Molecular Localization of LB-Q1, a DRw52- like T-Cell Recognition Epitope and Identification at the Genomic Level of Associated Shared Hybridizing Fragments*

Annemarie Termijtelen, Marcel G.J. Tilanus, Irma Engelen, Frits Koning, and Jon J. van Rood

ABS'IRACT: In this paper we report on the molecular localization of LB-Q1, a supertypic HLA class II determinant which we previously identified by the use of proliferative T cells. The population distribution shows that each of the DRw52 associated specificities DR3, DRS, and DRw6 may occur with and without LB-QI. DNA from nine DR3, six DRS, and 14 DRw6 homozygous B-cell lines was digested with the enzymes Taql, EcoRl, and PvuH. Using a DR[3 cDNA probe, shared hybridizing fragments were observed that correlate completely with the presence or absence of LB-Q1. T-cell recognition of LB-QI can be blocked with a monoclonal antibody (7.3.19.1) which in some haplotypes selectively reacts with the DR[MII chains, but cannot be blocked with a monoclonal antibody (I-LR2) reacting in those same haplotypes exclusively with DR[31 chains. Therefore, LB-Q1 maps to the DR[MII molecule. These data suggest the occurrence of relatively freqt.,ent previous recombinations between the two DR[3 chain geues present in DRw52 haplotypes.

ABBREVIATIONS HLA human leucocyte antigen HTC homozygous typing cell MHC major histocompadbility corn- MoAb monoclonal antibody

plex Facs fluorescence activated cell MLC mixed lymphocyte culture sorter TCGF T<ell growth factor B-LCL lymphoblastoid B-cell line EBV Epstein-Burr virus

I N T R O D U C T I O N

The HLA class II molecules are encoded within the HLA-D region of the human major histocompatibility complex (MHC). These molecules are composed of two noncovalently associated transmembrane glycoproteins of approximately 34,000 daltons (~ chains) and 29,000 daltons (/3 chains), respectively. Based on cellular, serologic, and biochemical analyses, the HLA-D region could be derided in three

From the Department of lmmunohaemotolo~ and Blood Bank, University Hospital. Leider. The Neth- erlands.

Address reprint requests to Dr. Jon J. van Rood, Department of lmmunohaematology ~ Blood Bank. University Hospital. P.O. Box 9600, 2300 RC Leiden, The Netherlands.

*This work was in part supported by the Dutch Foundation for Medical Research ¢Medigon) ¢grant nos. 13.83.01 and 13.29.76) and theJ.A. Cohen Institute for Radiopathology and Radiation Protection (IRS) (grant no. 3.3.1).

Received December 1 l, 1986: accepted May 13. 1987.

Human Immunology 19, 255-267 (1987) © Elsevier Science Publishing Co., inc., 1987 52 Vanderbflt Ave., New York, NY 10017

255 0198-8859187/$3.50

256 A. Termijtelen et al.

subregions, HLA-DP, -DQ, and -DR. Each subregion carries multiple o~ and/3 chai~ genes (reviewed in [1]).

The class II molecules are highly polymorphic. They provide the major stimuli in allogenelc mixed lymphocyte cultures (MLC) and serve as antigen presenting structures (restriction determinants) in T-cell responses to foreign antigens (re- viewed in [2]). The polymorphism o~" T-cell recognition epitopes may be re- sponsible for the MHC specific variation in immune responses. Therefore, the study of class II polymorphism is of considerable interest. Because there are multiple loci within each subregion and multiple class II (subregion) associated T-cell recognition epitopes [3-16], it is important to correlate the loci to products and function. Here we combine cellular and biochemical approaches in analyzing LB-Q1, a polymorphic epitope that induces allogeneic T-cell proliferation [17] and serves as restriction determinant for antigen specific T cells [18]. We show that LB-Q1 is a supertypic epitope representing a subtype of DRw52. This epitope is demonstrated to be encoded by the DR~SIII gene. Shared hybridizing frag- ments, correlating with the presence or absence of LB-Q1, were observed.

