Proc. Nail. Acad. Sci. USAVol. 85, pp. 227-231, January 1988Immunology

Transfer and expression of three cloned human non-HLA-A,B,Cclass I major histocompatibility complex genes in mutantlymphoblastoid cells

(additional human histocompatibility complex molecules/HLA-A,B,C-nufl mutant lymphoblastoid cells)

YOJI SHIMIZU*, DANIEL E. GERAGHTYt, BEVERLY H. KOLLERt, HARRY T. ORRt, AND ROBERT DEMARS*t*Laboratory of Genetics and Department of Human Oncology, University of Wisconsin, Madison, WI 53706; and tDepartment of Laboratory Medicine andPathology and the Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455

Communicated by Ray D. Owen, September 21, 1987 (received for review August 13, 1987)

ABSTRACT The HLA-A, -B, and -C class I human his-tocompatibility antigens and the genes that encode them havebeen isolated and characterized. Apparently complete class Inon-HLA-A,B,C genes have been identified on HindU-gener-ated 5.4-kilobase (kb), 6.0-kb, and 6.2-kb DNA fragmentsderived from lymphoblastoid cell line (LCL) 721. We studiedthe expressibility of these genes by subcloning them into thenonintegrating pHeBo vector and transferring the chimericplasmids into mutant LCL 721.221. This mutant was derivedfrom LCL 721 by means of immunoselections following Praymutagenesis that eliminated expressions of the HLA-A, -B, and-C a chains. The HLA-A,B,C-null phenotype of mutant721.221 made it possible to monitor the expression of class Igenes transferred into it by assaying cell surface binding ofmonoclonal antibodies BBM.1 and W6/32, which recognize.82-microglobulin and HLA class I a-chain epitopes, respec-tively. Increased binding of BBM.1 and W6/32 was clearlyobserved in transferents containing the class I gene of the6.0-kb DNA fragment but not in transferents containing theclass I genes of the 5.4- and 6.2-kb DNA fragments. However,one-dimensional gel electrophoresis of BBM.1 and W6/32immunoprecipitates made with ["S]methionine-labeled celllysates showed that transfer of each non-HL4-AB,C class Igene into 721.221 resulted in the appearance of an a chain thatcoprecipitated with P2-microglobulin. The three previouslyunreported a chains differed from each other in size and weresmaller than HLA-A, -B, and -C a chains. These observationsclearly show that these three cloned, nonallelic, non-HLA-A,B,C class I genes encode a chains that can be expressed inhuman cells.

Human class I histocompatibility (HLA) antigens are presenton most kinds of nucleated somatic cells and are part of thestructures that are recognized on target cells by autologousand allogeneic cytotoxic T lymphocytes (1). Each class Imolecule is formed by noncovalent association between an achain (-44 kDa) and 832-microglobulin (p2m; =12 kDa) (2).The f3 chain is encoded by a nonpolymorphic gene onchromosome 15 (3). In contrast, highly diverse polymor-phism in class I a chains has two sources: (i) the known achains are encoded by three closely linked loci, HLA-A, -B,and -C, in the major histocompatibility complex (MHC) onthe short arm of chromosome 6 (4); and (ii) each of the threeknown HLA class I loci is highly polymorphic. It is thoughtthat the polymorphism of class I antigens is important fortheir functions in immune response.The murine homologues of the HLA-A, -B, and -C antigens

are the H-2K, -D, and -L antigens. In addition, there are >26MHC-linked class I-related a-chain genes in the murine Qa,

Tia, H-2L, and H-2D regions (reviewed in refs. 5 and 6). achains encoded by these genes also associate with (32m, butthose that are known to be expressed are much less poly-morphic than the H-2K, -D, and -L a chains. The functionsof the non-H-2K,D,L antigens are unknown, but the expres-sions of some of them by only some somatic cells (e.g., Q10in liver and Tla by thymocytes and certain T-cell leukemias)suggest that they may have specialized roles in immunity.Serological evidence (see Discussion) suggests that non-HLA-A,B,C class I genes and antigens also exist in humans,but they have not been defined as fully as in the mouse. Thedefinition of such antigens is the goal of the researchdescribed below.Our laboratories have cloned the class I genes of the

