HLA-DPp residue 69 plays a crucial role in allorecognition

G. Diaz M. Catalfamo M.T. Coiras A.M. Alvarez D. Jaraquemada C. Nombela M. Sanchez-Perez J. Arroyo

Key words: allorecognition; epitopes; HLA-DP antigens; polymorphism: sitedirected mutagenesis

Acknowledgments: This work was supported by Grant SAF 97-0134 from the Cornision Interministerial de Ciencia y Tecnologia (ClCVr) from Spain, Grant BIOMED 3063 from the European Union and in part by Grant 94/0987 from the Fondo de lnvestigaciones Sanitarias (FIS) of the Spanish Ministry of Health. We would like to thank Dr. Marshall for providing monoclonal antibodies, Dr. E. Long for providing cDNAs and Rosa Perez, Maria Isabel Garcia and Amalia Vazquez for technical work. Gema Diaz has a fellowship from the Cornunidad Autonoma d e Madrid (CAM). Maria T. Coiras and Marta Catalfamo are MEC and FIS fellows respectively.

Received 5 January 1998, revised, accepted for publication 23 April 1998

Copyright ,B Munksgaard 1998 Tissue Antigens. iSSN 0001.2815

Tissue Antigens 1998: 52: 27-36 Printed in Denmark. Ail rights reserved

HLA-DPP residue 69 plays a crucial role i n a I lo recogn i t ion

Abstract: To investigate the conhibution to allorecognition of the individual polymorphic positions Glu 69 and Val 36 from the DPB1*02012 allele, DPB1*02012 cDNA was subjected to site-directed mutagenesis and alleles expressing Lys at 69 and Ala at 36 were generated. The lymphoblastoid cell line (LCL) 45.EM1, a previously generated mutant B-LCL which expresses normal levels of DPA mRNA but is not able to transcribe DPB, was transfected with wild-type or mutant DPB1'02012 cDNAs. The ability of two HLA- DPw2 alloreactive CD4+ cytotoxic T-lymphocyte (CTL) clones to lyse the panel of DPB1*02012 wild-type and site-directed mutant B-cell lines was tested. Both CTL clones (8.3 and 8.9) lysed the B-LCL 45.1, which is haploid for HLA and expresses wild-type DPB1*02012, and transfectants expressing Ala at 36 instead of Val, indicating that this polymorphic residue is not critical for T-cell recognition. However, the change of Glu to Lys at 69 prevented recognition by clones 8.3 and 8.9. These data demonstrate that the residue at peptide-binding position 69 is crucial for T-cell receptor recognition and suggest the requirement for a negatively charged residue at this position for allostimulation of these T-cell clones. The side chain of DPP- 69 is predicted to point into the peptide-binding groove, and the existence of positive (Lys) or negative (Glu) residues probably leads to substantial differences in the allo- or auto-DP-bound peptides or to differences in the conformation of the peptide-MHC complex, which would therefore be responsible for specific DPw2 allorecognition. The binding of a panel of monomorphic and polymorphic anti-HLA-DP monoclonal antibodies (mAbs) to these transfectants was also tested by flow cytometry. The changes at Glu 69 and Val 36 did not affect recognition by any of the monomorphic antibodies tested. However, the binding pattern of some of the polymorphic mAbs was clearly modified. Therefore, even though it is not crucial for T-cell allorecognition, polymorphic residue 36 must be involved in epitopes recognized by some polymorphic anti-DP antibodies, while residue 69 of the DPB molecule is crucial both for T-cell allorecognition and recognition by some mAbs.

The function of MHC class II molecules in the immune response is to bind peptides derived from foreign antigens and present them to T cells (1). Recognition of the allo-MHC by recipient T cells is one cause of allograft rejection (Z), a phenomenon that has been called allorecognition. Allorecognition involves a T-cell receptor (TCR) at

Authors' affillatlons:

G. Dlaz'. M. Catblfarno2. M.T. Coirasl. A.M. Alvare?. D. Jaraquernada'. C. Nombela'. M. Sbncher-P.5re~'.~, J. Arroyo'.'

'Departamento de Microbioiogla II. Facultad de Farmacia. Universidad Complutense, Madrid. Spain,

2Unitat d'lnmunologla. Departarnent de Biologia Celular i fisioiogia. Universitat Autdnoma de Barcelona. Bellaterra. Spain,

'Centro de Citometn'a de Flujo y Microscopla Confocal, Universidad Complutense. Madrid, Spain.