MATERIALS AND METHODS

LB-Q1 specific T-cell reagents. Responder lymphocytes were primed in MLC and expanded as described elsewhere [17]. At day 14 of culture, cells were plated in V-shaped microtiter plates (Sterilin, M25ARTL) adding 1, 5, or 10 activated cells and 25,000 irradiated (3000 Rads) cells from the original stimulator to each well. After 10 days, these cultures were transferred to flat bottomed microtiter plates (Greiner, 655160) and 25,000 irradiated stimulator cells (original stimu- lator~ were added to each well. Growing cultures were transferred to 24 well tissue culture plates (Costar, 3524) in 2 ml of tissue culture medium each. Every week 1 × 106 random, irradiated feeder cells were add~.d to each well, and twice a week, 50% of ,~e medium was replaced by fresh medium containing 20% TCGF (lymphocuk-T, Biotest Diagnostics). If cells were overgrowing 50% of the surface, cultures were divided over four wells. When approximately 2 × 106 to 5 × 106 cells were growing, the cultures were frozen in 1 × 106 aliquots and stored at - 196°C until further testing.

B-celllines (B-LCL). Epstein-.Barr virus (EBV) transformed B-cell lines were used for the preparation of DNA samples. The B-cell line panel consisted of DR3, DRS, and DRw6 homozygous typing cells (HTC), as listed in Table 4. Information on the MLC reactivity and the HLA-A, -B, and -C types of the DRw6 and most of the DR3 typing cells has been published [19, 20].

Proliferation assay. A total of 3 to 5 × 103 primed T cells was cocultured with 10 ~ irradiated (2000 Rads) stimulator cells in flat bottomed microtiter plates for 64 hr. Sixteen hours before harvesting, the cultures were labeled with 1/zCi 3H thymidine (NEN, spec. act. 6.7 Ci/mmoi).

Monoclonal antibodies (MoAbs). For inhibition studies the following MoAbs were used: Bl . l .G6 [21] (t3zm); B1.23.22 and B9.12.1 [21] (class I); B8.11.2 [22] (DR); PdV 5.2 (class II); 7.3.19.1 [23] (DRw52-1ike); I-LR2 [24] (DRw52-1ike); IVD12 [25] (DQw3); SPV-L3 [26] (DQ). All MoAbs were ascites, added to the cultures in a 1/300 dilution. This was a highly oversaturated concentration for all MoAbs, as defined by Facs IV binding analyses. Some of the MoAbs obtained from other investigators contained NAN3. These MoAbs were dialyzed against 3 l of Hanks BSS and 1.5 I of RPMI 1640 in three sequential changes. Prior to

Molecular Localization of LB-Q1 257

use, all MoAbs were diluted in RPM11640 and filtered tV, rough 0.45 ~m millipore filters.

MoAb inhibition studies. For inhibition of LB-Q1 reactive T ceils, 50,000 irra- diated (2000 Rads) stimulator cells per well were plated in flat bottomed micro- titer p!.~tes and incubated with MoAb 0.5 hr before addition of 5000 T cells per well. Cultures were incubated for 64 hr and harvested as described above.

DNA analysis. DNA was isolated from EBV transformed B-cell lines according to the method of Gross-Bellard [27]. Restriction enzyme digestions were per- formed as recommended by the manufacturer. The DNA fragments were sep- arated on an 0.7% agarose gel. Treatment of the gel and transfer to nitrocellulose have been described in detail elsewhere [28]. Southern hybridizations were per- formed under standard conditions following the formamide hybridization pro- tocol for gene screen plus nylon membranes (NEN). The DRy8 cDNA probe [29] was radiolabeled in a nick-translation assay [30], using ~-32P-dATP and ~x-3aP - dCTP (NEN). After hybridization, the filters were washed to final wash steps of 2 x SSC, 1% SDS at 65*(2 and 0.1 x SSC at room temperature and exposed to Kodak XS films.

