Tissuc Anrrpns 1993. If 165-168 Prinrcrl in Drnniark . All rrghrs reserved

C o p p r i a h r C, Munksaaard 1993 ~~

T I S S U E A N T I G E N S ISSN oooi-zais

Brief Communication

Characterization of a murine monoclonal antibody recognizing HLA-DQ5( l), DQ6( 1) and DQ4 antigens S. Walsh, C. Cayrol, D. Clemeni, P. Mouynet, A. Alam, J Tkaczuk, A. Cambon-Thomsen. Characterization of a murine monoclonal antibody recognizing HLA-DQS( I), DQ6( 1) and DQ4 antigens. Tissue Antigens 19-93: 41: 165-168. 0 Munksgaard, 1993

The production of monoclonal antibodies (mAb) recognizing monomorphic and polymorphic deter- minants on HLA class-I1 molecules has contrib- uted to the functional and biochemical analysis of the HLA molecules (1). Moreover. mAb to poly- morphic determinants provide useful tools for HLA typing.

We report here the production and characteriza- tion of a novel mAb, OHA TM904, specific for HLA-DQ5( l), DQ6( 1) plus DQ4 antigens. The hy- bridoma secreting this mAb has been generated by fusing splenocytes from immunized C3H mouse with the X63Ag8.653 myeloma cells, according to methods previously described (2). The C3H mouse was immunized four times intraperitoneally with the human lymphoblastoid cell line (B-LCL) BON expressing DR103, DQ5( 1) and DPw4 molecules, then alternatively with the DR103 transfectants over a period of 2 months, and boosted intra- venously with the combination of the two cell types 3 days before the fusion. Hybridoma supernatants were first tested by micro-ELISA assays (3), on the cell lines used as immunogens, DR 103 transfected L-cells, B-LCL BON and on the untransfected L- cells. The hybridoma secreting the OHA TM904

Susan Walsh', Corlnne CayroP, Danlile Clamant', Pascal8 Monynet', Antoina Alam', Jean Tkaczuk' and Anne Cambon-Thomsan' fentre de Recherches sur le Polymorphisme GCnbhque des populations humaines, Toulouse Cedex, France

Key words: murine mAb - W polymorphism - HLA serology - HLA translectants Received 10 November. revised. accepted for publication 14 December 1992

mAb was selected and found to react against the B-LCL BON (DR103, DQS(l), DPw4), but neither against the DR103 transfectants nor the un- transfected L-cells. The binding of OHA TM904 mAb to the B-LCL BON, DR103 transfectants, DQ6( 1) transfectants and DPw4 transfectants was tested by cytofluorometry. The OHA TM904 mAb clearly reacted with the B-LCL BON (Fig. 1A) and the DQ6(1) transfectants (Fig. IC), but did not recognize the DR103 transfectant (Fig. 1B) or the DPw4 transfectant (Fig. lD), suggesting that this mAb, OHA TM904, recognized a determinant of the HLA-DQ molecules. The specificity of this mAb was further analyzed on a panel of 34 human B-LCL using micro-ELISA assays. The OHA TM904 mAb was found to be polymorphic for DQ molecules since it bound to all cells expressing HLA-DQ5( l), -DQ6( 1) or -DQ4 antigens (Table 1). This mAb was also very weakly reactive with DQ8(3)-positive cells (< 10"!0 binding), as was re- ported during the 11 th International Histocompat- ibility Workshop (IHW) (4). The binding of the OHA TM904 mAb was also tested by micro-ELI- SA on a panel of HLA class-I1 transfectants (19 HLA-DR, 3 HLA-DQ and 2 HLA-DP transfec-

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Walsh et al.