'Centro de Secuenciaci6n Automatizada de DNA. Universidad Cornplutense. Madrid. Spain

Correspondence to: Or. Javier Arroyo Deparlamento de

Microbiologla II Facultad de Farmacia Universidad Complutense 28040 Madrid Spain Tel: +34 1-3941746 Fax: +34 1.3941745 E-mail: jarroyo@ eucrnax.sirn.ucm.es

27

Diaz et a1 : Functional analysis of polymorphic HL4-DPP residues

the T-cell surface, the MHC molecule, and peptides bound to the groove of this MHC, at the stimulator side (3, 4). Evidence exists of two pathways for allorecognition. The direct pathway involves direct recognition of MHC molecules on the surface of stimulator cells by the T cells (5). In the indirect pathway, T cells recognize pro- cessed’ alloantigens by self-antigen-presenting cells (6). Peptides should play an important role in both pathways (7).

An important reason for clarifymg the mechanisms responsible for allorecognition is to open the possibility of investigating immuno- therapies capable of modulating T-cell responses (8) and transplantation tolerance (9). This approach should permit advances in preventing the rejection of organ allografts. From a basic point of view it is also important to characterize the mechanisms responsible for a phenomenon that has long been a puzzle to immunologists.

The recent description of the three-dimensional structure of the HLA class II DR1 and DR3 molecules complexed to influenza hemagglutinin peptide (HA307-319) (10) and CLIP 81-104 (11) respectively.has been crucial in the identification of DR residues critical for peptide binding and T-cell recognition. The relative functional importance of each MHC residue has been extensively addressed using an approach consisting of site-directed mutagenesis, gene transfer and in zityo functional analysis of the newly created alleles. Combination of structural data from class II peptide complexes, from the recently described a-P TCR (12, 13), or from class II-pep tide-TCR complexes, together with MHC class II functional analysis data, should permit a more precise understanding of the molecular basis of T-cell recognition.

The contribution of polymorphic residues of MHC class 11 molecules, especially HLA-DR, to antibodybinding epitopes (14), antigen-specific T-cell recognition (1517) and allorecognition (18, 19), mostly residues included in the peptide-binding pockets, has been extensively studied. DPBl alleles are less well characterized and most have been recently described using oligonucleotide typing and sequencing technology. Despite the knowledge gained from these sequences, little is known about the role and importance of the polymorphic residues as regards to the function of HLA-DP molecules. Examination of the amino acid sequences of the HLA-DPP chain in different alleles reveals the existence of several poljmorphic positions clustered into six regions (A: residues 8, 9, 11; B 33, 35, 36; C 55, 56; D 65,69 E 76; and F 84 to 87). Some authors have shown that in most cases a correlation exists between T-cell recognition and the presence of particular polymorphic DPBl sequences. These studies tested allospecific T-cell clones for the recognition of stimu- lators carrying different DPBl alleles. In these reports, several anti- DP T-cell clones with different specificities were generated and the recognition of cell lines expressing known DP alleles was used to define several DP alloepitopes (2C-22).

By site-directed mutagenesis and functional analysis, our group (23) and others (24) have defined individual residues of HLA-DP critical for the binding of polymorphic antibodies. Moreover, we have also studied some aspects of HLA-DP allorecognition by char- acterizing HLA-DPw2 allospecific T-cell clones. These studies allowed us to define HLA-DPw2-specific cytotoxic T-lymphocyte (CTL) clones that are able to lyse only some of the HLA-DPw2- expressing target cells, depending on the cell type expressing the DP molecule (25). This is probably due to the presentation of different peptides by DPB1*02012 within the context of different cellular background.

In the present work we used site-directed mutagenesis to evaluate the contribution of the polymorphic residues Glu 69 and Val 36 from DPB1*02012 to T-cell allorecognition by HLA-DPw2 specific cytotoxic human T-cell clones. Residues Glu 69 and Val 36 of DPB1*02012 were respectively changed to Lys and Ala (present in DPB1*0401), and the binding of polymorphic anti-DP monoclonal antibodies (mAbs) and lysis mediated by two HLA-DPwZ-specific cytotoxic clones, 8.3 and 8.9, was elevated. Our data show that both residues are critical for the formation of antibody-binding epitopes. Furthermore, the residue DPP-69 is crucial for recognition of the DPB1*02012 molecule by allogenic T-cell clones.