Nomenclature for DR~ genes and gene products in DRw52 haplotypes. Bosch et al. [31] demonstrated that in DRw6 haplotypes the DRw52-1ike MoAb 7.3.19.1 precipitates a DRt8 chain which they referred to as DR~Sa. Sequential precipitation with MoAb B8.11.2 (DR backbone) demonstrated a second DR~ chain, referred to as DRASb. In similar experiments, Karr et al. [32] demonstrated that in DR5 and DRw6 haplotypes the MoAbs 7.3.19.1 and I-LR2 (both DRw52-1ike) pre- cipitate distinct DR/8 chains, referred to as DRt82 and DR/81, respectively. In DR3 homozygous cells the 7.3.19.1 determinant is present on both DR molecules [33].

The DR backbone determinant B8.11.2 is present on DR18I and DR/SIII in all DRw52 associated haplotypes. Previous findings showed that the B8.11.2 determinant was absent from DRtSIII in the DR3 haplotypes [34]. This obser- vation, however, was more recently ascribed to the fact that the affinity between the DR3 DR~8III molecule and the MoAb B8.11.2 is unfavorable for precipitation studies but not for functional assays [33]. Thus, it is presently believed that the DR318III molecule also carries the B8.11.2 epitope (Bontrop et al., submitted for publication). The DR18 chain genes from the DR3 haplotype have been cloned and linked. There are three DRI8 genes, but only two of them, the so-called t3I and ~IlI genes, appear to be able to encode intact DR/8 chains. The DRw6 haplotype has the same organization [35, 36]. A DRw6 ~8II! gene, transfected in mouse L cells, was shown to encode a product recognized by the MoAb 7.3.19.1 ([37], Berte et al., manuscript in preparation), implying that DR~8II! is homol- ogous to DR/Sa and DR182. The interpretation of these combined data is sum- marized in Table 1.

To avoid confusion we only refer to DRI8 genes and chains as to DRy81 and DR~111.

RESULTS

Population Genetics of LB-Q1 Associations betwec.n LB-Q1 and established HLA antigens were derived from a panel of 207 individuals, typed with 2-4 LB-Q1 specific T-cell reagents. All

258

TABLE 1

A. Termijtelen et al.

Clarification of terminology on DR/3 chain genes and products within DRw52

MoAb determinant

HLA-DR Gene Gene pro8uct 7.3. ! 9.1 I.LR2 B8.11.2

DR3 DR~I [35,36] DR~b [33] + NT + DR~III [35,36] DR~a [33] + NT +

DR5 DR~I [36,38] DR~I [32] - + + DRBI|I DRI82 [32] + - +

DRw6 DR~I [35,36] DR~Sb [31] DR~I [32] - + + DR~81II [35-37] DRBa [31] DRI82 [32] + - +

Shared

LB-Q! positive individuals were also DRw52 positive (Table 2). DRw52 is a supertyIoic DR specificity including DR3, DRS, DRw6, and DRw8 [39]. LB-Q1 was significantly associated with DR5 and DRw6, although four out of 39 DR5 positive and 38 out o f 72 DRw6 positive individuals were found that were negative for LB-Q1. DR3 and DRw8 were not significantly associated with LB- Q1 (Table 2). Nevertheless, 13 out of 47 DR3 positive, LB-Q1 positive individ- uals were found, including two of the DR3 homozygous B-LCL.

