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Both HLA-DR and HLA-DQ determinants contribute to HLA-Dw typing
Both HLA-DR and HLA-DQ Determinants Contribute to HLA-Dw Typing G. Sterkers, D. Zeliszewski, A. C. Freidel, L. Gebuhrer, H. Betuel, and J. P. Levy
ABSTRACT: The respoctivecontribution of HLA-DR and HLA-DQ genepreducts in the induction of allogeneicproliferative responses in primary mixed lympho.7~tereactionand, therefore, in HLADw typing, is still unclear or controversial. This is in part due to a stmng linkage disequilibrium betweenHI.A-DR and -DQ genes. We used DR- or DQ-restricted influenza-spuific T-cell clones to define DR and DQ products on a largepanel of aUogeneicantigen presenting cells. With this functionalscreeningassay, we identified two haplotypos upishunusual D R I l ~ associations. Cells of these haplotypes were then used as responderceils in mixed lymphocyteculture and stiraulated by homozygous typing cells displaying DR or DQ incompatibilities. Our results indicate that DR or DQ incompatibilities alone can give rise, in both cases, to strong T-cell proliferation in a mixedlymphecyte reaction. This was further verifiedby blockingexperimentsof secondarymixed lymphocyte reactions by HLA-spocifie monoclonalantihulies. Anti-DQ, but not anti.DR, antibodies inhibited DQ-incompatible responses. Conversely, anti-DR, but not anti-DQ, antikuties could block DR-incompatible mixed lymphocyte r~zctions. Together, the results suggest that both HLA-DR and DO. gone products can be involved in HLA-Dw typing. Finally, in dual DRand DQ-incompatible mixed lymphocyte reactioncombinations, HI.A-DR moleculesseem to have an immunodominant effect, because the response is mostly inhibited by anti-DR antibodies. lmmunodominance of HI,A-DR allodeterminants may, at least in part, explain some of the controversial conclusions reportedby others concem~ingthe role of HLA-DQ moleculesin HLADw typing. ABBREVIATIONS APC antigen presenting cells PBM peripheral blood roononuclear cells IL2 interleukin 2 MLR mixed lymphocyte reaction MoAb monoclonal antibody
HTC 3H-TdR 2-D-PAGE DNV
homozygous typing cell Tritiated thymidine two-dimensional polyacr,/lamide gel electrc~ phoresis double normalized value
INTRODUCTION Human class II antigens of the major histocompatibility complex (MHC) are highly polymorphic heterodimers encoded by genes of the human leukocyte antigen (HLA)-D region on chromosome 6. At least three sets of molecules
Fromthe Laboratoired'lmmunologieet Virologicdes Tumeurr, Inserm U 152, CNR$ UA 628, HOpital Cochin,27, rue du FaubourgSaintJacques, 75014 Paris,FrancefG.S., D.Z., J.P.L.) and the Laboratoire ¢?Hi~tocompatibilit#,CentreR~gionalde TransfusionSanguinede Lyon, Lyon, France(A.C.F., L.G., H.B.). Address reprints requeststo Dr. G. Sterkers, Laboratoired~lm~unologieet de Virdogiedes Turnouts, lmerm U152, CNRS UA628, HOpitalCochin,27, rue du FaubourgSaintJacques, 75014 Paris, France. ReceivedOctober27, 1986;acceptedAugust 3, 1987. HumanImmunology20, 233-247 (1987) © ElsevierSciencePublishingCo., Inc., 1987 52 Vanderbi/¢Ave.,New York,NY 10017
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G. Sterkers etal. encoded by .genes of HLA-DR, -DP, and -DQ subregion, respectively, have been identified within the HLA-D region of the MHC, based on serological, functional, and biochemical evidences. Serologically defined antigenic specificities have been assigned to different HLA-D gene products, i.e., DR1 through DRw14 on one of the DR molecules, and DQwl through DQw3 on D Q molecules. Hence, HLA-D molecules can be characterized, at least in part, using specific antibody reagents (see review [1-3]). Class lI MHC antigens are also known to induce the proliferation of alloreactive T lymphocytes in mixed lymphocyte culture. Therefore, HLA-D typing can be routinely achieved by measuring the proliferation of donor cells being typed when cultured with a panel of commonly accepted HLA homozygnus typing cells (HI'C). This mixed lymphocyte reaction (MLR) defines HLA-D specificities designated Dwl through Dw19 [4,5]. The Dw specificities are subtypic to the serologically defined DR and DQ antigens, in that several Dw specificities can be associated with the same serological specificity. Although HLA-DR, -DP, or .DQ molecules have been shown to induce the proliferation of alloreactive Tcell clones [6,7], their respective contribution in the induction of primary MLR remains controversial. The HLA-DP incompatibility is believed not to induce primary MLR reactivity [7]. In contrast, it is generally accepted that DR molecules are the major antigens involved in this functional assay. Finally, contradictory results have been reported concerning the role of D Q molecules [6,8-11]. Many experiments suggest that they may play a significant role in primary MLR [6]. However, it has been recently proposed that D Q antigens per se cannot stimulate T cells in primary cultures [8]. Moreover, a negative regulation of the T-cell proliferation in MLR by DQ molecules has been proposed [11]. On the whole, the DR and/or DQ molecular basis for MLR-defined HLA-D differences remains unclear. Two major difficulties must be circumvented to answer this question: (1) the poor definition of class II molecules by conventional typing methods that do not allow the assumption ofwbetber class II antigens are shared by unrelated individuals or not; (2) a strong linkage disequilibrium between DR and D Q genes, such that most DR-identical unrelated individuals are also D Q identical. Hence, combination pairs of responder/stimulator cells in MLR differing in either DR or D Q molecules are the exception. In our experimental approach to investigate the role of DQ molecules in Dwtyping, we used influenza-specificT-cell clones restricted to DR or D Q molecules to define HLA-D specificities on a large panel of donor cells used as antigen presenting cells (APC). Among this panel, we isolated two APC donors exhibiting an unusual DR/DQ association. Cells from these donors were used as responder cells in primary MLR and were stimulated by HTC presenting DR or D Q incompatibilities. In each case, the set of molecules involved in these proliferative responses was assessed by testing the inhibitory effect of different HLA-specific monoclonal antibodies (MoAb) in secondary MLR. The results lead to the conclusion that (1) both HLA-DQ and HLA-DR may be involved in primary MLR and, therefore, contribute to HLA-Dw typing; (2) HLA-DR seems to be immunodominant in combination pairs of responder/stimulator cells differing for both DR and DQ specificities.
MATERIALS A N D M E T H O D S Peripheral Blood Mononuclear Cells The peripheral blood mononuclear cells (PBM) were isolated from blood samples as previously described [12]. The HLA phenotypes v:ere determined according
HLA-DR and HLA-DQ Contributions to HLA-DwTyping
235
TABLE 1 Monoclonal antibodies used in this paper Name L243 L227 Genox 353" BT3.4 B7.21.I w6.32
Specificity
Reference
DR DR + DP
14 14
DQwl DQwI and DQ moleculesassoicatedto DR.4and w8
15 16
DP HLA-A,B,C
17 18
"Genox3.5l, whichrecognizesDQwl-positivemolecu/es,wasonlyusedwhenstimulatorcellshadthisspec~cit~. to methods described elsewhere [ 13] by using local reagents and reagents defined by the 9th International Histocomp~tibility Workshop. Influenza Viruses Influenza A/Texas and E/Singapore viruses were kindly provided by Dr. C. Hartnoun, Pasteur Institute, Paris [12]. Monoclonal Antibodies Anti-HLA MoAb used in the experiments to inhibit secondary MLRs are described in Table 1. These MoAb were used as ascitic fluids. Their specificity has been extensively described elsewhere [14-18]. Culture Medium Cells were cultured in RPMI 1640 supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, 2 x 10"s bem-mercaptoethanol, antibiotics, and 5% (v/v) heat-inactivated pooled human AB serum as previously described [12]. T-Cell Clones Influenza AfI'exas virus-specific T-cell clones were used to define HLA-D functional molecules on a large panel of donor's PBM used as A.PC.These proliferative clones were obtained as previously described [ 12]. Their specificity and restriction have been extensively documented elsewhere [19-21]. Their main characteristics are indicated in Table 2. T-Cell Clone Proliferation Assay T-cell clone proliferation assay was performed as previously described [ 11]. Briefly, 1 x 104 washed cloned cells were cultured with I x 105 irradiated (4000-rad) PBM as APC previously infected with the influenza A/Texas virus. Cultures were made in 0.2-ml round bottomed microculture plates and were incubated for 3 days at 37°(; in 5o~ CO2 atmosphere. Cell proliferation was estimated by adding 0.8/zCi of tritiased thymidine (3H-TdR, 5 Ci/mmol) to each well for the last 6 hr. Cells were then harvested, and the radioactivity was incorporated in DNA counted.