Material and methods

Cell lines and culture

Lymphoblastoid cell line (LCL) 45.1 was derived from the B-cell line 721 by mutagenesis with gamma radiation (300 rad) and selection with a class I-specific mAb plus complement, rendering a deletion of the short arm of one of the number six chromosomes, and therefore being a mutant haploid for HLA (remaining haplotype: A2,BS,DRl,DQurl,DPw2 (DPAI *0103, DPBl*O2012)) (26,n). LCL 127 was derived from LCL 721 using a similar procedure. It is also HLA haploid but the deletion occurred in the other number six chromosome. The remaining haplotype is A l , B8, Cw 7, DR3, DQw2, DPw4 (28). LCL 139 was derived by gamma irradiation of 45.1 and selection with anti-DR mAb L243. This cell line is DQwl, DPw2, but DR negative (28). LCL 45.EM1 was derived from 45.1 by ICR191 irradiation and subsequently selected by resistance to lysis by HLA- DPwZ-allospecific CTL clone 8.9 (25). This cell line is HLA-DPw2 negative (29). LCLs were cultured in RPMI 1640 medium (Gibco Lab- oratories, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories Inc, Middlesex, UK), 2 mL-glutamine, 100 U/ml penicillin and 100 pglml streptomycin. Cells were incubated at 37°C in a humidified incubator with 5% CO2.

28 Tissue Antigens 1998: 52: 27-36

Diaz et al : Functional analysis of polymorphic HLA-DPP residues

CTL populations

mA-DPw2-specific CTL clones 8.3 and 8.9 were used in the cytotoxicity assays. These CTL were cloned by limiting dilution from mA-DPw2-specific bulk reagents and were maintained in culture as described (30). Cryopreserved clones were expanded, after thaw- ing, by stimulation with HLA-DPwZpositive irradiated peripheral mononuclear blood leukocytes in media consisting of IMDM with 10% human serum, penicillin, streptomycin, and 15% phytohemag- glutinininduced T-cell supernatant. Cytotoxicity was assayed after 4-6 days of incubation at 37°C in 6% C02.

Cell-mediated lysis

Cytotoxicity was measured in a standard 51Cr release assay as previously described (30). In brief, effectors were incubated at 37°C for 4 h with 51Cr-labeled targets (5x103/well in duplicate wells) in a final volume of 150 p1 in round-bottom microtiter plates (Costar, Cambridge, MA). Spontaneous release was determined in the absence of effector cells and maximal release was determined in the presence of 5% Triton X-100.

DNA constructions and transfection

Full-length DPBl cDNAs (wild type and site-directed mutants) were subcloned by ligation of the Sma I-Hind III cDNA inserts into the plasmid pREP4 (Invitrogen corporation, San Diego, CA) previously digested with Pvu I1 and Hind 111. B-LCL 45.EM1 was transfected with these constructions by electroporation with a BTX-600 Electro Cell Manipulator (BTX Inc., San Diego, CA). Cells were washed twice in RPMI and resuspended at 3x1O6 cells per 250 p1 HeBS, pH 7.2, (20 m~ HEPES, 6 m~ dextrose, 0.7 m~ Na2HP04, 5 m~ KCI, 137 mM NaC1) on ice. They were then mixed with DNA (15-20 lg) resuspended in 20 p1 HeBS. The mix was pulsed twice for 30 s at 325-350 V, 50 pF and 360 a, maintained at room temperature for no more than 5 min, and plated onto 35 mm dishes with 2 ml of complete RPMI. After 48 h of culture, cells were assessed by flow cytometry for recovery of HLA-DP expression. Subsequently, stable transfectants were obtained by selection for 2-3 weeks in the presence of 300 pg of Hygromycin B (Sigma Chemical Co, St. Louis).

Sitedirected rnutagenesis

Mutagenesis of DPB1*02012 cDNA was carried out as previously described (23). Sma I-Xho I full-length DPP cDNA was inserted in MUmp19 and mutagenesis was performed using the Muta-Gene M13 in vitm Mutagenesis Kit (Bio-Rad, Inc., Richmond, CA) as de-

scribed by the manufacturers. Mutagenesis to change the codon for Glu 69 to Lys has been described (23). The primer used to change the codon for Val 36 to Ala was: 5'-GTG CAG CGA CAG C T T CGC __ GCG C T T GAG GAG GGC CAA CAT-3' (mismatches with the wild- type DPB1*02012 sequence are underlined). The mutations were confirmed by DNA sequencing on an automated DNA sequencer (ALF, Pharmacia), using the Autoread Sequencing Kit supplied by the manufacturer.