Epstein-Barr virus-transformed lymphoblastoid cell line(LCL) 721. The combined use of 'y ray-induced mutants andtransfer of cloned class I genes has resulted in identificationof the HLA-A, -B, and -C genes in LCL 721 (refs. 7-10; alsounpublished data). In addition, nucleotide sequencing indi-cates that 5.4-kilobase (kb) (D.E.G., B.H.K., and H.T.O.,unpublished observations), 6.0-kb (11), and 6.2-kb (D.E.G.,B.H.K., and H.T.O., unpublished observations) HindIII-generated DNA fragments contain intact non-HLA-A,B,Cclass I genes (12). We describe here evidence for theexpressivity of these genes, which for simplicity we call"5.4", "6.0," and "6.2" genes, in a system developed forassaying the expression of normal and altered class I genes inhuman lymphocytes. Each gene was inserted into the non-integrating pHeBo vector (13) and introduced into mutanthuman LCL 721.221 (called ".221"; see below), which isdevoid of HLA-A, -B, and -C expression but in whichtransferred HLA-A, -B, and -C genes are expressed.

Excepting H-2Db (14), mouse class I a chains-Qa and TIas well as H-2K, -D, and -L--are associated with 132m at thecell surface. Therefore, lacking antibodies that we knewwould recognize non-HLA-A,B,C class I a chains, weassayed expression of transferred human class I genes withmonoclonal antibody (mAb) BBM.1, which binds to free ora chain-associated 82m (15). We also used mAb W6/32,thinking it possible that this mAb, which recognizes deter-minants formed by association of HLA-A, -B, or -C a chainswith f32m (16), might also bind to non-HLA-A,B,C class Imolecules. The known class I a chains become associatedwith f32m during their posttranslational processing within thecell, and neither chain is present at the lymphoblastoid B-cellsurface unless associated with the other. Therefore, weexpected that appearance of class I a chains after genetransfer into .221 might be manifested (i) by the appearance

Abbreviations: P2m, P2-microglobulin; HPRT, hypoxanthine/gua-nine phosphoribosyltransferase; LCL, lymphoblastoid cell line;MHC, major histocompatibility complex; mAb, monoclonal anti-body.tTo whom reprint requests should be addressed.

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within the transferent cells of electrophoretically detectablemolecules that were immunoprecipitated with BBM.1, W6/32, or both; and (ii) by increased flow cytometer-assayedbinding of BBM.1 or W6/32 to the surface of transferentcells. These assays revealed that each of the S.4, 6.0, and 6.2class I genes encodes a characteristically sized a chain, oneof which (product of 6.0) is readily detectable at the cellsurface.

EXPERIMENTAL PROCEDURESCells, Media, and Mutagenesis. The origin and y-ray mu-

tagenesis of the Epstein-Barr virus-transformed lymphoblas-toid B-cell line 721 and the isolation of HLA antigen-lossmutants have been described (17). y rays deleted the entireshort arm of one chromosome 6 in mutant .45 (18), whichretains just one copy of the MHC [HLA-A2; -B5; Cm§].Complementary deletion mutant .114 was isolated by select-ing against the HLA-A2 phenotype (19) and retains only theHLA-AJ; -B8; Cp§ haplotype. Mutant .184 TGr3 was derivedfrom .45 and is a hypoxanthine/guanine phosphoribosyltrans-ferase-negative (HPRT-) derivative (10) of .184, which hashomozygous deletions of the HLA-A and -B loci but stillexpresses the maternal HLA-C allele; TGr refers to thiogua-nine resistance (20). Details of the derivation and propertiesof HLA-A,B,C-null mutant .221 will be described elsewhere.Stock cultures were maintained at about 3-10 x 105 cells perml in RPMI 1640 medium (21)/heat-inactivated fetal bovineserum, 85:15 (vol/vol).Recombinant DNA Constructions and Gene Transfers. The

following human class I genes, all cloned from LCL 721, wereinserted as HindIII-generated DNA fragments into theHindIIl site of the pHeBo expression vector (10): HLA-Al[the 4.7-kb fragment from cosmid 23.1 (12)]; HLA-A2 [the5.1-kb fragment from plasmid pHLA-2a (22)]; HLA-5.4, -6.0,and -6.2 [5.4-kb, 6.0-kb, and 6.0-kb HindIII generated frag-ments, the cloning of which is described elsewhere (refs. 11and 12; also unpublished data)].The HLA-B8 gene was cloned in the HPRT-bearing vector