256
G. Stefkers et al. TABLE 2 T-cell clone characteristics Restricting
Phenotype of efficient
phenotype~
Clones
molecule; b
allogeneicAPO
References d
DR2short/DQwl/DwFJO
Illg Illa ie lh
DQ DQ DR DR
DwFJO DwFJO DwFJO DwFJO
20 20 20 20
DRwS/DQ-/Dw8.1
TA4 TA10
DR DR
Dw8.1 Dw8.1
19 19
Original donor
~The six T-cell clones used in this paper were issued from two different HLA phenotyped donors. ZThe restricting molecule of each clone was determined by the ability of well-characterized MoAb to inhibit their specific responses. q'he correlation of these restrictions with HLA specificities was determined by the ability of HLA-phenotyped allogenic APC to present the specific antigen m each clone. q'he reference of each c!one is indicated.
Mixed Lymphocyte React/on The 1 x 105 effector cells were cultured for 6 days in round-bottomed microtiter plates as above, together with 1 x 10~ irradiated stimulator cells in 0.2 ml of culture medium supplemented with 5% human AB serum. The 3H-TdR incorporation was determined as above. Secondary Alloreactive T-Cell Responses The PBM (1 x 106/ml) were cultured for 7 days with 4000-rad-irradiated aHogeneic PBM (I x 106) under the same culture conditions as described above. Recombinant IL2 (10 IU/ml), purchased from Jansen Pharmaceutica, Belgium, was then added to cultures for another period of 7 days. After this time, living cells were recovered, washed twice in culture medium, and resuspended in fresh culture medium in the absence of exngeneous IL2. Then 1 x 104 cells were reseeded for 3 days into 0.2-ml round-bottomed microtiter plates together with I x 10~irradiated allogeneic PBM. Cell proliferation was estimated by measuring 3H-TdR uptake, as described above. Blocking Experiments The specificity of the secondary alloreactive response was evaluated by testing the ability of anti-HLA MoAb to inhibit cell proliferation. In these experiments, ascitic fluids were added to cultures on day 0 in secondary MLR at a final dilution of 1/100. RESULTS Cells from Donor 49 Share Identical or Highly Related HLA-DR But Not HLA-DQ Products with Cells of HLA-DRw8/DQ Blank/Dw8.1 Haplotypes Two influenza-specific proliferative T-cell clones, TA4 and TA10, obtained from a HLA-DRwS/DQ blank ( - )/Dw8.1 donor, were used to define functional HLA-
HLA-DR and HLA-DQ Contributions to HLA-Dw Typing
257
D molecules on a large panel of PBM donors. These clones (Table 2) were previously shown to recognize influenza A/Texas antigens in association with a HLA-DR molecule. Indeed, their specific proliferation could be inhibited by anti-DR but not by anti-DQ or DP MoAb [19]. Using a !argo panel of DRw8positive cells as APC, we also showed that this HLA-DR product is closely associated with the HLA-DwS.I specificity. Indeed, antigen-specific proliferation of TA4 and TA10 cells could be induced by APC from all HLA-DRwS/DQ/ Dw8.1 donors but not by APC from HLA-DRwS/DwS.2 individuals [19]. As indicated in Table 3, cells from donor 49 (DRwS/DQwl) did not type '-~s Dw8.1 nor Dw8.2, because the 9th Workshop or local HLA DRw8/DwS.1 or Dw8.2 HTC could induce their proliferation in primary MLR. Yet, APC from donor 49 were able to induce antigen-specific proliferation of TA4 and TA10 clones (Table 4). Consistent with previous observations [18], the HLA-DRwS/DQ/ Dw8.1 homozyguns-typingcell SID could serve as APC for the clones, whereas allogeneic DRw8/Dw8.2 cells could not. These obse~ations suggested that cells from donor 49 have, at lea~t, one HI.A-DR product highly related or identica| to those of HLA-Dw8.1 haplotypes. In contrast, HLA-DQ p.~>ducts on APC from donor 49 are necessarily different from that of the HLA-Dw8.1 HTC SID. Indeed, segregation studies of HLA specificities within the family of donor 49 indicated that the HLA-Dw8 haplotype from APC 49 codes for a HLA-DQwl molecule, whereas HTC $1D is HLA-DO.~,- blank (DQw-) like all Dw8.1 ceils from our panel. Thus, TA4 an~[ TA10 clones allowed us to describe an unusual haplotype consisting of a functional HLA-DR molecule, identical or closely related to that present in the HLA-DRw8/DQ-/Dw8.1 haplotype, associated with a HLA-DQwl molecule. The originality of the DR/DQ association on ceils from donor 49 was confirmed by the fact that nine other HLA-DRwS-positive ceils, which could present antigen to clones TA4 and TA10, were all DRwS/DQw/ Dw8.1. A DQ-Incompatible, DR-Compatible HLA.Dw8.1 Homozygous T y p i n g Cell Induces t h e Proliferation of Cells from Donor 49 in Primary and Secondary Mixed Lymphocyte Response: This Proliferation Is Inhibited by anti-DQ Monoclonal Antibodies Because HLA-DRw8/DQ-/Dw8.1 HTC from donor SID share identical or highly related HLA-DR but not D Q products with cells from donor 49, this represented a suitable model to test the ability of DQ molecules to stimulate T cells in primary response. Cells from donor 49 were used as responders and HTC SID as stimulators in primary MLR. Results in Table 3 indicate that T cells from donor 49 strongly proliferated, in primary MLR, when stimulated by the DR-compatible, DQ-incompatible HLA-DwS.1 HTC SID. A strong proliferation was also observed when cells 49 were stimulated by the FJO HTC, which differs from cells 49 for both DR and D Q products, as indicated below. Cells from donor 49 also strongly responded in secondary MLR when challenged by the same stimulator,/ ceils as in primary cultures (Figure IA,B). It is noteworthy that, under our experimental conditions, these secondary responses were devoid of alloreactivity against third-party cells. The FJO HTC could not induce the proliferation of 49 anti-SID primed cells (Figure 1B), nor could SID cells activate 49 anti-FJO primed lymphocytes (Figure 1A). The specificity of the secondary response allowed us to investigate further the stimtL!ato~ molecules involved by testing the effect of anti-HLA MoAb on these proliferative responses. Figure 1A illustrates that an~i-DR, but not anti-DQ, MoAb significantly inhibited the proliferation in combination pairs of effector/stimulator cells rais-
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HLA-DR and HLA-DQ Contributions to HLA-DwTyping TABLE
4
Cells 49 share H L A - D R but not H L A - D Q DRw8/DQblank/Dw8. I cells~
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Influenza virus
APC Panel number
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+ +
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22,361 - 1~460 2,201 -2_ 773 141803 ± 39
DRw8;DQw-;Dw8.1 DRw8;DOw3;Dw8.2 DRwI3;DQwl;Dwl8 DRw8;DQwl; DwDR3; DQw2;Dw3 DR2sh;DQwl;DwFJO DR2sh;DQwl;DwFjO
+ + + -
+
3,507 :~ 943 1,226 ± 589 901 ~ 311 t01773 • 481 817 *-. 186 1,388 4. 305 1,217 _*69
2,406.4- 1,241 276 ± 52 277 ± 168 141788 ± 1,482 509 ± 259 892.4- 313 1,321 ± 518
+ + +
"Cloned T cells were stimulated by HLA-phenotyped irradiated APC, and the telavant influertr.z AFt'exas antigen, or the irrelevant B Singapore virus as com~oL b[~H]-Thymidine incorporation w ~ ,~etermined on the third day of culture. Results ate expressed as mean counts per minute ± SD of triplicate culture. Posidvc f~ales are underlined.