A : LCL BON (DR103, W 5 , DPw4)

T- 12 1 T+

oHATM804 1 I - -

C : DQ6 Transfectant T-

B : DR103 Transfectant

1 T-

T*

L D : DFW4 Transfectant

T-

T*

Figure 1. Flow cytometry analysis of the OHA TM904 binding to various cell lines. The cells used are: panel A, the 6-LCL BON (DR103, DQS(l), DPw4); panel B, the DR103 transfectants; panel C, the D Q q I ) transfectants; panel D. the DPw4 transfectants. The control antibody, for the B-LCL, the DR and DQ transfectants, was the anti-DR. DQ 9.3.Fl0 mAb and, for the DP transfectant, the anti-clw-I1 IVA12 mAb. As a negative control, FIX-rabbit anti-mouse Ig was w d alone. The histograms plot cell number against fluorescence intensity on a logarithmic scale; 1OooO cells were analyzed for each plot by a Coulter Electronic Cytometer (Coulter Epics C).

tants) obtained from the 11 th IHW. It did not bind to any DR or DP transfectants but it strongly reacted with DQ6( 1) and DQ4 transfectants (Table 2). All these results show that the OHA TM904

mAb (IgG1, not cytotoxic) recognized the DQS( l ) , DQ6(1) and DQ4 molecules.

In a previous report, biochemical analysis of the OHA TM904 mAb had shown that this mAb

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Brief communication

Table 1. Reactivity' of OHA TM904 mAb with B-LCL'. using micro-ELISA

OHA dass-ll Phenotype

V 10th B-LCL ws DR w DP TM904

HOM2 JESTHOM ROU BB SER F. SER C. OROU AMAL WJR076 BOU DEM BOR cox DUCAF

DEU YAR PE l l7 JHAF SP00l 0 EM21 BM1 6 KOSE EMJ054 ABO AMAIA OBB WIN ALB MAN BTB MADURA OKB SMI

aBL

9005 1 9004 1

1 103 10317 103I7 103

9010 15(2) 901 2 16(2)

2 9007 214

214 9022 17(3) 9019 17(3) 9020 17(3) 9025 4 9026 4 9028 4 9030 4 9036 l l ( 5 ) 9043 l l ( 5 ) 9038 12(5) 9056 13114 9097 13(6) 906 1 146) 9064 146) 9052 7 9095 7

7 7

9067 8 9069 8 9075 9

711 0

W ) - 8 1 4 8 1 4 8

4 8 - 1

Y l ) 213 112 - 8 1 - 8 6(1) , - 8 6(1) 4 8 1 214 8 518 314 8 1 I 3 4 8 2 3 1

1 2 2 2 1 7(3) 4 1

4 1 1

8(3) 8(3) 7(3) 3 1

2 8 1

5(1)

1 7(3) 7(3) 2 Y l ) 2 8 6(1) 314 8

4 8 1

Y l ) 7(3) 9(3) 4 1 219 4 1 2 1 1 2 2 1 4 4 6 4 4 6 9(3) 4 1 112 4 8

-

-

-

Results are scored as fdlows: 1: < 11% binding compared to the binding of the antiDR.W 9.3.FlO mAb; 2: 11-20% binding: 4: 2 1 4 0 % binding; 6: 41-8096 binding; 8 > 80% binding. ' The B-LCL were obtained from the 10th IHW or were locally derived (7.

immunoprecipitates the a- and P-DQ chains as well as the associated intracellular invariant chain (5) . Immunoblotting experiments were performed with cellular extracts from the B-LCL BON (DR103, DQ5, DPw4) and the patterns obtained were com- pared to those of the anti-PDQ monomorphic Tu22 mAb (6, 10). The OHA TM904 mAb reacted with the a/ p-DQ dimer, in non-denaturing conditions where the u/p complex was not dissociated, as well as the control Tu22 mAb; in denaturing conditions ( 5 min, lOOOC) where the u and p chains were separated, the OHA TM904 mAb recognized the isolated p-DQ chain in the same way as the anti- DQ Ti22 mAb (data not shown). These data indi- cate that the OHA TM904 mAb recognizes a poly- morphic epitope present on the P-DQ chains.