Monoclonal antibodies

The mAbs used in this work and consensus specificities were B7/ 21: DP monomorphic (31, 32); ILRI: DPw2, 3, 4.2, DR5 (33); DP1l.l: DPw2, DPw4 (34); NFLD.M58 DPB1*0201, 0301, 0402, 0601, 0901, 1001, 1401, 1601, 1701, DRB1*1101 (35); NFLD.M60: DPB1*0201, 0301, 0402, 0601, 0901, 1001, 1401, 1701, DRB1*1101 (36); NFLD.M63: DPB1*0301, 0402, 0601, 0901, 1401, DRB1*1101 (36); NFLD.M66: all DPBl* except 0202, 0401, 0402 and 1501; weak on DPB1*02012 (36); NFLD.M68: DP monomorphic (36); NFLD.M69: DP+DR monomorphic (36, 37); NFLD.M70 DPA1*0201 (36); NFLD.M73: DPB1*0101, 0201, 0301, 0402, 0601, 0901, 1001, 1401, 1601, 1701, DRB1*1101 (w. Marshall, personal communication); NFLD.M75: DPB1*0301, 0402, 0601, 0901, 1401, DRB1*1101 (M? Marshall, personal communication); NFLD.M77 DPB1*0301, 0601, 0901, 1001, 1401, 1701 (33); and NFLD.Ml01: DPB1*0201, 0301, 0402, 0601, 0901, 1001, 1401, 1601, 1701, DRB1*1101 (w. Marshall, personal communication).

lmmunofluorescence staining, flow cytometric analysis

(FCA) and data processing

Recognition by mAbs was measured by indirect immunofluor- escence staining: 2x1O6 cells were incubated with the specific mAb for 30 min at 4"C, washed three times with phosphate-buf- fered saline (PBS), and then incubated for another 20 rnin with fluoresceinated goat antimouse Ig (G, A, M) (Sigma Immunochem- i d s , St. Louis, MO). Cells were then washed at least three times with PBS and diluted in 300 p1 of the Same buffer plus 0.005% of propidium iodide in order to discriminate dead cells. FCA was done on a FACScan flow cytometer (Beckton-Dickinson, San Jose, CA). 10,OOO cells were acquired per sample and all of the analysis was carried out using propidium iodide-negative cells. B7/21 was chosen as the reference antibody for comparison with the others. The staining of each transfectant with each mAb was expressed as a percentage relative to B7/21 using the following formula: (MCF for mAb-MCF for negative control)/(MCF for B7/21-MCF for negative control), where MCF is mean channel fluorescence.

Tissue Antigens 1998: 52: 27-36 29

Diaz et a1 : Functional analysis of polymorphic HLA-DPP residues

Results

The functional role of Glu 69 and Val 36 of the DPB1*02012 chain in allorecognition was examined by testing the ability of transfected B cells expressing mutated HLA-DP molecules to be recognized by two HLA-DPw2-allospecific cytotoxic T-cell clones. Binding of these transfectants to a panel of anti-DP polymorphic mAbs allowed US

to evaluate the participation of these residues in the epitopes recog nized by these antibodies.

DPB1*0201 cDNA was subjected to site-directed mutagenesis, changing the residues Glu 69 and Val 36 in the DPB1*0201 chain to Lys and Ala. Lys and Ala are found at the corresponding positions in the DPB1*0401 allele, which is not recognized by HLA- DPwZ-specific T-cell clones. LCL 45.EM1, a B-LCL that we have previously characterized, was used as recipient for the transfection experiments. This cell line expresses normal levels of DPA mRNA but is not able to transcribe DPBl(29). The advantage of using this cell line is that only the DPBl cDNA is required to express normal levels of HLA-DPw2 (DPA1*0103/DPB1*0201).

Transfectants expressing wild-type (45EMl/DPB1*02012) or site-directed mutant HLA-DP molecules (45.EMl/DPB1*02012-K69 and 45.EMl/DPBl*02012-A36) were obtained by transfection of the 45.EM1 cell line with wild-type or mutant DPB1*02012 cDNAs. Cells expressing comparable high levels of HLA-DP molecules, measured by FCA of the binding of the monomorphic anti-HLA-DP B7/21 mAb, were selected.

100

80

60 cn

s ?

