Quantitative expression of epitopes defined by murine monoclonal antibodies on DR molecules from different HLA haplotypes

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1 Bioscience Reports 5, i (1985) 923 Printed in Great Britain Quantitative expression of epitopes defined by murine monoclonal antibodies on DR molecules from different HLA haplotypes Michelle LETARTE 1, Jane ADDIS, Sonia ITURBE and Dieter PETSCHE Division of Immunology, Research Institute, The Hospital for Sick Children, Toronto, and 1Department of Immunology, University of Toronto, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8 (Received 26 July 1985) Monoclonal antibodies 50D6 and 21r5, reactive with human class II molecules, were analyzed quantitatively by flow cytometry and cellular radioimmunoassay for their binding to cells of different HLA-DR types. Monoclonal antibody 50D6 bound equally to cells of all DR types tested except DR7, where no reactivity was observed. Monoclonal antibody 21r5 was reactive with all cells. However, the percentage of DR molecules at the cell surface expressing 21r5 epitope varied with the DR type and increased as follows: DR3-- DR7 < DR2 < DR5 < DR4 < DR1. MAb 50D6 reacted with an epitope spatially related to but distinct from the 21w4 epitope present on all DR molecules. The 50D6 epitope was shown to be present on isolated DR 1 molecules. The majority of murine monoclonal antibodies (MAb) reactive with HLA class II molecules do not recognize polymorphic determinants, i.e. epitopes restricted to molecules of a single DR, DQ or DP specificity and usually defined with human allosera (Bodmer & Bodmer, 1984). While analyzing several murine anti-hla-dr MAb produced in our laboratory, we observed that antibodies such as 21w4 reacted equally with all HLA-DR molecules while others such as 21r5 reacted with subsets of HLA-DR molecules with varying levels of expression amongst chronic lymphocytic leukemia cells (Addis et al., 1982). We have now studied by flow cytometry and cellular radioimmunoassay the quantitative expression of 21r5 epitope on HLAhomozygous B lymphoblastoid lines in an attempt to correlate the level of 21r5 with the HLA-DR phenotype. We have also analyzed the quantitative binding of MAb 50D6, shown in preliminary experiments to be unreactive with DR7 cells. Materials and Methods Cells Cells were obtained from the Human Genetic Mutant Cell Repository, Camden, New Jersey, and maintained in RPMI 1640 medium supplemented

2 924 LETARTE ET AL. with 20% fetal bovine serum (Gibco). All cell lines were tested frequently for mycoplasm contamination and shown to be negative. Cells were washed several times in phosphate buffered saline ph 7.4 prior to assay. Monoclonal an tibodies The production and purification of the IgG1 MAb 21r5 and 21w4 has been reported (Addis et al., 1982). MAb 50D6 was derived from a fusion described previously (Letarte et al., 1985): it is an IgG2a antibody which was purified by affinity to Protein A Sepharose and labelled as previously described (Letarte et al., 1985). Flow cytometry analysis Cells (5 X 105, viability > 98%) were incubated for 1 h at 4~ with saturating levels of MAb 21w4, 21r5, 50D6 or control, washed 2X and incubated for 1 h with fluorescein isothiocyanate - F(ab')2 goat anti-mouse IgG (Tago, Burlingame, CA). Fluorescence was read at 488 nm using an Epics V flow cytometer (Coulter Electronics, Hiateah, FL). A minimum of cells were analyzed. The fluorescent signal, which is logarithmically amplified over 3 cycles on the 256 channel abscissa (LIGFL, see Fig. 1), is such that an increase of 25.6 channels corresponds to a doubling in fluorescence intensity. The relative fluorescence index (RFI) is used to convert the log scale to the linear scale in order to compare the test sample to the control sample. 2 [Channel number of mean LIGFL of test sample RFI = Channel number of mean LIGFL of control sample] The RFI values were calculated using the E.A.SY. 1.2 program 'IMMUNO'; the graphics were done using the E.A.SY. 1.2 program 'GRAF3D' (Coulter Electronics). Cellular radioirnmunoassay (CRIA ) The quantitative cellular radioimmunoassay of HLA-DR antigens has been described previously (Addis et al., 1982). Because of the high levels of HLA- DR antigens on homozygous cell lines (Falk et al., 1985), the number of target cells was reduced to 2.5 X 104 cells and ox red blood cells were added to facilitate pelleting of cells. 12SI-F(ab')2-rabbit-anti-mouse IgG-Fc (RAM- Fc) was used at a concentration of 500 ng per well in the second incubation. All assays were done in microtitre plates, in triplicate, and all experiments were repeated 3 times. A saturating level was calculated from the plateau of the saturation curve in ng of RAM-Fc bound per cells. Ratios 21r5 : 2 lw4 and 50D6 : 21w4 were calculated from the saturation levels measured in the same experiment.