In order to determine the possible amino-acids

Table 2. Reactivity' of OHA TM904 mAb with transfectantsD. using micro-ELISA

Transfectants No 11th WS OHA TM904

L 8400 OR1 01 81 02 DRlO3 81 04 OR1 501 81 09 DR301 8112 DR401 8114 OR402 8116 DR403 8117 DR404 8118 OR405 81 20 DR406 81 22 OR403 81 24 OR1 1 81 25 OR701 81 29 OR801 81 30 OR1001 81 31

DRB4'0101 8137 DRBS0101 8106 ORBS01 02 8110

8202 wA1'0103

8204 DQA1.0201

8206 OQA1.03

8301 DPBl'0201 OPE1 '0401 8303

DRB3'0201 a1 34

wB1'0201 } DaB1'0401 }

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 a

1

6

1 1

Results are scored as fdlows: 1: < 11% binding compared to the binding of the anti4R.W 9.3.FlO mAb: 2: 11-20% binding; 4: 2140% binding: 6: 41 -80% binding; 8 > 80% binding. Transfected L-cells were obtained from the 11 th IHW

which could correlate with the specificity of the OHA TM904 mAb, we compared the sequences of the reactive p-DQ chains to the non-reactive p-DQ chains. We found that the amino-acids glycine- arginine (G-R) at position 54-55 of the D l domain of the p-DQ chains are present on the molecules recognized by the OHA TM904 mAb (DQ4, 5, 6) and absent on the non-reactive DQ molecules (DQ2, 3) (Fig. 2). These findings suggest that resi- dues G-R at position 54-55 of the p-DQ chains could influence the reactivity of the OHA TM904 mAb.

It is interesting to note that alleles of the HLA- DQ molecules are those most clearly defined sero- logically; in fact, more than 30 polymorphic HLA- DQ mAb cover the full range of DQ specificities, some of them being monospecific (7). Moreover, several other mAb have been previously described, having similar specificities as OHA TM904: TAL 1.2, SHI (9W901), SH2 (9W927), KSll (10W3091), TSU 802 (lIWO856), TSU TM901 (ALlDQ61.1. l), IKE (1 1 W0857), FER TM903 (1B6) (4, 6, 7, 8). One of them, TAL 1.2, has been reported as reacting with the P-DQ chains, in

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Walsh et al.

DQBl '0501 DQB1.0502 DQBl 05 03 DQB1.0601 DQB1.0602 DQB1.0603 DQBl'O604 WBl'OZ01 DQBl.0301 DQBl'0302 0481.0303 DQB1'0401 DQBl*O4OZ

OEA a 9 0 4

1 10 20 30 40 s ! O 70 80 90

Figure 2. Primary sequences of the f31 domaii of the &DQ chains. The residues correlating with the specificity of OHA TM904 mAb are boxed. The standard one-letter amino-acid code and the WHO official HLA nomenclature for naming the alleles are used (9).

Western blot, as well'as the OHA TM904 mAb (6). These data suggest that the epitope(s) recognized by these mAb, present on DQ1 and DQ4 mol- ecules, are immunogenic for mice and quite access- ible to mAb binding.

We have reported here the production and char- acterization of an IgGl murine mAb, OHA TM904, that recognizes a polymorphic determi- nant of the P-DQS( I), -DQ6( 1) and -DQ4 chains. Analysis of the p-DQ chain sequences suggests that residues glycine-arginine at positions 54-55 of the P I domain influence the reactivity of the OHA TM904 mAb.

Acknowlidgments

We thank H. Brun and G. Cassar for immunofluor- escence analyses and F. Moro for technical help. S . W. had a fellowship from INSERM and Fondation pour la Recherche Medicale.

References

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1988: 57: 991-1028.

4. Inoko H. Bodmer JG. Heyes JM, Drover S, Trowsdale J. Marshall WH. Joint report on the transfectant/monoclonal antibody component. In: Tsuji K, Aizawa M. Sasazuki T, eds. HLA 1991, vol. I . Oxford: Oxford University Press,

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biol 1986 171: 77-92. Address: Dr Anne Cambon-Thomsen Centre de Recherches sur le Polymorphisme Gcnctique des populations humaines CNS UPR 8291, CHU Purpan 31300 Toulouse Cedex France Telephone: 61.15.84.00 Telefax: 61.49.90.36

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