40

20

0

b

6/ 1 3/ 1 1/1 0.3/1 . U T

The functional involvement of residues Glu 69 and Val 36 of the the DPP chain (DPB1*02012) in T-cell recognition was examined by assessing the ability of these transfectants to be lysed by two different HLA-DPw2-alloreactive cytotoxic T-lymphocytes, 8.3 and 8.9 (25, 30). As shown in Fig. 1, both CTL clones were able to lyse the HLA-DPw2 B-cell line 45.1 (haploid for HLA and expressing HLA- DPBlW2012) and transfectant 45.EMl/DPB1*02012 (wild type), but were unable to lyse B-cell line 127, which is haploid for HLA and expresses DPB1*0401. However, clone 8.3 did not recognize cell line 139, as we previously described (25), even though it is HLA-DPw2 positive. Lysis of the the DPBlW2012 wild type and site-directed mutant transfectants revealed that HLA-DPP residue Glu 69 is es- sential for allorecognition mediated by clones 8.3 and 8.9, since none of the clones were able to lyse transfectants expressing mutant HLA-DPP molecules with Lys instead of Glu at this position (Fig. 1). These results suggest a role for the charge of this residue (Lys positive or Glu negative) in HLA-DP allorecognition. The broad effect on T-cell recognition caused by the Glu to Lys substitution cannot be explained by a great alteration in the conformation of the DPP chain, since recognition by all the monomorphic and several polymorphic anti-DP mAb tested (as shown below) was not affected by the mutation. However, cells expressing Ala at 36 were lysed, as well as 45.1 or 45.EMl/DPB1*02012 (both expressing the DPw2 wild-type molecule). Therefore, residue Val 36, in spite of being one of the four polymorphic positions of the P l domain different for DPB1*02012 and DPB1*0401 alleles, and of being allocated in the floor of the putative peptide-binding groove, is not crucial for DP

45.1 -I- 127 * 139

45.EMl 45.EMl/DPB-K69 45.EMllDPB-AS

Y

9/1 311 1/1 0.311 E/T

Fig. 1. Cytotoxic reactivity of HLA-DPw2-specific alloreactive CTL clones 8.3 (Panel A) and 8.9 (Panel B) for B-cell lines expressing i) wild-type HLA-DPw2 (DPA1*0103/ DPBl*02012): LCLs 45.1, 139 and 45.EMl/DPBl*02012; ii) wild-type HLA-

DPw4 (DPB1*0401): LCL 127; or iii) mutant HLA-DPw2 (DPA1*0103/ DPB1*02012-A36 or -K69) molecules. E/T effector to

target ratio.

30 lissue Antigens 1998: 52: 27-36


allele weakly although previous consensus specificity did not in- clude this allele.

The binding of other polymorphic mAbs was clearly modified by the mutations. The DPP-36 Val to Ala substitution increased the binding of NFLD.M60 (Fig. 3). Staining of the HLA-DPw2 wild-type molecule with this mAb was 60% of that of B7/21 staining, while the Val to Ala change increased binding to 200%. A similar effect was seen with NFLD.M66 and M70. Although for these mAbs binding to the wild type was 30% and 20% of the B7/21 binding, respectively, Ala at 36 instead of Val doubled this binding to 60% (for NFLD.MG0) and 40% (for NFLD.M70). For NFLD.M63, however, Ala

15% to the mutant transfectant) (Fig. 2 and 3). These data show that residue Val-36 of HLA-DPB1*02012 is important for the epitope recognized by these antibodies, although probably not as a sole component since mutation did not cause any total loss or gain in antibody binding.

plays an important role in the epitope recognized by mAbs NFLD.M63, M66, ~ 7 0 and M.75 (Fig. 2 and 3), since in all cases binding was clearly increased by the change from Glu to Lys at 69. This effect was higher for NFLD.M63 and M75, in which binding related to B7/21 increased from 30% and 40% in the wild type to 14&150% in the mutants (Fig. 2 and 3).

+ 0 - ’ $ $ 3 ? 2 8 E 5 % 2 f 36 produced a decrease in mAb binding (30% to the wild-type and n 5 5 p 5 4 5 5 5 2 G 2 2 2 2 2 2 2 2 2

ANTI-HLA-DP mAb Fig- 2* Binding Of the panel Of monomo@ic and ~ l ~ o ~ h i c anti-HLA-DP mAbs to transfectants expressing wild-type, DPB- K69 or DPB-A36 mutant HLA-DP molecules (DPAl*0103/ DPB1*02012). The staining of each transfectant is expressed as a percentage relative to B7/21 mAb by the following formula: (MCF for mAb-MCF for negative conkol)/(MCF for B7121-MCF for negative conh-oh as ex. plained in Material and methods. Data represent means S E M .

FCA Of mAb binding showed that DPP-K69

allorecognition, at least for the tested HLA-DPw2-allospecific CTL clones.