Hybr id i z ing Fragment s Associated wi th LB-QI

Three different restriction enzymes could generate polymorphic D N A fragments that correlated with the presence or absence of LB~Q1 when a DR/3 chain c D N A probe was used as the hybridization probe. UsingTaqI, the DR/3 probe hybridized with an 8.0-kb band if LB-Q1 was present (n = 14) and with a 7.7-kb band if LB- Q1 was absent (n= 15, Table 3). Results of a representative experiment are depicted in Figure 1. The upper bands in this blot are specific for LB-Q1. In the DR3 homozygous cells CAA, GYS, and WIL and the DRw6 homozygous cells KT11, KRA, CRI, and LIA, which are all LB-Q1 negative, the hybridizing band of 7.7 kb is present. The LB-Q1 positive cells WT49 (DR3 homozygous), Jp , WAP, HLF (DR5 homozygous) 307, APD, O H , and GEV (DRw6 homozygous) share the 8.0 kb hybridizing band. In each lane, the second band from the top correlates with the DR type of these B-cell lines [40]. I f EcoRl was used as restriction enzyme, a band of approximately 3.9 kb correlated with the absence of LB-Q1 (n= 10), whereas a 2.5 kb band was shared between all the LB-Q1

TABLE 2 Association of LB-Q1 with DRw52 and the DRw52 related HLA-DR Srecificities

Group 1 Group 2 + + + _ _ + _ _ r ~ pb

LB-Q1 DRw52 62 0 89 56 0.386 0.0001 LB-Q1 DR3 13 49 34 111 -0.015 NS LB-QI DRw5 35 27 4 141 0.615 0.0001 LB-Q1 DRw6 34 28 38 107 0.264 0.0004 LB-Q1 DRw8 2 60 9 136 -0.037 NS

~Correlation coefficient. ~Fisher's exact p.

Molecular Localization of LB-Q1

T A B L E 3 Shared hybridizing fragments, associated with LB-Q1

259

DR~ cDNA hybridization

B - L C L Laboratory ~ HLA-DR LB-QI Taqi Pvull EcoRi

WIL DR3 - 7.7 b 4.7 +4.9 3.9 HAR DR3 - 7.7 4.7 + 4.9 3.9 AVL DR3 - 7.7 4.7 + 4.9 3.9 CAA DR3 - 7.7 4.7 +4.9 3.9 EYS DR3 - 7.7 4.7 + 4.9 3.9 GYS DR3 - 7.7 4.7 + 4.9 3.9 BL DR3 - 7.7 n.t. 3.9 QBL DR3 + 8.0 4.8 2.5 WT49 WER DR3 + 8.0 4.8 2.5 ATH DR5/DRwl 1 + 8.0 4.8 2.5 THR DRS/DRwl 1 + 8.0 4.8 2.5 WAP DRS/DRwl I + 8.0 4.8 2.5 JP DRS/DRwl I + 8.0 4.8 2.5 JVM DRS/DRwl I + 8.0 4.8 2.5 HLF SVE DRS/DRwl2 + 8.0 4.8 2.5 GP SVE DRw6/DRw13 - 7.7 4.7 +4.9 n.t. HHK DRw6/DRw13 - 7.7 4.7 + 4.9 3.9 WDV DRw6/DRwl 3 - 7.7 4.7 + 4.9 3.9 KRA GSW DRw6/DRwl 3 - 7.7 4.7 + 4.9 3.9 KTI 1 TSU DRw6/DRwl 3 - 7.7 4.7 +4.9 n.t. DLO HAN DRw6/DRw 13 - 7.7 4.7 + 4.9 n.t. APD DRw6/DRw 13 + 8.0 4.8 n.t. FVB NIH DRw6/DRwI3 + 8.0 4.8 n.t. CB BSH DRw6/DRw 13 + 8.0 4.8 n.¢. 307 Ki$ DRw6/DRw 13 + 8.0 4.8 n.¢. OH TSB DRw6/DRwI4 + 8.0 4.8 n.t. GEV FES DRw6/DRwI4 + 8.0 4.8 n.t. CRI LAY DRw6/DRwI4 - " 7.7 4.7 + 4.9 n.t. LIA LAY DRw6/DRwI4 - 7.7 4.7 + 4.9 n.t.

arhe laboratories from which HTCs were obtained are indicated at the relevant places with their identification codes used in the Ninth International Histocompatibility Workshop [38|. 6Specific fragment size in kilobases.

