monomorphic and poly- morphic DR and DQ determinants were able to mediate in ADCC. The antibodies detect- ing polymorphic determinants mediated ADCC only against cells of the proper HLA- DWDQ haplotype.

Discussion

The objective of this study was to investigate if mouse monoclonal antibodies directed to HLA-class I1 molecules are able to mediate in ADCC. Fc-receptors on human peripheral blood cells specific for all human Ig-(sub)- classes have been described (reviewed in Moretta et al. 1981) whereas ADCC can be mediated by antibodies of the IgG, IgM and

IgE immunoglobulin class (Kovithavongs & Dossetor 1982).

Furthermore, binding of mouse immuno- globulins to human Fc-receptors has been demonstrated (Tax et al. 1984) indicating that mouse monoclonal antibodies might mediate ADCC. The results in this study indicate that all antibodies tested, irrelevant of the Ig- (sub)class, mediated ADCC. However, not all antibodies mediated ADCC to the same extent. Some MnAbs such as B8.11.2 and 7.5.10.1 are quite efficient at mediating ADCC (Table 1, Figure 1) while others such as LDl . l and PdVS.2 are relatively ineffi- cient. It is unlikely that these results are due to differences in antibody concentrations since similar results at two different antibody concentrations were obtained (not shown). Furthermore, depending on the HLA-haplo- type of the target cells, differences in the per- centage of target cells killed were observed. For example, both the 7.3.19.1 and the I-LR2 MoAb, which both react with DRw52-like de- terminants, strongly mediate ADCC against DR3+ cells, intermediately against DRw6+ cells and only weakly against DR5+ cells. Similarly, the PD3 MoAb strongly mediated ADCC against all target cells tested except those of the HLA-DR2 and DR7 haplotype. In the latter case this may result from the fact that PD3 binds only weakly to DR2 and DR7 positive cells (Koning et al. 1986), however, the I-LR2 MoAb binds equally well to DR3, DRS and DRw6 positive cells (Koning et al. 1986) but nevertheless differential ADCC is observed with this MoAb. Since the MoAb 7.5.10.1 mediates ADCC to the same extent against all cell lines tested (Figure l), it is un- likely that this is thc result of differences among the cell lines with regard to susceptibil- ity to ADCC mediated lysis.

Two (not completely unrelated) factors may therefore be of importance for inhibition stud- ies carried out with anti-class 11 MoAbs: a) not all antibodies mediate ADCC to the same

174 KONING ET AL.

extent and b) depending on the HLA-haplo- type, a given antibody can mediate different levels of ADCC. These characteristics of anti- HLA-class I1 antibodies might result in differ- ential inhibition patterns of functional assays, depending on the antibodies andlor the HLA- haplotype of the stimulator/antigen present- ing cell used if in the assay cells are present which can exert ADCC. Therefore, in order to exclude the possibility of ADCC influen- cing inhibition studies during functional as- says, proper controls should be included in the assay. These could be either control anti- bodies directed against HLA-class I1 mol- ecules which, although able to mediate ADCC, do not inhibit stimulation of respon- der T-cells or, alternatively, testing if the re- sponder T-cells are able to kill class I1 positive stimulator cells by ADCC.

Acknowledgments

The authors wish to thank the Drs. Malissen, Capra, Ziegler, Spits, Nadler, Tanigaki, John- son, and Radka for the generous gift of mono- clonal antibodies. This work was in part sup- ported by the Dutch Foundation for Medical Research (FUNGO) which is subsidised by the Dutch Foundation for the Advancement of Pure Science (ZWO), and the J. A. Cohen Institute for Radiopathology and Radiation Protection (IRS).

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Address: Dr. E Koning Laboratory of Immunogenetics Building 5 Room Bl-04 National Institutes of Health Bethesda, Maryland 20892 USA