Clone 8.3 did not recognize cell line 139, as we previously described (25), in spite of the fact that it is HLA-DPw2 positive, probably because of differences in the peptide-MHC complex recognized

Discussion

by each clone. No differences were found, however, in the pattern of recognition of 45.EMU DPB1*02012, 45.EM1/ DPB1*02012-K69 or 45.EM1/ DPB1*02012-A36 transfectants by both CTL clones, 8.3 and 8.9.

The binding of a large panel of anti-DP mAbs (see Material and methods) to the wild-type and K69 and A36 DPB1*02012 bansfec- tants was also tested. This permitted us f i s t to rule out the possibility of a great alteration in the molecule as a consequence of the mutations and, secondly and more importantly, it allowed us to map epitopes for antibody binding. FCA of the binding of these mAbs to the transfectants revealed, as shown in Fig. 2, that changes in Glu 69 and Val 36 did not affect recognition by the rnonomorphic mAbs for HLA-DP assessed (B7/21, NFLD.M67, NFLD.M68 and NFLD.M69) or by some polymorphic mAbs (ILRl, DP11.1, NFLD.M58, NFLD.M73, NFLD.M77 and NFLD.Ml01). B7/21 was chosen as the reference antibody and data are shown as binding Percentages compared to binding of this mAb (as indicated in Ma- terial and methods). Our data revealed that polymorphic antibodies mLD.M63, M66, M70, M75 and M77 recognized the DPB1*02012

We have evaluated the importance of polymorphic residues 36 and 69 of the DPP chain in allorecognition. The data reported here indi- cate that residue 69 of DPB1*02012 plays a crucial role in HLA- DPw2 allorecognition, since replacement of residue Glu 69 to Lys completely abolished allorecognition by two different HLA-DPw2- spedic T-cell clones, whereas a change of polymorphic DPP 36 did not affect T-cell recognition. Moreover, we have demonstrated that both residues are involved in the epitopes recognized by several polymorphic DP r d b s .

From the sequences of the alleles recognized by these mAbs, binding epitopes have been previously tentatively identified (35,37). For NFLD.M63 and M75 the proposed epitope was DE at 5556, and AV at 86-87 for NFLD.M66. Our results clearly suggest that, although not as the sole component and probably with DE at 55-56 or AV at 86-87, the charge of residue 69 is critical for the epitopes recognized by these antibodies. For NFLD.M70, the proposed epitope was ascribed to the DPA1*0201 chain (37). Our data demonstrate that polymorphic positions 36 and 69 on the DPP chain also



B7121

NFLD.M66

NFLD.M60

NFLD.M70

NFLD.M63

NFLD.M75

FLOURESCENCE INTENSITY

Fig. 3. Flow cytometric histograms of the binding of polymorphic mAbs NFLD.MG0, M63, M66, M70 and M75, whose binding is affected by the mutations, to the wild-type (45.EMll DPB1*02012) (WT) and mutant HLA-DP transfectants (45.EM1/

DPB1*02012-A36 and 45.EMlIDPB1*02012-K69). Histograms of the staining of these transfectants to the reference rnonornorphic mAb B7/21 are also included. Controls correspond to the non-specific binding of the FITC rabbit antirnouse Ig.

influence this recognition. A similar effect has previously been described for other anti-DP mAbs (34). The tentative epitope proposed for NFLD.M77 (37) included L11 and FV 3536. Our results clearly demonstrate, in contrast to previous reports, that V36 is not involved in the epitope recognized by this mAb. Finally, residue 36 is

part of the NFLD.M6O epitope, probably with DE 55-56, which was previously identified as the epitope recognized by these mAbs by comparing sequences of the alleles recognized by this antibody.

When compared to the three-dimensional structure of the class I1 histocompatibility HLA-DR1 (38), residues 69 and 36 of HLA-DPP

32 Tissue Antigens 1998: 52: 27-36


correspond to residues 71 and 38 of DR1, because DPP has a deletion following amino acid 23 relative to the DRP chain.

Although the exact structures of DR1-HA 306-318 (10) and DR3- CLIP 81-104 (11) complexes have been established, the structure of the DP molecule is not known. Considering the alignment of polymorphic and conserved residues between DR and DP, and taking into account that peptides isolated from DP2 (39) are similar to those isolated from DR and DQ in length and properties, it is possible to model a putative peptide-binding site for DP2 based on known DR structures. Very recently, Chicz et al., studying self-pep tides bound to DP2, and modeling DP on the DR molecule, described a hypothetical peptide-binding site for DP2 (39).