posi t ive cells tested (n = 8). For example, as shown in Figure 2, the hybridizing band o f 3.9 kb is found with the DR3 homozygous, LB-Q1 negative cells AVL, C A A , H A R , BL, and EYS, whereas the D R 3 homozygous cell Q B L and the DR5 homozygous cells A T H , T H R , JP, J V M , and WAP, which are all LB-Q1 posidve, share the corresponding 2.5-kb band. The H L A - D R associated RFLP obtained with Pvul I as restriction enzyme has been described e lsewhere [41]. In the present study we demonst ra te that the presence o f a specific 4.8-kb band cor- relates with LB-Q1 (n = 14), whereas the absence o f this band, but the alternative presence o f 4.7 and 4.9-kb bands are observed in all LB-Q1 negative cells (n = 13). N o discrepancies were found when the above described RFLPs were compared to each o ther or to the cellular typing o f LB-Q1 (Table 3).

I n h i b i t i o n S tud ie s w i t h M o A b s

The molecular localization o f the surface componen t responsible for the T-ceU proliferat ion was studied using monoclonal reagents in an inhibition assay (Table

260 A. Termijtelen et al.

DR3 DR5 D Rw6

÷ ÷ ÷ ÷ ÷ + _ _ + ÷ _ _ LBQ1 4.

6.7

4.3

2.3

FIGURE 1 Hybridization ofa cDNA DRg pr0~ toTaq~ aigested cellular DNA from different DR homozygous B-cell lines. The upper arrow points at the LB-QI specific 8.0- kb band, the lower arrow at the 7.7-kb band present in the absence of LB-Q1.

4). The LB-Q1 specific T-cell reagent c21 was stimulated by either an LB-Q1 positive Dwl8 or an LB-Q1 positive Dw3 HTC. Neither anti-class I and anti- /32 microglobulin nor anti-DQw3 MoAbs were found to be inhibitory. However, anti-DR MoAbs did inhibit.

The results with the antibody pair 7.3.19.1 and I-LR2 are of further interest, because, in the DR5 and DRw6 haplotypes, precipitation studies demonstrated ~hat these MoAbs can distinguish between DRgI and DRgIII gene products (Table 1). In DR3 homozygous cells, 7.3.19.1 reacts with both DRg chains whereas no precipitation data of I-LR2 are available. As shown in Table 4, only the 7.3.19.1 MoAb was inhibitory. These results were extended using a total of four DRg, three DRw6, and three DR3 LB-Q1 positive stimulator cells (Figure 3). In this experiment, the DQ monomorphic MoAb SPV-L3 was included. Although all the MoAbs used showed good binding to the cells according to FACS analysis, only the DR monomorphic MoAb B.8.11.2 and the DRw52-1ike MoAb 7.3.19.1 inhibited. Four other LB-Q1 specific T-cell reagents showed identical inhibition patterns when tested (data not shown).

DISCUSSION In the present study we analyzed ~ supertypic determin~t LBQ~, which we previously defined as an allogeneic T-cell recognition epitope [ 17]. It was recently shown that in eight DR3 homozygous B-cell lines a protein and DNA po|ymor-

Molecular Localization of LB-Q1 261

kb

9.5

6 .7

4.3

2.3

F I G U R E 2

DR3 DR5

4. m m 4. + 4. 4. 4" LBQ1

Hybridizat ion o f a c D N A DRI3 p robe to EcoRI digested cellular D N A from different D R homozygous B-cell lines. Arrows indicate the 3.9 and the 2.5 kb fragments associated with the absence or presence of LB-Q1.