3 EPITOPES OF DR MOLECULES 925!1~ ! LIGFL (channel number) Fig. 1. Analysis by flow cytometry of the levels of 50D6, 21w4 and 21r5 on B lymphoblastoid lines of different DR phenotypes. The six cell lines listed in Table 1 were incubated with saturating levels of either 50D6, 21w4 or 21r5 MAb as described in Materials and Methods. The frequency of cells per channel is plotted versus the Logto integrated green fluorescence (LIGFL). The control histograms were equivalent to that shown for the DR7 cell line stained with 50D6 MAb. More than 95% of cells were reactive in each of the positive histograms shown. Results and Discussion The 50D6 epitope is not expressed on cells of DR7 phenotype Fig. 1 demonstrates that the 50D6 MAb was reactive with cells expressing either DR 1, 2, 3, 4 or 5 phenotype but unreactive with cells of DR7 phenotype. The latter cells were normal in their expression of HLA-DR antigens as seen by their high reactivity with MAb 21w4. In all positive histograms shown in Fig. 1, >95% of cells were reactive with either 50D6, 21r5 or 21w4 MAb, indicating that all the cells in the samples were expressing all 3 determinants.

4 926 LETARTE ET AL. 32- Ii 2 g 24- ~16- < rc 8- / / d /,e / / 8 4 g g (3 16~E g v % 50D6 culture supernatant ng 50D6-1gG added Fig. 2. Quantitation of 50D6 cell surface expression by CRIA. (A) GM3098 cells (DR3 ; e) or GM3155 cells (DR7 ; zs) were incubated for 2h in 50/11 of 50D6 culture supernatant at the concentrations indicated. Saturating levels of 12SI-RAM-Fc (500 ng/well; 600 c.p.m./ng) were added for 2 h and cells washed and counted; results are in ng RAM-Fc bound per 2.5 x 10 4 ceils. (B) The same cells were incubated directly with t2si-50d6 IgG (5000 c.p.m./ng); results are in ng 50D6- IgG bound per 2.5 x 104 cells. Table 1. Quantitative expression of DR molecules bearing 21r5 and 50D6 epitopes on ceils of different DR phenotype Cells were incubated with saturating levels of MAb 21r5, 50D6 or 21w4 and then analysed quantitatively by flow cytometry or cellular radioimmunoassay (CRIA) Cell line DR Relative fluorescence Ratios Ratios type index (RFI) 21r5:21w4 50D6 : 21w4 21r5 21w4 50D6 From From From From RFI CRIA RFI CRIA values levels values levels GM GM GM GM GM GM Cells of DR7 phenotype were minimally reactive in the CRIA either in the indirect assay with 12q-RAM-Fc or in the direct assay with D6 IgG when compared to cells of DR3 phenotype (Fig. 2). The levels of 50D6 bound to cells of different HLA-DR phenotype are compared in Table 1. As expected for the DR7 cells reacting equally well