matched for HLA-DR and DQ molecules (49 anti-FJO). In this combination, DR products are clearly different according to serological analysis. Moreover, products are different according to functional analysis, because DQ-restxicted clones from the donor FJO can be stimulated by APC from donor FJO but not from donor 49 (data not shown). In contrast, anti-DR MoAb had no significant effect on the 49 anti-SID combination, which is only mismatched for HLA-DQ molecules. This response was slightly, but significantly (more than 40°~) and reproducibly, inhibited by the BT3.4 anti-DQ MoAb. Together, these results indicate that HLA-DQ molecules can have a high stimulatory effect per se in primary MLR and, therefore, play a role in HLA-Dw typing. Cells from Donors 41.6 and 41.9 Share Identical or Highly Related HLA~DQ But Not HI,A-DR Products with Cells of the HL -DR2 Shox~YDQwl/DwFJO Haplotype We have previously described nine influenza-specific T-cell clones issued from a HLA-DR2sh/DQwl/DwFJO homnzygnus individual [20,21]. As recalled in Table 2, clones Ilia and lllg were shown to be restricted by a HLA-DQ molecule, because their ~pecific proliferation is inhibited by anti-DQ but not by anti-DR or anti-DP MoAb [20]. The other seven clones are restricted by DR products, because their proliferation is inhibited by and-DR, but not by anti-DQ, MoAb [19]. Using a large panel of DR2-posidve cells as APC, we could show that six out of the seven DR-restricted clones and the two DQ-restricted clones recognize antigen together with I-ILA class II products closely associated with the DR2sh/DQwl/DwFJO haplotype. Indeed, these clones could be activated by all HLA-DR2sh/DQwl/DwFJO but none of the DIC2 long/DQwl/Dw2, Dw12, or DR2-negadve APC from the panel [20]. This also indicated, as already suggested
240
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HLA-DR and HLA-DQ Contributions to HLA-Dw Typing
241
by others [6], that HLA-DQwl-positive molecules are more highly polymorphic than expected from serology. Surprisingly, cells from HLA-DR2 long positive donors from one family, 41.6 and 41.9, could present antigen to the DQ-restricted IIIa and IIIg but not to the DR-restricted T-cell clones as illustrated by the results obtained with clones Ie and TS0 (Table 5). According to previous results, and as also shown in Table 5, HLA-DR2 long (DR2)/DQwl/Dw2 homozygous cells PRU or HLA-DR2 long (DR2)/DQwl/Dwl2-positive cells 86 could not present antigen to the HI.A-DR or -DQ-restricted clones. Together, these results indicate that 41.6 and 41.9 APC, which type as HLA-DR2 long (DR2)/DQwl/Dw blank (Dw-), share highly related or identical HLA-DQ but not HLA-DR products with the HLADR2sh/DQwl/Dw~O haplotype. In a panel of 20 HLA-DR2-positive APC, cells from donors 41.6 and 41.9 issued from the same family were the only two that could stimulate the IIIa and IIlg DQ-restricted clones (data not shown). This confirms that this DR/DQ association is very unusual. Finally, it is worth notice that although all HLA-DR2-posidve ceils used in this study are also HLA-DQwl positive, HLA-Dw2 or Dwl2 APt: could not stimulate the DQ-restricted T-cell clones. Thus, the HLA-DQ polymorplfism described by IIla and lllg is more precise than indicated by serological analysis. It also reinforces the idea that T-cell clones are fine probes for HLA class II polymorphism analysis. Inhibition Studies of the 41.6 Secondary Response Against HLA-Dw2 or DwFJO Homozygous T y p i n g Cell by HI.A-Specific Monoclonal Antibodies As shown in Table 3, 41.6 and 41.9 HLA-DR2 + cells, which have an unusual association of DR and D Q products, did not type as Dw2, Dwl2, or DwFJO, because the 9th Workshop or local HLA-DR2-pusidve HTC could induce their proliferation in primary MLR. Because they could be activated by HLA-Dw2 donor PRU and HLA-DwFJO HTC, with which they were shown to share identical or related DR and DQ products, respectively, it could be concluded again that both DR and DQ can separately stimulate T cells in primary culture. This was further investigated by tesdng the inhibitory effect of anti-I-H.A MoAb on the secondary response of 41.6 cells activated in three different combinations. The 41.6 cells were primed with (1) donor MAM HTC (HLA-DR1/DQwl/Dwl), which differ for HLA-DR, as defined by serology, and for DQ products, because they could not present antigen to the two IIIa and IIlg DQ-restricted influenzaspecific T-cell clones as 41.6 cells did; (2) FJO HTC (DR2sh/DQwl/DwFJO), which differs in HLA-DR, as described above; (3) donor PRU HTC (DR2/DQwl/Dw2), which differs for HLA-DQ (see above). The 41.6 cells were restimulated by the homozyguus priming cells. Again, these secondary responses were devoid of alloceactivity against third-party cells (Figure 2). Blocking experiments by anti-HLA MoAb were performed as described under Materials and Methods. In the combination pair of responder/stimulator cells displaying a difference in DR (41.6 anti-FJO), the secondary response was clearly inhibited by anti-DR MoAb (Figure 2B). Conversely, the 41.6 response against PRU cells, induced by a difference in DQ, was strongly inhibited by and-DQ antibodies (Figure 2C). Finally, in the 41.6 anti-MAM combination, where respoeder and stimulator cells differ for both DR and DQ products, the response was mostly inhibited by the anti-DE antibodies (Figure IA). This inhibition was similar to that seen in combination B (41.6 anti-FJO) where responder and stimulator cells differ only in HLA-DR molecules. The inhibition obtained by anti-DQ antibodies in this corn-
242
G. Sterkers et al.
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HLA°DR and H L A - D Q Contributions to H L A - D w Typing
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G. Sterkers et al. bination might not be significar~t. This phenomenon is currently observed even when cloned T cells are used as effectors and may be attributed to nonspecific effects of HLA-specific MoAb in vitro. It may also be argued that anti-DQ MoAb may exhibit cross-reactivity with DR molecules. Although this cannot be formally ruled out, this possibility seems very unlikely based on available biochemical and serological informations concerning these MoAb. Together again, the results suggest that HLA-DR-linked determinants have an immunodominant effect in primary MLR in combination pairs mismatched for both DR and DQ.
DISCUSSION The use of influenga-specific T-cell clones as probes allowed us to describe two haplotypes displaying an unusual DR/DQ association among a large panel of APC from HLA-D~w8 and HLA-DR2 donors. One of these haplotypes is expressed by cells from donor 49 (DRw8/DQwl/Dw-) which, on functional and genetic criteria, seem to share one DR but not DQ product with the HLA-DRw8/DQ/ Dw8.1 haplotype. This conclusion is reinforced, at the protein level, by the observation that cells from donor 49 raised the same HLA-DR pattern as HLADw8.1 cells in two-dimensional polyacrylamide gel (2-D-PAGE) analysis (to be published). The other haplotype is represented by cells from 41.6 and 41.9 (DR2/DQwl/Dw-) donors, which share D Q but not DR products with the HLADR2sh/DQwl/DwFJO haplotype. Here again, 2-D-PAGE analysis of proteins from 41.6 and 41.9 cells reveaied that they have the same D Q beta patterns as cells of the FJO haplotype but the same DR beta patterns az cells of DR2/DQwl/Dw2 haplotype, t Previous molecular studies have occasionally identified D N A sequence differences in the beta-chain migrating identically in 2-D-PAGE for DR molecules from cells of different Dw subtypes [22-24]. Although sequences of HLA class II products studied here are not available, our results are consistent with the conclusions raized from our 2-D-PAGE and functional studies. Cells from the donors with these unusual DR/DQ associations were then used to investigate the role of DQ, DR, or DQ and DR differences in the induction of primary MLR in different responder/stimulator cell combinations. Our results demonstrate, that although a DR incompatibility is sufficient to induce a strong primary MLR, the DQ incompatibility alone can also induce a strong T-celi proliferation in primary cultures. Hence, both HLA-DQ and HI.A-DR molecules might participate in HLA-Dw typing. To confirm the involvment of HLA-DQ allodeterminants and to exclude the participation of other HLA class II molecules in some combination pairs, we used anti-HLA class 1I MoAb to inhibit secondary MLR. Blocking experiments were performed in secondary, rather than in primary, MLR because of the frequent nonspecific effects of anti-HLA antibodies in primary MLK [25]. Our results clearly show selective patterns of blocking by the different antibodies in the different combinations. The DR-incompatible combinations were strongly inhibited by anti-DR antibodies. Conversely, DQ-incompatible combinations were significantly inhibited by anti-DQ antibodies and little affected by anti-DR. These observations were consistent with the above conclusion concerning the participation of both DR and DQ molecules in HLA-Dw typing. However, in DR- and DQ-incompatible combinations, the response was strongly and repeatedly inhibtLotteauV, FreidelC, DavidV, GebuhrerL, BetuelH, TeytonL, CharronDJ: Structuralvariants of the DR beta 1, beta 2, beta 3, DQ alpha,and DQ beta productscontributeto the molecularand functionaldiversityof the HLA-DR2haplotypes.6th InternationalCongressof Immunology,1986.