pHPT32 (23) and was transferred in the resultant plasmidpHPT(B8) as described (10). An HLA-C allele was identifiedon a 7.6-kb EcoRI-generated DNA fragment in cosmid 37.1(12) by using a locus-specific probe (22) prepared from the 3'untranslated region of the HLA-C gene (24). This DNAfragment was excised from the cosmid and inserted into theEcoRI site of pHPT32 to form plasmid pHPT(C), which wasused to transfer the HLA-C gene.These plasmids were introduced into HLA-class I-null

mutant .221 (see below) by means of electroporation, andhygromycin-resistant (pHeBo plasmids) or HPRT+ (pHPT 32plasmids) transferents were isolated as described (10).DNA and RNA Analysis. Southern blotting analysis of

total-cell DNA and extrachromosomal Hirt-extract DNA wasperformed as described (10) by using a "general" class I(HLA-B7) cDNA probe (25). Blot-hybridization analysis wasperformed with cytoplasmic RNA and either the HLA-B7cDNA probe and nonspecific conditions or the locus-specificprobes and specific conditions (22).mAbs and Antibody-Binding Assays. Hybridomas BBM.1

and W6/32 were obtained from the American Type CultureCollection. BBM.1 binds to 182m that is either free orassociated with an a chain (15). W6/32 binds to a determinant

§Two expressed HLA-C alleles are presented in LCL 721, but fourtyping laboratories have failed to attain consistent HLA-C typingwith alloantisera. Therefore, Cm denotes the HLA-C allele in thematernal haplotype ofLCL 721 (present in .184TGr3, below) and Cpdenotes the allele in the paternal haplotype. Cx (Fig. 1B) indicatesthat the identity of the LCL 721 haplotype from which the clonedHLA-C gene was derived is unknown.

formed by the association of 82m with HLA class I a chains(16). Binding of the mAbs, used at saturating concentrations,was assayed with a Coulter Electronics EPICS C flowcytometer as described (10).

Immunoprecipitations. Radiolabeling of cell proteins, im-munoprecipitation, and one-dimensional electrophoresiswere performed as described by Jones (26) with the followingmodifications. Cells (107) in logarithmic phase were centri-fuged and resuspended in 2 ml of methionine-free RPMI 1640medium containing 5% fetal calf serum. L-[35S]Methionine(Amersham) was added (100-150 ,Ci; 1 Ci = 37 GBq) to thecells, which were placed in a 37°C CO2 incubator for 4-5 hr,with mixing every 30 min. Preparation of cell extracts was asdescribed (26). Labeled cell extracts were precleared twicebefore immunoprecipitation by incubating 100 ,ul of extractwith 20 ,ul of packed and prewashed Pansorbin (Calbiochem)for 15 min at 4°C. For each immunoprecipitation, 100 ,ul ofthemAbs BBM.1, W6/32, or OKT4 (anti-CD4, used here as anegative control), 200 ,ul of Pansorbin, and 100 A1l of pre-cleared extract were used. Electrophoresis of labeled im-

Log fluorescence

FIG. 1. HLA-A,B,C-null mutant .221 expresses transferred classI genes. Binding of mAb BBM.1 (anti-.82m) (A-C) or ofmAb W6/32(D and E) was assayed with a flow cytometer; fluorescence inten-sities are displayed on a horizontal logarithmic scale. (A) LCL .45 hasone copy of the MHC (HLA-A2, -BS, -Cm) and .184 TGr3 hashomozygous deletions of the HLA-A and -B loci but expressesHLA-Cm. Mutant .221 was derived from .184 TGr3 and lacksexpressions of HLA-A, -B, and -C. (B) Transferents (Tr) pHPT(HLA-B8) -. .221 [Tr(B8)] and pHPT (HLA-C) -- .221 [Tr(C)]contain cloned HLA-B8 and -Cx§ genomic genes transferred into .221by means of electroporation. Transferent pHeBo (5.4) -- .221[Tr('5.4')] contains the transferred HLA-5.4 class I gene introducedinto .221 with the pHeBo vector. (C) Transferents pHeBo (6.0) -*

.221 [Tr('6.0')] and pHeBo (6.2)--.221 [Tr('6.2')] contain transferredHLA-6.0 and -6.2 class I genes, respectively. (D and E) Binding ofmAb W6/32 (anti-HLA-A,B,C). See A-C for explanation of the celllines.