TABLE 4 Inhibition of LB-Q1 specific proliferation of c21 by the addition of different MoAbs to the cultures

Stimulator cells

MoAb Specificity APD (Dwl8 HTC) QBL (Dw3 HTC)

- - 26,670 ~ 19,960 B 1. I.G6 ~zm 25,600 16,370 BI.23.22 class 1 25,360 15,000 B9.12.1 class I 24,380 20,296 B8.11.2 DR 180 13_l q PdV5.2 class l! 1,98.5 14_~5 7.3.19.1 DRw52-1ike 225 18._~5 I-LR2 DRw52-1ike 18,490 13,zt¢~0 IVD12 DQw3 ~ 18,590 14,490

qn counts per minute. Inhibited reactions are underlined. 6Because APD and QBL are DQw3 negative, this MoAb was included as a negative control.

262 A. Termijtelen et al.

A(n=31 ~ in=/.! Cin=3}

~ so N~

FIGURE 3 C21 was stimulated with DR3 positive (A), DR5 positive (B), or DRw6 positive (C) LB-Q1 positive stimulator cells in the presence of no MoAb (control), 7.3.19.1 (DRw52-1ike), B8.11.2 (DR mono- morphic), I-LR2 (DRw52-1ike), or SPV-L3 (DQ mono- morphic). Standard deviations reflect variations found between different stimulator cells.

phism could be detected correlating with LB-Q1 [33]. We now demonstrate that LB-Q1 is a polymorphic determinant "included" in DRw52: it is only expressed by less than half of the DRw52 positive donors in our panel. Hybridizing DR/3- DNA fragments correlating with the presence or absence of LB-Q1 were shared between DR3, DR5, and DRw6 homozygous B-cell lines (Figures 2 and 3, Table 3). This finding implies that it is possible to type for this T-cell recognition epitope in the absence of T-cell reagents. Similarly, it was recently shown that an HLA- DRw53-1ike restriction element for cloned, antigen specific, T cells was present in five out of five DR4 positive, but only in five out of seven DR7 positive individuals. The presence or absence of this restriction element correlated with the presence or absence of a Dgw53-1ike DR/3-DNA hybridizing fragment [42].

tn the DRw52 group of haplotypes, a number of observations have already been reported as to the complexity of the DR molecules. There are two DR// genes,/3I and/3III, that encode intact DR/3 chains. Studies with MoAbs and alloan,isera demonstrated that DRw52 is a heterogeneous antigen, ,~ot defined by one single epitope and that DRw52-1ike epitopes can be present on both DR/3 chains ([32, 43, 44] and Table 1). DRw52-1ike epitopes were detected on separate DR molec~,les using cloned T-cell lines [45]. In addition, a polymorphism at the DR/3III gene, as defined by oligonucleotide genotyping, splits DRw52 in DRw52a and DRw52b [46].

In our inhibition studies, we included the DRw52-1ike MoAbs 7.3.19.1 [24] and I-LR2 [25]. By means of two-dimensional gel electrophoreses the DRw52- like determinants defined by these MoAbs were previously found to reside on different DR molecules on DR5 and DRw6 homozygous B-cell lines. Whereas 7.3.19.1 precipitates the DR/3III molecule, the I-LR2 determinant is only present on the DR/3I molecule (Table I). Only 7.3.19.1 could inhibit proliferation of LB-Q1 specific reagents against DR5 and DRw6 positive stimulator cells. The capacity of I-LR2 to inhibit T-cell proliferation was demonstrated using Dw6 specific cloned T cells (unpublished observation). Thus we conclude that the T- cell recognition epitope LB-Q1 is in DR5 and DRw6 haplotypes, if present, located on the DR/3III molecule. In DR3 homozygous cells, 7.3.19.1 is present on both DR molecules. I-LR2, according to Facs analyses, binds to DR3 positive cells, but it is not known whether it binds in this haplotype to one or both DR/3 chains. Again only 7.3.19.1 could inhibit proliferation. The most likely expla- nation for this finding is that also in the DR3 haplotypes, LB-Q1 can be present on DR/3III but not on DR/3I, whereas I-LR2 is present on DR/3I and absent from DR/31II, comparable to the situation in the DR5 and DRw6 haplotypes.