HLA-DR and HLA-DQ Contributions to HLA-Dw Typing
245
ited by anti-DR MoAb, whereas anti-DQ had little or no effect. This may argue against the role of D Q allodeterminants in primary MLIL Yet, as already suggested, it can be postulated that in the case of dnal DR-DQ incompatibility, HLADR determinants may be immunodominant [10,11,26]. Controversial dam have been reported concerning the role of D Q products in Dw typing. Isolation of DQ-specific alloreactive T-ceB clones from bulk cultures of allostimulated lymphocytes strongly supports the participation of DQ determinants in Dw typing [6]. However, we (data not shown) and others {11] have reported similar HLA-Dw specificities in HLA-DR identical combination pairs displaying different HLA-DQ specificities. Thus, HLA-Dw specificities cannot be assigned to HLA-DQ molecules, at least in some combinations. In addition, several observations indicate that D Q molecules may preferentially activate suppressor T cells [9-11]. The D Q molecules would thus negatively regulate allogeneic responses in vitro [10]. Our experimental approach, using T-cell clones as fine probes to characterize unusual DR/DQ associations on functional criteria in some haplotypes, leads to the conclusion that in DR-compatible combination pairs HLA-DQ molecules can induce an allogeneic response in primary MLR. However, this observation was only made in two combinations of DQ-incompatible responder/stimulator cells. It may not be true in all combinatorial situations. Moreover, in the MLK typing assay using HTC, an arbitrary cutoff between positive and negative responses has been determined [27,28]. Indeed, complete or ahnost complete idendty of HLA-D-stimulating ailoantigens between cells from unrelated individnals is an exception [28]. It is, therefore, possible that several different epitopes are required on HLA-DQ molecules to induce a response strong enough to be considered as positive. This may not be the case in all cell combinations. It may also explain the immunodominant role of DR epitopes in most dual DR- and DQincompatible situations. Further expernnents are needed to explore this possibility. Finally, no evidence is presented here concerning the role of DP molecules in HI.A-Dw typing, because DP speciticides of cells used were not available. However, it should be stressed that B7.21.1 DP MoAb [16] could not inhibit secondary alloreactive responses in our experiments. This MoAb was shown to inhibit DP but not DR- or DQ-spocific T-cell clones obtained in our laboratory (not shown). Hence, the results suggest either that all combinations used in the present studies are DP compatible, which is quite unlikely, or conversally, that DP molecules poorly contribute to T-cell proliferation induction in DQ- and/or DR-incompatible combinations.
ACKNOWLEDGMENTS
The authors would like to thank Dr. R. Accolla for his gift of BT3.4 MoAb, and C. Hannoun for providing influenza viruses. We also thank Y. Hanin for her excellent technical assistance, and C. Pellet for typing this manuscript. REFERENCES 1. Bach FH: The HLA class II genes and products: The HLA-D region, lmmanol Today 6:89, i985. 2. Shackelford IDA,KanfmanJK, Korman AJ, StromingerJL: HLA DR antigens: Structure, separation of subpopulations, gane dotting, and function, lmmunol Rev 66:133, 1982.
246
G. Sterkers et al. 3. Kaufman JK, Auffmy C, Korman AJ, Shackelford DA, Sir~minger JL: The class !I molecules of the human and routine major histocompatibility complex. Cell 36:1, 19S4. 4. Bach FH, Hirsharn K: Lymphocym interaction: A potential histocompatibilitytest in vitro. Science 143:813, 1964. 5. Gross-Wilde H, Doxiadia I, Bmndt H: Joint Report Cellular part (HTC) 9th International Histocompatibility Workshop. In Albert ED, Baur MP, Mayr WR, eds: HistocompatibilityTesting 19S4. Berlin, Springer-Verlag, in press. 6. Reinsmoen NL, Bach FH: Clonal analysis of HLA-DR and -DQ associated determinants: Their contribution to Dw specificities. Hum lmmunol 16:329, 1986. 7. Shaw S, Johnson AH, Shearer GM: Evidence for a new segregant series of B-cell antigens which are encoded in the HLA-D region and stimulate secondary allogeneic proliferative and cytotoxic responses. J Exp Med 152:565, 1980. 8. Navarrete C, Jaraquemada D, Hui K, Awad J, Okoye R, Festenstein H: Different functions and associations of HLA-DR and HLA-DQ (DC) antigens shown by serological, cellular, and DNA assays. Tissue Antigens 25:130, 1985. 9. Fainboim L, Jaraquemada D, Festenstein H, Sachs JA: MHC specified lymphocyte activating and suppressor activating determinants in human mixed lymphocyte reactions. Scand J lmmunol 14:655, 1981. 10. OttenhoffTHM, Elferink DG, Hermans J, de Vries RRP: HLA class I! restriction repertoire of antigen-spocific T cells. 1. The main restriction determinants for antigen presentation are associated with HLA-D/DR and not with DP or DQ. Hum Immunol 13:105, 1985. 11. Jaraquemada D, Navarrete C, Oilier W, Awad J, Okoye R, Festenstein H: HLA-Dw specificity assignments are independent of HLA-DQ, HI,A-DR, and other class I1 specificities and define a biologically important segregant series which strongly activates a functionally distinct T-cell subset. Hum Immunol 16:259, 1986. 12. Sterkers G, Henin Y, Lepage V, Fradelizi D, Hannoun C, Levy Jp: Influenza A hemagglutinin specific T-cell clones strictly restricted by HLA-DR1 or HLA-DR7 molecules. EurJ Immunol 14:125, 1984. 13. Betuel H, Freidel AC, Gebuhrer L: HLA antigens and haplotypes in families of leukemic patients, eventual recipients of a bone marrow graft, in Touraine JL, Gluckman E, Grisceili C, eds: Bone Marrow Transplantation in Europe. vol. II, Amsterdam, Excerpta Medica, 1981, p. 255. 14. Lampson LA, Levy R: Two populations of In-like molecules on a human B-cell fine. J Immunol 125:293, 1980. 15. Brodsky FM, Parham P, Bodmer WF: Monoclonal antibodies m HLA-DRw determinants. Tissue Antigens 16:30, 1980. 16. Corte G, Calabri F, Damiaul G, Bargellesi A, Tosi R, Sorrendno R: Human la molecules carrying DCI determinants differ in both alpha and beta subunits from Ia molecules carrying DR determinants. Nature 292:357, 1981. 17. Watson AJ, De Mars R, Trowbridge IS, Bach FH: Detection of a novel human class 11 HLA antigen. Nature 304:358, 1983. 18. Barnstable DJ, Bodmer WE, Brown G, Galfre G, Milstein C, Williams AF, Ziegler A: Production of monoclonal antibodies to group A erythrocytes, HLA, and other human cell surface antigens. New tools for genetics analysis. Cell 14:9, 1978. 19. Henin Y, Sterkers G, Gomard E, Gebuhrer L, Freidel AC, Lepage V, Stern MH, Michon J, Betuel H, Levy JP: Functional subdivision of HLA-DRw8 with influenzaspecific cloned cell lines. Immunogenetics 22:407, 1985.