5 EPITOPES OF DR MOLECULES 927 with 50D6 and control MAb, a RFI value of 1 was observed. When the ratios of 50D6 and 21w4 MAb bound to the cells were calculated, negligible ievels (2-3%) were obtained for DR7 cells, confirming the absence of 50D6 epitope on the surface of these cells. For all other cell types, the RFI values were similar ranging from 49.8 to 65.4, indicating that these cells expressed equivalent amounts of 50D6 epitope on their surface. The ratios 50D6:21w4 calculated from the RFI values ranged from 1.29 to 1.41 whereas the ratios calculated from CRIA saturating levels ranged from 0.54 to Because these ratios gave such consistent values, we favour the hypothesis that divalent binding was obtained between 50D6 MAb and goat anti-mouse IgG while monovalent binding was observed between 50D6 MAb and RAM-Fc (see Fig. 2). Monovalent binding was obtained in both assays with 21w4 MAb. When the 50D6 MAb was analysed on several DR7 cell lines expressing either Dw7, Dwl7 or Dwl 1, all were unreactive with 50D6, extending our observations of the absence of 50D6 epitope to all DR7 cell lines tested (R. W. Karr, unpublished observations). Furthermore, when the 50D6 MAb was tested in the cytotoxicity assay against B lymphocytes isolated from a selected panel of 30 individuals, reactivity was seen for all cells except the DR7 homozygous typing cells (H. Mervat, unpublished observations). Two other anti-hla-dr MAb unreactive with DR7 cells and reactive with cells of other DR phenotype were analysed in the First International Workshop on Monoclonal Antibodies to Human MHC Class II Antigens (Steel, 1984). The quantitative binding of MAb 21r5 is correlated with the DR phenotype of the cell Fig. 1 demonstrates the differential reactivity of 21r5 MAb with HLA-DR homozygous cell lines. The histograms are presented in the order of increasing levels of 21r5 binding (DR7 = DR3 < DR2 < DR5 < DR4 < DR1). The RFI values calculated in Table 1 suggest that DR7 and DR3 cells express low levels, DR2 and DR5 intermediate levels, and DR 1 and DR4 cells, high levels of 21r5 epitope. The ratios 21r5:21w4 obtained by comparing RFI values or CRIA saturation levels for each DR type are remarkably similar and further support the view that 21r5 epitope is expressed on only certain subsets of DR molecules. The quantitative variation in the level of expression of an epitope being correlated with HLA-DR phenotype is to our knowledge a new observation. The functional significance of the levels of certain subsets of DR molecules remains to be established. Our results confirm that MAb binding measurements performed with the flow cytometer can be readily quantitated and that the RFI values calculated are comparable to the relative levels measured in the CRIA under saturating conditions. Relative localization on DR molecules of 21w4, 2.!r5 and 50D6 ep#opes We have previously demonstrated that MAb 2 l r5 and 2 lw4 recognize two distinct epitopes of ttla-dr molecules (Addis et al., 1982). We have also shown that MAb 21w4 binds to an HLA class II epitope present on several species including pig, sheep and mouse (Falk et al., 1983). In the murine

6 928 LETARTE ET AL. species, MAb 21w4 recognized an Ia.7-1ike epitope present on I-E (x chain (Letarte et al., 1982; Falk et al., 1983). Since 2 lw4 reacts equally well with all DR molecules and since cr chains are highly homologous (Kaufman et al., 1983), it is likely that the 21w4 epitope is located on DR c~ chains; however, reactivity of 21 w4 MAb with isolated a chains has not yet been demonstrated. The 21r5 epitope is expressed only on certain subsets of DR molecules, and since 2 or 3 ~ chains are present for each DR haplotype, this epitope might be localized only on some of the r chains. The reactivity of MAb 21 r5 with isolated ~ chains has not been shown and thus the possibility exists that 21r5 MAb could recognize a conformational determinant requiring both chains for expression. Potentiation of binding to the 2 lr5 epitope following binding of 21w4 MAb to cells has been observed and would imply that flexibility of the DR molecule is important in the binding of 21r5 NAb (Addis et al., 1982). Fig. 3 demonstrates that MAb 21w4 can block completely the reactivity of 50D64gG to cells of DR3 or DR1 type. MAb 77-34, reactive with DQwl 1 O0 - ~--& A & &~,~'-..._ A iiil."- ZX 20- [] c I - - " = 100- O..,.~ D --~-'4Z]~---"~[3 ~ E] ~ E]..., D # "\A~. ~ ~o'-~ o A i~ A 80- ORI I 0 Log~o competing antibody dilution Fig. 3. Blocking of 50D6 IgG binding by 21w4 MAb. GM3098 cells (DR3) or GM3104 ceils (DR1) (1 x 105 cells) were incubated for 16 h at 4 C with serial dilutions of MAb culture supernatants. 12sI-50D6-IgG (60 ng/well) was added without prior washing of the cells for 1 h incu~ bation. Results are in % 12sI-50D6-IgG bound in the absence of competing MAb. The 100N values were and c.p.m, respectively.