Molecular Localization of LB-Q1

OR genes DR haptotypes

DR3

DR5

DRw61W13

ORw61w14

•l I I l l l

, 3 ÷

E2ZX2-

- C - g " ' - I I - k -

I ÷ b-- E=X/XZa-

E=Z=3-

263

FIGURE 4 A schematic repre- sentation of the observed combi- nations of DR~ genes in diftbrent DRw52 hapbtypes. The genes marked + are LB-Q1 positive, the genes marked - are LB-Q1 neg- ative.

As shown in Table 3, the DRw6/DRwl 3, DRw6/DRwl4, and DR3 haplo~pes can all occur in the presence or absence of LB-Q1. The majority of DR5 positive individuals is also LB-Q1 positive, but four out of 39 DR5 positive individuals were found that were LB-Q! negative (Table 2). We assame that the serolo#cally defined DR specificities DR3, DR5, and DRw6 are encoded by DR~I, based on the following observation: the DR/31 gene is more polymorphic than the DRI3III gene; the nucleotide sequences of the first domain of DRt3I are different for DR3, DR5, and DRw6 haplotypes, whereas the nucleotide sequence of the DR/3III gene of a DR3 haplotype is identical to that of a DRw6 haplo~pe [36]. On the other hand, LB-Q1 was in the present study shown to be encoded by DR/3III. As depicted in Figure 4, at least eight different DR haplo~'pes can now be constructed, because DR3, DR5, DRw6/w13, and DRw6/wl4 may all occur with and without LB-Q1.

The association of each single DRI3I gene with the two different DRt3III genes could first be explained by at least four mutations resulting in a loss of the LB- Q1 determinant in part of the DR3, DR5, DRw6/w13, and DRw6/w14 haplo- types. Second, gene conversion-like events r~,~y have been involved; the DRt3m gene from one DR haplo~pe may have functioned as a donor gene for the stretch of DNA encoding LB-Q! (or the loss of LB-Q1) received by an other DR haplotype. Third, recombinations between DR~I and DR~III genes could be responsible for the DR/3I-DRI3IH combinations depicted in Figure 4. Such re- combinations have recently been suggested [46]. In that study, a DRt3III poly- morphism, DRw52a and DRw52b, was described based on oligunucleodde gen- otyping. DRw52a and DRw52b were associated with DR3, 5, and w6 in a similar manner as LB-Q1. As a possible explanation for these associations, recombina- tions between/3I and/3III were postulated. The present DNA data demonstrate that in the change from LB-Q1 posifivity to negativity (or vice versa) not on|y a single epitope, i.e., LB-Q1, but also a number of restriction sites must have been involved. These restriction sites will encompas a relatively large stretch of DNA, which makes mutation and gene conversion very unlikely. Therefore, we feel that our data strongly support the latter hypothesis, implying a number of previous recombinations between DR/3I and DR/3III genes present in DRw52 haplorypes. It is interesting to note that such recombinations have been restricted to the DRw52 haplotypes because none of the other DR specificities (i.e., DR1, 2, 4, 7, w9, or wl0) were, to our knowledge, ever observed in association with se- rologically or cellularly detectable DRw52-1ike determinants.

264 A. Termijtelen et al.

ACKNOWLEDGMENTS We thank Drs. J. Gorski, M.L Bosch, R.E. Bontrop, R.R.P. de Vries, and J. D'Amaro for stimulating discussions and Mrs. L Suwandi-Thung for technical assistance. We also thank Ms. T. van Westerop and Mrs. M. Hartog for typing the manuscript. We are grateful to Dr. B. Malissen, (B1.LG6; BI.23.22; B9.12.1; BS.11.2), Dr. L Nadler (I-LR2), Dr. J.D. Capra (IV D12), and Dr. H. Spits (SPV L3) (DQ) for generously making available MoAbs, and to Dr. E. Long for kindly providing the DRI3 cDNA probe.

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