HLA-DR and HLA-DQ Contributions to HLA-Dw Typing
247
20. Zeliszewski D, Sterkers G, Freidel AC, Gebuhrer L, Bemel H, Levy JP: Functional definition of HLA-DR and -DQ epitope~ specific for DIL?.-short, DwFJO haplotype. Immunogenetics 23:385, 1986. 21. Zeliszewski D, Sterkers G, Chnppin J, Freidel AC, Gebuhter L, Betuel H, Le',3'JP: An antiviralT-cell clone defines a F~nctional super tyi, i¢ specificity shared by different HI.A-DR molecules fi'om DR2-~hort, DRwll, and DRwI3 haplotypes. Immunogenetics 25:84, 1987. 22. Capra G, Accolla RS: Stroct,,ral analysis of human la antigens reveals the existence of a fourth moleculc.t"subs,.t distinct from DP, DQ. and DR molecules. J Exp Med 165:47, 1987. 23. Cairns JS, Curtsinger JM, DaM CA, Freeman S, Alter BJ, Bach FH: Sequence polymorphism ofHLA Di~.bem I allelesrelating roT-cell recognized determinants. Nature 317:167, 1985. 24. Gruner JP, Watonn AJ, Bach FH: Dw/LD related molecular polymorphism of DR4 beta-chains. J E'~p Med 157:1687, 1983. 25. Sterkers G, Henin Y, Kalil J, Bagot M, Levy JP: Influence of HLA class I and class ii specific monoclonal antibodies on DR-restricted lymphoprolifemtiverespomes. 1. Unseparated populations of effector cells. J lmmunol 131:2735, 1983. 26. Bach FH, Reinsmoen NL: The role of HI.A-DR and HLA-DQ products in T*lymphocyt~ activation and in contributing to "Dw specificities." Hum Immunol 16:271, 1986. 27. Re~nsmoen HI,, Bach FH: Five HLA-D clusters associated with HLA-DR4. Human lmmuunl 4:249, 1982. 28. Morling N, Jakobson BK, Platz P, Ryder I.P, Svejgaard A, Thomsen M: Typing for human alloantigens with the primed lymphocyte typing (PLT) technique with notes on the interpretation of PLT data. Hum Immunol 2:233, 1981. 29. Ryder LP, Thomsen M, Plata P, Sveigaard A: Data reduction in LD typing. In Kissmeyer-Nielsen F, ed: Hismcompatibility Testing 1975. Copenhagen, Munksgaard, 1975, p. 567.
ABSTRACT: The respoctivecontribution of HLA-DR and HLA-DQ genepreducts in the induction of allogeneicproliferative responses in primary mixed lympho.7~tereactionand, therefore, in HLADw typing, is still unclear or controversial. This is in part due to a stmng linkage disequilibrium betweenHI.A-DR and -DQ genes. We used DR- or DQ-restricted influenza-spuific T-cell clones to define DR and DQ products on a largepanel of aUogeneicantigen presenting cells. With this functionalscreeningassay, we identified two haplotypos upishunusual D R I l ~ associations. Cells of these haplotypes were then used as responderceils in mixed lymphocyteculture and stiraulated by homozygous typing cells displaying DR or DQ incompatibilities. Our results indicate that DR or DQ incompatibilities alone can give rise, in both cases, to strong T-cell proliferation in a mixedlymphecyte reaction. This was further verifiedby blockingexperimentsof secondarymixed lymphocyte reactions by HLA-spocifie monoclonalantihulies. Anti-DQ, but not anti.DR, antibodies inhibited DQ-incompatible responses. Conversely, anti-DR, but not anti-DQ, antikuties could block DR-incompatible mixed lymphocyte r~zctions. Together, the results suggest that both HLA-DR and DO. gone products can be involved in HLA-Dw typing. Finally, in dual DRand DQ-incompatible mixed lymphocyte reactioncombinations, HI.A-DR moleculesseem to have an immunodominant effect, because the response is mostly inhibited by anti-DR antibodies. lmmunodominance of HI,A-DR allodeterminants may, at least in part, explain some of the controversial conclusions reportedby others concem~ingthe role of HLA-DQ moleculesin HLADw typing. ABBREVIATIONS APC antigen presenting cells PBM peripheral blood roononuclear cells IL2 interleukin 2 MLR mixed lymphocyte reaction MoAb monoclonal antibody
HTC 3H-TdR 2-D-PAGE DNV
homozygous typing cell Tritiated thymidine two-dimensional polyacr,/lamide gel electrc~ phoresis double normalized value
INTRODUCTION Human class II antigens of the major histocompatibility complex (MHC) are highly polymorphic heterodimers encoded by genes of the human leukocyte antigen (HLA)-D region on chromosome 6. At least three sets of molecules
Fromthe Laboratoired'lmmunologieet Virologicdes Tumeurr, Inserm U 152, CNR$ UA 628, HOpital Cochin,27, rue du FaubourgSaintJacques, 75014 Paris,FrancefG.S., D.Z., J.P.L.) and the Laboratoire ¢?Hi~tocompatibilit#,CentreR~gionalde TransfusionSanguinede Lyon, Lyon, France(A.C.F., L.G., H.B.). Address reprints requeststo Dr. G. Sterkers, Laboratoired~lm~unologieet de Virdogiedes Turnouts, lmerm U152, CNRS UA628, HOpitalCochin,27, rue du FaubourgSaintJacques, 75014 Paris, France. ReceivedOctober27, 1986;acceptedAugust 3, 1987. HumanImmunology20, 233-247 (1987) © ElsevierSciencePublishingCo., Inc., 1987 52 Vanderbi/¢Ave.,New York,NY 10017
233 0198-8859/87/$3.~0
234
G. Sterkers etal. encoded by .genes of HLA-DR, -DP, and -DQ subregion, respectively, have been identified within the HLA-D region of the MHC, based on serological, functional, and biochemical evidences. Serologically defined antigenic specificities have been assigned to different HLA-D gene products, i.e., DR1 through DRw14 on one of the DR molecules, and DQwl through DQw3 on D Q molecules. Hence, HLA-D molecules can be characterized, at least in part, using specific antibody reagents (see review [1-3]). Class lI MHC antigens are also known to induce the proliferation of alloreactive T lymphocytes in mixed lymphocyte culture. Therefore, HLA-D typing can be routinely achieved by measuring the proliferation of donor cells being typed when cultured with a panel of commonly accepted HLA homozygnus typing cells (HI'C). This mixed lymphocyte reaction (MLR) defines HLA-D specificities designated Dwl through Dw19 [4,5]. The Dw specificities are subtypic to the serologically defined DR and DQ antigens, in that several Dw specificities can be associated with the same serological specificity. Although HLA-DR, -DP, or .DQ molecules have been shown to induce the proliferation of alloreactive Tcell clones [6,7], their respective contribution in the induction of primary MLR remains controversial. The HLA-DP incompatibility is believed not to induce primary MLR reactivity [7]. In contrast, it is generally accepted that DR molecules are the major antigens involved in this functional assay. Finally, contradictory results have been reported concerning the role of D Q molecules [6,8-11]. Many experiments suggest that they may play a significant role in primary MLR [6]. However, it has been recently proposed that D Q antigens per se cannot stimulate T cells in primary cultures [8]. Moreover, a negative regulation of the T-cell proliferation in MLR by DQ molecules has been proposed [11]. On the whole, the DR and/or DQ molecular basis for MLR-defined HLA-D differences remains unclear. Two major difficulties must be circumvented to answer this question: (1) the poor definition of class II molecules by conventional typing methods that do not allow the assumption ofwbetber class II antigens are shared by unrelated individuals or not; (2) a strong linkage disequilibrium between DR and D Q genes, such that most DR-identical unrelated individuals are also D Q identical. Hence, combination pairs of responder/stimulator cells in MLR differing in either DR or D Q molecules are the exception. In our experimental approach to investigate the role of DQ molecules in Dwtyping, we used influenza-specificT-cell clones restricted to DR or D Q molecules to define HLA-D specificities on a large panel of donor cells used as antigen presenting cells (APC). Among this panel, we isolated two APC donors exhibiting an unusual DR/DQ association. Cells from these donors were used as responder cells in primary MLR and were stimulated by HTC presenting DR or D Q incompatibilities. In each case, the set of molecules involved in these proliferative responses was assessed by testing the inhibitory effect of different HLA-specific monoclonal antibodies (MoAb) in secondary MLR. The results lead to the conclusion that (1) both HLA-DQ and HLA-DR may be involved in primary MLR and, therefore, contribute to HLA-Dw typing; (2) HLA-DR seems to be immunodominant in combination pairs of responder/stimulator cells differing for both DR and DQ specificities.