7 EPITOPES OF DR MOLECULES 929 molecules (Falk et al., 1985), did not block any binding to DR1 cells which also express DQwl molecules. MAb 21r5 could block to some extent the reactivity of 50D6-IgG to DR1 cells where the level of 21r5 epitope is high, but could not block the reactivity to DR3 cells where the level of 21r5 epitope is low. Fig. 4 illustrates that 50D6 MAb can block 30% of the binding of 21w4 IgG to DR1 cells whereas 21r5 MAb could not. These results suggest that the 50D6 epitope is spatially related but not identical to the 21w4 epitope. The ratio 50D6:21w4 is equal on cells of different DR types, and because the degree of homology between c~ chains is much higher than that observed with (3 chains, we would like to suggest that the 50D6 epitope is located on DR oe chain. Sequence analysis of DR7 chain and comparison with other DR c~ chain sequences might reveal a deletion or mutation of a few amino acids unique to DR7 c~ chain. When the 50D6 reactivity was analysed by Western Blot, no binding to separated o~ or polypeptide chains was observed; only the dimeric DR molecules obtained under mild dissociating conditions were reactive with 50D6 (Steel, 1984). Thus we cannot rule out the possibility that both c~ and ~ chains are required for expression of the 50D6 epitope. The 50D6 epitope is present on isolated DR molecules To confirm the presence of 50D6 epitope on isolated DR molecules, extracts prepared from DR1 cells (Falk et al., 1985) were fractionated on 21w4-IgG Sepharose. The bound and eluted fractions, assayed by inhibition of CRIA, were shown to express 81%, 95% and 96% of the 21w4, 21r5 and 50D6 activity, respectively, present in the original extract. When the DR1 molecules eluted from the 21 w4-igg column were radiolabelled and immunoprecipitated with 50D6 and 21r5 MAb, patterns characteristic of DR 100 A ,(,k---at,~-r~-..7..:, ' /x'-~a Z5 Zx A---- A 80 o~ ~3 C DR1 ~, 21w4 21 O I I I Loglo competing antibody dilution Fig. 4. Partiai blocking of 21w4-IgG binding by 50D6 MAb. GM3104 cells (DR1) were incubated as described in Fig. 3 and w4-IgG (200 ng/well) was added. Results are in % 12sI-21w4-IgG bound in the absence of competing antibody (100% = c.p.m.).

8 930 LETARTE ET AL. molecules were obtained. The c~ chains were resolved as acidic spots with pi ranging from 4.3 to 4.9 while the ~ chains which are more basic migrated with pi ranging from 5.8 to 6.8 (Fig. 5). These patterns could not be distinguished from the 21w4 immunoprecipitates. No reactivity of isolated DR1 molecules with MAb (anti-dqwl) could be observed. Furthermore DQwl molecules isolated by affinity to IgG-Sepharose (Falk et al., 1985) were devoid of reactivity to 50D6, 2 l r5 and 21 w4 MAb. Fig. 5. Two-dimensional gel analysis of 50D6 and 2 lr5 immunoprecipitates of DR1 molecules. The DR1 molecules were purified from a taurocholate extract of GM3104 cells by affinity to 21w4-IgG Sepharose (Falk et al., 1985). The 21w4 eluate was labeled with 12sI and immunoprecipitated with either 50D6 or 21r5 MAb and subjected to two-dimensional gel electrophoresis (as described by Shackelford & Strominger, 1980). The a chains are acidic and migrate towards the anode, whereas the /3 chains are more basic and migrate towards the cathode. These results indicate that the 50D6 epitope is present on isolated DR1 molecules and is absent from DQwl molecules. Since the immunoprecipitation patterns obtained could not be distinguished from that seen with 2 l w4, reactive with all DR1 molecules, or 21r5, reactive with 70-80% of DR1 molecules (Falk et al., 1985), our data suggest that all DR1 molecules express the 50D6 epitope. It is also likely that all DR molecules of other phenotypes also carry the 50D6 epitope with the exception of DR7 molecules. Acknowledgements This research was funded by grants from the Medical Research Council and the National Cancer Institute of Canada. M. Letarte is a Terry Fox

9 EPITOPES OF DR MOLECULES 93 i Research Associate of the National Cancer Institute. We thank Dr. Helena Mervat, Canadian Red Cross, Toronto, for testing 50D6 MAb on a panel of typing cells. We also thank Dr. Robert W. Karr, V.A. Medical Centre, Iowa City, for unpublished observations on the reactivity of 50D6 MAb. References Addis JBL, Tisch R, Falk JA & Letarte M (1982) J. Immunol. 129, Bodmer J & Bodmer W (1984) hnmunol. Today 5, Falk JA, Addis JBL & Letarte M (1983) Human Immunol. 7, Falk JA, Lewis WH & Letarte M (1985) Human Immunol., in press. Kaufman JF, Auffray C, Korman AJ, Shackelford DA & Strominger JL (1984) Cell 36, Letarte M, Meghji G & Lorimer G (1982) Molec. Immunol. 19, Letarte M, Iturbe S & Quackenbush EJ (1985) Molec. Immunol. 22, Shackelford DA & Strominger JL (1980) J. Exp. Med. 151, Steel CM (1984) Disease Markers, vol 2, Human MHC Class H Antigens: Genetics, Structure and Function, Wiley & Sons Publishers.

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