The crystal structure of the DR1-HA (10) and DR3-CLIP (11) complexes demonstrate that both residues, DRP-71 and DRP-38, are important for peptide binding. There are five pockets in the DR1 molecule that accommodate the side chains of the HA 3M-318 p e p Fig. 4. Location of the residues DPP-36 and DPp-69 in the hypo-

~~

tide in the DR1-HA peptide complex. Arg p71 is part of pocket 4 and pocket 7 within the DR1 peptide-binding site (10). Both pockets accommodate peptide side chains 4 and 7 (numbering the N-ter- minal anchor residue in position Pl). DRP-71 also contacts the CLIP peptide in pockets 6 and 7 of the DR3-CLIP complex. Furthermore, Dessen et al. have recently described the X-ray crystal structure of DR4 complexed to a peptide from human collagen II protein (40). Details of a predicted salt bridge between DRP-71 and aspartic acid at P4 peptide position suggest that this residue participates in both peptide binding and TCR activation. The charge of this amino acid in the peptide-binding site appears to be critical in discriminating DR molecules linked to increased susceptibility to rheumatoid arthritis. These data should support the hypothesis that the charge of the equivalent DPP-69 position plays an important role in allorecog nition. DRP-38 does not contact HA peptide in DR1-HA complex, but does contact CLIP in the DR3-CLIP complex at pocket 9. Fur- thermore, this residue, as suggested by Chicz et al. (39), may contact peptide in DP2-peptide complexes as part of pocket 7.

Modeling the DP molecule on DR, DPP-69 (equivalent to DRP- 71) should be located in the a-helix of the DPP chain with the side chain poin,ting into the peptide-binding groove (Fig. 4). It may par- ticipate in the formation of the peptide-DP complexes by interacting with the peptide at pockets 4 and 7, and possibly interacting with the TCR. DPP-36 (equivalent to DRP-38) may interact with peptides at pockets 7 and 9.

In view of the location of residues 69 and 36 of the DPP molecule, the role of these amino acids would be to determine the binding of peptides that are directly or indirectly involved in the epitope. The fact that residues in the peptide-binding groove determine antibody binding through their influence on the binding or orientation of peptides has been shown previously (41). Using mouse L-cell trans-

thetical three-dimensional structure of the HLA-DP molecule. Posi- tions DPP-36 and DPP-69 correspond to residues 38 and 71 of DR1 as a consequence of a deletion in DPP following amino acid 23 relative to the DRB chain.

fectants, we reported that the DPP-69 Glu to Lys substitution abol- ishes the binding of M66 to this transfectant (23). Interestingly, in B cells the effect was the opposite, probably due to differences in the peptides presented by these two cell lines. Previous reports have also shown differences in the recognition of HLA class I1 molecules by the same mAb depending on the cell background (37).

Consistent with our T-cell allorecognition data, mutations at position 71 of HLA-DRP, corresponding to DP position 69 of HLA- DPP, completely abolish recognition by several DR-allospecific T- cell clones (18, 19), as well as the DR-restricted recognition of a pertussis toxin peptide (42) and influenza hemagglutinin peptides (16).

The effect of the Glu to Lys substitution on T-cell allorecognition could be explained by: a) an alteration in the interaction between the DP-peptide complex and the TCR, or b) the involvement of this residue in MHC-peptide interactions. The effect on peptide binding could be exerted on: a) the peptide display, by changing the nature of the peptide presented or by preventing the binding of any peptide to the DP molecule, or b) a change in the conformation of the DP- bound peptide and therefore in the conformation of the DPB1*02012 molecule-peptide complex.

Three-dimensional structures of the TCR and its orientation in the TCR-self-peptide or viral-peptide MHC class I complex receptors have been described (12, 13). These authors reported a central role for V, and V, CDR3s in peptide recognition and CDRs 1 and 2 contacting both the peptide and the a-helix of the MHC molecule.



However, although models for TCR-MHC class II complexes have been proposed (43, 44), only the fine structure of the crystal of this complex will permit us to know whether position 69 of DP is or is not directly interacting with the TCR.

Using site-directed mutagenesis on HLA-DR molecules, studies by Signorelli et al. (16) and Zeliszewski et al. (17) evaluated the role of the DR7 and DRll 71 residues in peptide binding and antigen- specific T-cell recognition. They demonstrated that the change in charge at this position, despite being crucial for T-cell recognition, has relatively little impact on quantitative peptide binding. The role of position HLA-DP Glu 69 in peptide binding has not yet been evaluated. However, the above findings make the possibility of changes in the orientation or conformation of the peptide in the DP molecule more likely than decreased peptide-binding capacity as the phenomenon responsible for the abolition of HLA-DPwZ-specific T- cell allorecognition observed by us when the Glu to Lys change at 69 was introduced into the DPB1*0201 molecule.