MATERIALS A N D M E T H O D S Peripheral Blood Mononuclear Cells The peripheral blood mononuclear cells (PBM) were isolated from blood samples as previously described [12]. The HLA phenotypes v:ere determined according
HLA-DR and HLA-DQ Contributions to HLA-DwTyping
235
TABLE 1 Monoclonal antibodies used in this paper Name L243 L227 Genox 353" BT3.4 B7.21.I w6.32
Specificity
Reference
DR DR + DP
14 14
DQwl DQwI and DQ moleculesassoicatedto DR.4and w8
15 16
DP HLA-A,B,C
17 18
"Genox3.5l, whichrecognizesDQwl-positivemolecu/es,wasonlyusedwhenstimulatorcellshadthisspec~cit~. to methods described elsewhere [ 13] by using local reagents and reagents defined by the 9th International Histocomp~tibility Workshop. Influenza Viruses Influenza A/Texas and E/Singapore viruses were kindly provided by Dr. C. Hartnoun, Pasteur Institute, Paris [12]. Monoclonal Antibodies Anti-HLA MoAb used in the experiments to inhibit secondary MLRs are described in Table 1. These MoAb were used as ascitic fluids. Their specificity has been extensively described elsewhere [14-18]. Culture Medium Cells were cultured in RPMI 1640 supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, 2 x 10"s bem-mercaptoethanol, antibiotics, and 5% (v/v) heat-inactivated pooled human AB serum as previously described [12]. T-Cell Clones Influenza AfI'exas virus-specific T-cell clones were used to define HLA-D functional molecules on a large panel of donor's PBM used as A.PC.These proliferative clones were obtained as previously described [ 12]. Their specificity and restriction have been extensively documented elsewhere [19-21]. Their main characteristics are indicated in Table 2. T-Cell Clone Proliferation Assay T-cell clone proliferation assay was performed as previously described [ 11]. Briefly, 1 x 104 washed cloned cells were cultured with I x 105 irradiated (4000-rad) PBM as APC previously infected with the influenza A/Texas virus. Cultures were made in 0.2-ml round bottomed microculture plates and were incubated for 3 days at 37°(; in 5o~ CO2 atmosphere. Cell proliferation was estimated by adding 0.8/zCi of tritiased thymidine (3H-TdR, 5 Ci/mmol) to each well for the last 6 hr. Cells were then harvested, and the radioactivity was incorporated in DNA counted.
256
G. Stefkers et al. TABLE 2 T-cell clone characteristics Restricting
Phenotype of efficient
phenotype~
Clones
molecule; b
allogeneicAPO
References d
DR2short/DQwl/DwFJO
Illg Illa ie lh
DQ DQ DR DR
DwFJO DwFJO DwFJO DwFJO
20 20 20 20
DRwS/DQ-/Dw8.1
TA4 TA10
DR DR
Dw8.1 Dw8.1
19 19
Original donor
~The six T-cell clones used in this paper were issued from two different HLA phenotyped donors. ZThe restricting molecule of each clone was determined by the ability of well-characterized MoAb to inhibit their specific responses. q'he correlation of these restrictions with HLA specificities was determined by the ability of HLA-phenotyped allogenic APC to present the specific antigen m each clone. q'he reference of each c!one is indicated.
Mixed Lymphocyte React/on The 1 x 105 effector cells were cultured for 6 days in round-bottomed microtiter plates as above, together with 1 x 10~ irradiated stimulator cells in 0.2 ml of culture medium supplemented with 5% human AB serum. The 3H-TdR incorporation was determined as above. Secondary Alloreactive T-Cell Responses The PBM (1 x 106/ml) were cultured for 7 days with 4000-rad-irradiated aHogeneic PBM (I x 106) under the same culture conditions as described above. Recombinant IL2 (10 IU/ml), purchased from Jansen Pharmaceutica, Belgium, was then added to cultures for another period of 7 days. After this time, living cells were recovered, washed twice in culture medium, and resuspended in fresh culture medium in the absence of exngeneous IL2. Then 1 x 104 cells were reseeded for 3 days into 0.2-ml round-bottomed microtiter plates together with I x 10~irradiated allogeneic PBM. Cell proliferation was estimated by measuring 3H-TdR uptake, as described above. Blocking Experiments The specificity of the secondary alloreactive response was evaluated by testing the ability of anti-HLA MoAb to inhibit cell proliferation. In these experiments, ascitic fluids were added to cultures on day 0 in secondary MLR at a final dilution of 1/100. RESULTS Cells from Donor 49 Share Identical or Highly Related HLA-DR But Not HLA-DQ Products with Cells of HLA-DRw8/DQ Blank/Dw8.1 Haplotypes Two influenza-specific proliferative T-cell clones, TA4 and TA10, obtained from a HLA-DRwS/DQ blank ( - )/Dw8.1 donor, were used to define functional HLA-
HLA-DR and HLA-DQ Contributions to HLA-Dw Typing
257
D molecules on a large panel of PBM donors. These clones (Table 2) were previously shown to recognize influenza A/Texas antigens in association with a HLA-DR molecule. Indeed, their specific proliferation could be inhibited by anti-DR but not by anti-DQ or DP MoAb [19]. Using a !argo panel of DRw8positive cells as APC, we also showed that this HLA-DR product is closely associated with the HLA-DwS.I specificity. Indeed, antigen-specific proliferation of TA4 and TA10 cells could be induced by APC from all HLA-DRwS/DQ/ Dw8.1 donors but not by APC from HLA-DRwS/DwS.2 individuals [19]. As indicated in Table 3, cells from donor 49 (DRwS/DQwl) did not type '-~s Dw8.1 nor Dw8.2, because the 9th Workshop or local HLA DRw8/DwS.1 or Dw8.2 HTC could induce their proliferation in primary MLR. Yet, APC from donor 49 were able to induce antigen-specific proliferation of TA4 and TA10 clones (Table 4). Consistent with previous observations [18], the HLA-DRwS/DQ/ Dw8.1 homozyguns-typingcell SID could serve as APC for the clones, whereas allogeneic DRw8/Dw8.2 cells could not. These obse~ations suggested that cells from donor 49 have, at lea~t, one HI.A-DR product highly related or identica| to those of HLA-Dw8.1 haplotypes. In contrast, HLA-DQ p.~>ducts on APC from donor 49 are necessarily different from that of the HLA-Dw8.1 HTC SID. Indeed, segregation studies of HLA specificities within the family of donor 49 indicated that the HLA-Dw8 haplotype from APC 49 codes for a HLA-DQwl molecule, whereas HTC $1D is HLA-DO.~,- blank (DQw-) like all Dw8.1 ceils from our panel. Thus, TA4 an~[ TA10 clones allowed us to describe an unusual haplotype consisting of a functional HLA-DR molecule, identical or closely related to that present in the HLA-DRw8/DQ-/Dw8.1 haplotype, associated with a HLA-DQwl molecule. The originality of the DR/DQ association on ceils from donor 49 was confirmed by the fact that nine other HLA-DRwS-positive ceils, which could present antigen to clones TA4 and TA10, were all DRwS/DQw/ Dw8.1. A DQ-Incompatible, DR-Compatible HLA.Dw8.1 Homozygous T y p i n g Cell Induces t h e Proliferation of Cells from Donor 49 in Primary and Secondary Mixed Lymphocyte Response: This Proliferation Is Inhibited by anti-DQ Monoclonal Antibodies Because HLA-DRw8/DQ-/Dw8.1 HTC from donor SID share identical or highly related HLA-DR but not D Q products with cells from donor 49, this represented a suitable model to test the ability of DQ molecules to stimulate T cells in primary response. Cells from donor 49 were used as responders and HTC SID as stimulators in primary MLR. Results in Table 3 indicate that T cells from donor 49 strongly proliferated, in primary MLR, when stimulated by the DR-compatible, DQ-incompatible HLA-DwS.1 HTC SID. A strong proliferation was also observed when cells 49 were stimulated by the FJO HTC, which differs from cells 49 for both DR and D Q products, as indicated below. Cells from donor 49 also strongly responded in secondary MLR when challenged by the same stimulator,/ ceils as in primary cultures (Figure IA,B). It is noteworthy that, under our experimental conditions, these secondary responses were devoid of alloreactivity against third-party cells. The FJO HTC could not induce the proliferation of 49 anti-SID primed cells (Figure 1B), nor could SID cells activate 49 anti-FJO primed lymphocytes (Figure 1A). The specificity of the secondary response allowed us to investigate further the stimtL!ato~ molecules involved by testing the effect of anti-HLA MoAb on these proliferative responses. Figure 1A illustrates that an~i-DR, but not anti-DQ, MoAb significantly inhibited the proliferation in combination pairs of effector/stimulator cells rais-
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HLA-DR and HLA-DQ Contributions to HLA-DwTyping TABLE
4
Cells 49 share H L A - D R but not H L A - D Q DRw8/DQblank/Dw8. I cells~
Autologous SID
175 49 F~O
products with
Influenza virus
APC Panel number
239
Cloned T cells6
Genottpe
A
B
TA4
TAI0
D R w 8 i DQw-; D w S . l DR4/DQw3;DwI3 DRWSI D Q w - i Dw8.1
+ +
+
29~628 ± 2~!72" 325 ± 1,797 24t221 :~- 36.5.5
22,361 - 1~460 2,201 -2_ 773 141803 ± 39
DRw8;DQw-;Dw8.1 DRw8;DOw3;Dw8.2 DRwI3;DQwl;Dwl8 DRw8;DQwl; DwDR3; DQw2;Dw3 DR2sh;DQwl;DwFJO DR2sh;DQwl;DwFjO
+ + + -
+
3,507 :~ 943 1,226 ± 589 901 ~ 311 t01773 • 481 817 *-. 186 1,388 4. 305 1,217 _*69
2,406.4- 1,241 276 ± 52 277 ± 168 141788 ± 1,482 509 ± 259 892.4- 313 1,321 ± 518
+ + +
"Cloned T cells were stimulated by HLA-phenotyped irradiated APC, and the telavant influertr.z AFt'exas antigen, or the irrelevant B Singapore virus as com~oL b[~H]-Thymidine incorporation w ~ ,~etermined on the third day of culture. Results ate expressed as mean counts per minute ± SD of triplicate culture. Posidvc f~ales are underlined.
matched for HLA-DR and DQ molecules (49 anti-FJO). In this combination, DR products are clearly different according to serological analysis. Moreover, products are different according to functional analysis, because DQ-restxicted clones from the donor FJO can be stimulated by APC from donor FJO but not from donor 49 (data not shown). In contrast, anti-DR MoAb had no significant effect on the 49 anti-SID combination, which is only mismatched for HLA-DQ molecules. This response was slightly, but significantly (more than 40°~) and reproducibly, inhibited by the BT3.4 anti-DQ MoAb. Together, these results indicate that HLA-DQ molecules can have a high stimulatory effect per se in primary MLR and, therefore, play a role in HLA-Dw typing. Cells from Donors 41.6 and 41.9 Share Identical or Highly Related HLA~DQ But Not HI,A-DR Products with Cells of the HL -DR2 Shox~YDQwl/DwFJO Haplotype We have previously described nine influenza-specific T-cell clones issued from a HLA-DR2sh/DQwl/DwFJO homnzygnus individual [20,21]. As recalled in Table 2, clones Ilia and lllg were shown to be restricted by a HLA-DQ molecule, because their ~pecific proliferation is inhibited by anti-DQ but not by anti-DR or anti-DP MoAb [20]. The other seven clones are restricted by DR products, because their proliferation is inhibited by and-DR, but not by anti-DQ, MoAb [19]. Using a large panel of DR2-posidve cells as APC, we could show that six out of the seven DR-restricted clones and the two DQ-restricted clones recognize antigen together with I-ILA class II products closely associated with the DR2sh/DQwl/DwFJO haplotype. Indeed, these clones could be activated by all HLA-DR2sh/DQwl/DwFJO but none of the DIC2 long/DQwl/Dw2, Dw12, or DR2-negadve APC from the panel [20]. This also indicated, as already suggested
240
G . S t e r k e r s e t al.
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HLA-DR and HLA-DQ Contributions to HLA-Dw Typing
241
by others [6], that HLA-DQwl-positive molecules are more highly polymorphic than expected from serology. Surprisingly, cells from HLA-DR2 long positive donors from one family, 41.6 and 41.9, could present antigen to the DQ-restricted IIIa and IIIg but not to the DR-restricted T-cell clones as illustrated by the results obtained with clones Ie and TS0 (Table 5). According to previous results, and as also shown in Table 5, HLA-DR2 long (DR2)/DQwl/Dw2 homozygous cells PRU or HLA-DR2 long (DR2)/DQwl/Dwl2-positive cells 86 could not present antigen to the HI.A-DR or -DQ-restricted clones. Together, these results indicate that 41.6 and 41.9 APC, which type as HLA-DR2 long (DR2)/DQwl/Dw blank (Dw-), share highly related or identical HLA-DQ but not HLA-DR products with the HLADR2sh/DQwl/Dw~O haplotype. In a panel of 20 HLA-DR2-positive APC, cells from donors 41.6 and 41.9 issued from the same family were the only two that could stimulate the IIIa and IIlg DQ-restricted clones (data not shown). This confirms that this DR/DQ association is very unusual. Finally, it is worth notice that although all HLA-DR2-posidve ceils used in this study are also HLA-DQwl positive, HLA-Dw2 or Dwl2 APt: could not stimulate the DQ-restricted T-cell clones. Thus, the HLA-DQ polymorplfism described by IIla and lllg is more precise than indicated by serological analysis. It also reinforces the idea that T-cell clones are fine probes for HLA class II polymorphism analysis. Inhibition Studies of the 41.6 Secondary Response Against HLA-Dw2 or DwFJO Homozygous T y p i n g Cell by HI.A-Specific Monoclonal Antibodies As shown in Table 3, 41.6 and 41.9 HLA-DR2 + cells, which have an unusual association of DR and D Q products, did not type as Dw2, Dwl2, or DwFJO, because the 9th Workshop or local HLA-DR2-pusidve HTC could induce their proliferation in primary MLR. Because they could be activated by HLA-Dw2 donor PRU and HLA-DwFJO HTC, with which they were shown to share identical or related DR and DQ products, respectively, it could be concluded again that both DR and DQ can separately stimulate T cells in primary culture. This was further investigated by tesdng the inhibitory effect of anti-I-H.A MoAb on the secondary response of 41.6 cells activated in three different combinations. The 41.6 cells were primed with (1) donor MAM HTC (HLA-DR1/DQwl/Dwl), which differ for HLA-DR, as defined by serology, and for DQ products, because they could not present antigen to the two IIIa and IIlg DQ-restricted influenzaspecific T-cell clones as 41.6 cells did; (2) FJO HTC (DR2sh/DQwl/DwFJO), which differs in HLA-DR, as described above; (3) donor PRU HTC (DR2/DQwl/Dw2), which differs for HLA-DQ (see above). The 41.6 cells were restimulated by the homozyguus priming cells. Again, these secondary responses were devoid of alloceactivity against third-party cells (Figure 2). Blocking experiments by anti-HLA MoAb were performed as described under Materials and Methods. In the combination pair of responder/stimulator cells displaying a difference in DR (41.6 anti-FJO), the secondary response was clearly inhibited by anti-DR MoAb (Figure 2B). Conversely, the 41.6 response against PRU cells, induced by a difference in DQ, was strongly inhibited by and-DQ antibodies (Figure 2C). Finally, in the 41.6 anti-MAM combination, where respoeder and stimulator cells differ for both DR and DQ products, the response was mostly inhibited by the anti-DE antibodies (Figure IA). This inhibition was similar to that seen in combination B (41.6 anti-FJO) where responder and stimulator cells differ only in HLA-DR molecules. The inhibition obtained by anti-DQ antibodies in this corn-
242
G. Sterkers et al.
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HLA°DR and H L A - D Q Contributions to H L A - D w Typing
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244
G. Sterkers et al. bination might not be significar~t. This phenomenon is currently observed even when cloned T cells are used as effectors and may be attributed to nonspecific effects of HLA-specific MoAb in vitro. It may also be argued that anti-DQ MoAb may exhibit cross-reactivity with DR molecules. Although this cannot be formally ruled out, this possibility seems very unlikely based on available biochemical and serological informations concerning these MoAb. Together again, the results suggest that HLA-DR-linked determinants have an immunodominant effect in primary MLR in combination pairs mismatched for both DR and DQ.