Chiz et al. recently identified self-peptides bound to DP2 in B cells (39). Based on the assumption that DP could be modeled on DR, anchor residues were tentatively assigned by these authors using alanine-substituted analogs of these peptides. Residues F62, V57, S60 and L65 from’ peptide I1p 52-65 (derived from interferon- induced protein 1-8D) were predicted as anchor residues reaching into the hypothetical P6, P1, P4 and F’9 pockets respectively of the peptide-binding site of the DP2 molecule. As previously stated, DPP-69 should form part of pocket 4, and therefore could be crucial in the interaction between P4 residue (analog to S60 from peptide IIP 53-65) of an hypothetical allopeptide and the DP2 molecule. Although there are few cases in which allorecognition has been described as being independent of the peptide presented by the MHC (all0 or self) (45), in most alloresponses peptides must play an important role in the molecular mechanism responsible for the allorecognition of class II MHC molecules (7, 46). Our data suggest that this would also be the case for allorecognition of HLA-DP molecules, where the Glu to Lys substitution at DPB1*02012 could be involved in changes in the shape and charge of pockets 4 and 7 of the DP molecule that accommodates side chains of the presented peptide, leading to conformational changes in the peptide-HLA-DP- TCR complex that would be responsible for the lack of T-cell allorecognition.

In spite of the significant differences in the pattern of recognition mediated by T-cell clones 8.3 and 8.9, as deduced from the discrimi- nation in T-cell allorecognition of the cell line 139, neither of the HLA-DPw2-allospecific CTL clones were able to recognize the mutant DPa/DPP-K69 molecule. This suggests a pivotal role for the polymorphic position 69 in alloresponses.

Several reports have previously suggested the importance of Glu at 69 in allorecognition. Potolicchio et al. (47) showed that HLA- DPBl mismatches at position 69 are associated with high helper T- lymphocyte precursor frequencies in unrelated bone marrow transplant pairs. In a very recent report with a group of 19 potential bone marrow transplant recipients and 34 matched unrelated do- nors, Nicholson et al. (48) reported that alloresponses to HLA-DP detected in the primary mixed lymphocyte reaction in most cases correlates with the presence of Glu at DPP-69, although strong responses were also observed when DPB1*0301 (Lys 69) was the only difference in the stimulator cells. Our results clearly demonstrate and reinforce the previously suggested importance of DPP residue 69, the nature of the amino acid at position DPP-69 being critical for alloreactivity since only the change of Glu (negatively charged) to Lys (positively charged) is responsible for the lack of HLA-DPw2- specific T-cell allorecognition.

The absence of effects following substitution at DPB1*02012 residue 36 could be explained if this position is not in contact with the peptide presented by the DP molecule, in spite of being located inside the peptide-binding groove, or, in the event of it contacting the peptide, if substitution at this position has little or no effect on the determinants recognized by the TCR. The second explanation is more likely, since Chiu et al. (39) predicted this position interacting with a DP2 peptide. Our data support HLA-DP allorecognition, a fact previously described for DR-restricted T-cell recognition (15, 49): polymorphic residues on the floor of class II molecules affect T-cell recognition less frequently than substitutions in the a- helix. Thus, we could expect that polymorphic positions on the floor contribute to T-cell allorecognition to a lesser extent than residues in the a-helix.

Some reports have suggested the association of specific HLA-DP alleles and susceptibility to several diseases. Although the DQ antigen is the major determinant of susceptibility to celiac disease, the possibility has been reported that the charge of the amino acid at position 69 in DPP could play an important role in susceptibility to this disease (50). This position was also implicated in susceptibility to pauciarticular juvenile rheumatoid arthritis (51). Susceptibility to beryllium disease (52) and to “hard metal disease” (53) have been suggested to be specifically related to Glu 69 from DPB1*0201. Our results may shed new light on the involvement of HLA-DP-specific residues such as DPP-69 in these HLA-DP-associated diseases. Moreover, understanding the mechanisms that contribute to allorecognition should afford new ways to design peptide immunotherapy for autoimmune diseases and transplantation.

34 nssue Antigens 1998: 5 2 27-36

Diaz et al : Functional analysis of polymorphic HU-DPP residues

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HLA-DPp residue 69 plays a crucial role in allorecognition