DISCUSSION The use of influenga-specific T-cell clones as probes allowed us to describe two haplotypes displaying an unusual DR/DQ association among a large panel of APC from HLA-D~w8 and HLA-DR2 donors. One of these haplotypes is expressed by cells from donor 49 (DRw8/DQwl/Dw-) which, on functional and genetic criteria, seem to share one DR but not DQ product with the HLA-DRw8/DQ/ Dw8.1 haplotype. This conclusion is reinforced, at the protein level, by the observation that cells from donor 49 raised the same HLA-DR pattern as HLADw8.1 cells in two-dimensional polyacrylamide gel (2-D-PAGE) analysis (to be published). The other haplotype is represented by cells from 41.6 and 41.9 (DR2/DQwl/Dw-) donors, which share D Q but not DR products with the HLADR2sh/DQwl/DwFJO haplotype. Here again, 2-D-PAGE analysis of proteins from 41.6 and 41.9 cells reveaied that they have the same D Q beta patterns as cells of the FJO haplotype but the same DR beta patterns az cells of DR2/DQwl/Dw2 haplotype, t Previous molecular studies have occasionally identified D N A sequence differences in the beta-chain migrating identically in 2-D-PAGE for DR molecules from cells of different Dw subtypes [22-24]. Although sequences of HLA class II products studied here are not available, our results are consistent with the conclusions raized from our 2-D-PAGE and functional studies. Cells from the donors with these unusual DR/DQ associations were then used to investigate the role of DQ, DR, or DQ and DR differences in the induction of primary MLR in different responder/stimulator cell combinations. Our results demonstrate, that although a DR incompatibility is sufficient to induce a strong primary MLR, the DQ incompatibility alone can also induce a strong T-celi proliferation in primary cultures. Hence, both HLA-DQ and HI.A-DR molecules might participate in HLA-Dw typing. To confirm the involvment of HLA-DQ allodeterminants and to exclude the participation of other HLA class II molecules in some combination pairs, we used anti-HLA class 1I MoAb to inhibit secondary MLR. Blocking experiments were performed in secondary, rather than in primary, MLR because of the frequent nonspecific effects of anti-HLA antibodies in primary MLK [25]. Our results clearly show selective patterns of blocking by the different antibodies in the different combinations. The DR-incompatible combinations were strongly inhibited by anti-DR antibodies. Conversely, DQ-incompatible combinations were significantly inhibited by anti-DQ antibodies and little affected by anti-DR. These observations were consistent with the above conclusion concerning the participation of both DR and DQ molecules in HLA-Dw typing. However, in DR- and DQ-incompatible combinations, the response was strongly and repeatedly inhibtLotteauV, FreidelC, DavidV, GebuhrerL, BetuelH, TeytonL, CharronDJ: Structuralvariants of the DR beta 1, beta 2, beta 3, DQ alpha,and DQ beta productscontributeto the molecularand functionaldiversityof the HLA-DR2haplotypes.6th InternationalCongressof Immunology,1986.
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ited by anti-DR MoAb, whereas anti-DQ had little or no effect. This may argue against the role of D Q allodeterminants in primary MLIL Yet, as already suggested, it can be postulated that in the case of dnal DR-DQ incompatibility, HLADR determinants may be immunodominant [10,11,26]. Controversial dam have been reported concerning the role of D Q products in Dw typing. Isolation of DQ-specific alloreactive T-ceB clones from bulk cultures of allostimulated lymphocytes strongly supports the participation of DQ determinants in Dw typing [6]. However, we (data not shown) and others {11] have reported similar HLA-Dw specificities in HLA-DR identical combination pairs displaying different HLA-DQ specificities. Thus, HLA-Dw specificities cannot be assigned to HLA-DQ molecules, at least in some combinations. In addition, several observations indicate that D Q molecules may preferentially activate suppressor T cells [9-11]. The D Q molecules would thus negatively regulate allogeneic responses in vitro [10]. Our experimental approach, using T-cell clones as fine probes to characterize unusual DR/DQ associations on functional criteria in some haplotypes, leads to the conclusion that in DR-compatible combination pairs HLA-DQ molecules can induce an allogeneic response in primary MLR. However, this observation was only made in two combinations of DQ-incompatible responder/stimulator cells. It may not be true in all combinatorial situations. Moreover, in the MLK typing assay using HTC, an arbitrary cutoff between positive and negative responses has been determined [27,28]. Indeed, complete or ahnost complete idendty of HLA-D-stimulating ailoantigens between cells from unrelated individnals is an exception [28]. It is, therefore, possible that several different epitopes are required on HLA-DQ molecules to induce a response strong enough to be considered as positive. This may not be the case in all cell combinations. It may also explain the immunodominant role of DR epitopes in most dual DR- and DQincompatible situations. Further expernnents are needed to explore this possibility. Finally, no evidence is presented here concerning the role of DP molecules in HI.A-Dw typing, because DP speciticides of cells used were not available. However, it should be stressed that B7.21.1 DP MoAb [16] could not inhibit secondary alloreactive responses in our experiments. This MoAb was shown to inhibit DP but not DR- or DQ-spocific T-cell clones obtained in our laboratory (not shown). Hence, the results suggest either that all combinations used in the present studies are DP compatible, which is quite unlikely, or conversally, that DP molecules poorly contribute to T-cell proliferation induction in DQ- and/or DR-incompatible combinations.
ACKNOWLEDGMENTS
The authors would like to thank Dr. R. Accolla for his gift of BT3.4 MoAb, and C. Hannoun for providing influenza viruses. We also thank Y. Hanin for her excellent technical assistance, and C. Pellet for typing this manuscript. REFERENCES 1. Bach FH: The HLA class II genes and products: The HLA-D region, lmmanol Today 6:89, i985. 2. Shackelford IDA,KanfmanJK, Korman AJ, StromingerJL: HLA DR antigens: Structure, separation of subpopulations, gane dotting, and function, lmmunol Rev 66:133, 1982.
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20. Zeliszewski D, Sterkers G, Freidel AC, Gebuhrer L, Bemel H, Levy JP: Functional definition of HLA-DR and -DQ epitope~ specific for DIL?.-short, DwFJO haplotype. Immunogenetics 23:385, 1986. 21. Zeliszewski D, Sterkers G, Chnppin J, Freidel AC, Gebuhter L, Betuel H, Le',3'JP: An antiviralT-cell clone defines a F~nctional super tyi, i¢ specificity shared by different HI.A-DR molecules fi'om DR2-~hort, DRwll, and DRwI3 haplotypes. Immunogenetics 25:84, 1987. 22. Capra G, Accolla RS: Stroct,,ral analysis of human la antigens reveals the existence of a fourth moleculc.t"subs,.t distinct from DP, DQ. and DR molecules. J Exp Med 165:47, 1987. 23. Cairns JS, Curtsinger JM, DaM CA, Freeman S, Alter BJ, Bach FH: Sequence polymorphism ofHLA Di~.bem I allelesrelating roT-cell recognized determinants. Nature 317:167, 1985. 24. Gruner JP, Watonn AJ, Bach FH: Dw/LD related molecular polymorphism of DR4 beta-chains. J E'~p Med 157:1687, 1983. 25. Sterkers G, Henin Y, Kalil J, Bagot M, Levy JP: Influence of HLA class I and class ii specific monoclonal antibodies on DR-restricted lymphoprolifemtiverespomes. 1. Unseparated populations of effector cells. J lmmunol 131:2735, 1983. 26. Bach FH, Reinsmoen NL: The role of HI.A-DR and HLA-DQ products in T*lymphocyt~ activation and in contributing to "Dw specificities." Hum Immunol 16:271, 1986. 27. Re~nsmoen HI,, Bach FH: Five HLA-D clusters associated with HLA-DR4. Human lmmuunl 4:249, 1982. 28. Morling N, Jakobson BK, Platz P, Ryder I.P, Svejgaard A, Thomsen M: Typing for human alloantigens with the primed lymphocyte typing (PLT) technique with notes on the interpretation of PLT data. Hum Immunol 2:233, 1981. 29. Ryder LP, Thomsen M, Plata P, Sveigaard A: Data reduction in LD typing. In Kissmeyer-Nielsen F, ed: Hismcompatibility Testing 1975. Copenhagen, Munksgaard, 1975, p. 567.