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1 2 3 4 5 6 7 8

-66.0'4~E__ 4 -43.0

-24.0

1 2 3 4 5 6 7 8F': --o

IYf.4

-66.0

: =__-43~~4.0

.:: -~~24.0

_ _ ~~~~18.4- ~~~~~14.3

FIG. 2. Immunoprecipitation with mAb BBM.1 (anti-p2m) shows that the HLA-5.4, -6.0, and -6.2 class I genes encode non-HLA-A,B,C achains. Extracts of cells labeled in medium containing [35S]methionine were subjected to immunoprecipitation with mAb BBM.1 (lanes 2, 4,6, and 8) or with negative control mAb OKT4 (lanes 1, 3, 5, and 7) and then to one-dimensional electrophoresis in a NaDodSO4 gel followedby autoradiography. (Left) Comparison ofimmunoprecipitates made from HLA-A,B,C-null mutant .221 (lanes 1 and 2) with immunoprecipitatesmade from .221 transferents containing transferred HLA-5.4 (lanes 3 and 4), -6.2 (lanes 5 and 6), or -6.0 (lanes 7 and 8) class I genes. (Right)Comparison of a .221 transferent containing a transferred 6.0 gene (lanes 7 and 8) with .221 containing only the transferred pHeBo vector (lanes3 and 4) or a transferred HLA-AJ gene (lanes 5 and 6). LCL .114 is a deletion mutant that expresses one copy (HLA-Al, B8, C) of the MHC(lanes 1 and 2). Note the presence of two BBM.1-precipitable molecules just below the actin position in lane 4. Sizes are shown in kDa.

munoprecipitates was performed as described (26) withNaDodSO4/10% polyacrylamide gels for 3 hr at 40 mA. Gelswere stained for 30 min in 0.2% Coomassie blue/20%omethanol/10% acetic acid, destained overnight in twochanges of 20% methanol/10% acetic acid, washed in dis-tilled water for 30 min, and soaked in 1 M sodium salicylatefor 30 min before drying.

RESULTSHLA-A,B,C-Null Mutant .221. LCL mutant .184 TGr3 is

HPRT- and is phenotypically HLA-A,B-null because of yray-induced homozygous deletions of the HLA-A and -B loci(10, 20). The HLA-A2 gene cloned in the pHeBo vector,

1 2 3 4 5 6 7 8

- -~~~~~~~~66.0'-_43 0 "W. -43.0_~ q_W

-24.0;:

-14.3

FIG. 3. Immunoprecipitability by mAb W6/32 indicates that thea chains that are endogenously expressed in .221 (lanes 1 and 2) orthat are expressed in .221 transferents containing transferred HLA-5.4 (lanes 3 and 4), -6.0 (lanes 5 and 6), or -6.2 (lanes 7 and 8) genesare related to the HLA-A, -B, and -C a chains. [35S]Methionine-labeled cell extracts were immunoprecipitated with control mAbOKT4 (lanes 1, 3, 5, and 7) or with W6/32 (lanes 2, 4, 6, and 8) andsubjected to one-dimensional electrophoresis in a NaDodSO4 gel.Note the two W6/32-precipitable molecules just below the actinposition in lane 2. Sizes are shown in kDa.

which does not insert into chromosomes of Epstein-Barrvirus-transformed LCLs, is fully expressed after transfer into.184 TGr3 (10). To obtain a completely class I-null cell line,we treated .184 TG13 (107 cells) with yrays (300 R; 1 R = 0.258mC/kg) and used complement plus mAb W6/32 to select forclass I-null derivatives. Mutant .221 was found among thesurvivors, and complement-dependent microcytotoxicitytests confirmed that it was refractory to specific lysis by mAbW6/32 plus complement (data not shown).The properties of mutant .221 will be described in detail

elsewhere (Y.S. and R.D., unpublished data) and can besummarized as follows: (i) flow cytometry showed that .221specifically binds little or no mAb BBM.1 (Fig. 1A) or mAbW6/32 (Fig. 1D); (ii) apparently intact HLA-5.4, -6.0, and-6.2 genes are present (there was slight expression of twonon-HLA-A,B,C class I a chains) (Figs. 2 Right and 3); (iii)class I mRNA detectable with the HLA-B7 cDNA probe wasnot detected in .221 (Fig. 4); and (iv) LCL 721-derivedgenomic HLA-Al, -A2, -B5, -B8, and -Cx genes cloned into

1 2 3 4 5 6

*1~~~~~~~~~~~~~~~FIG. 4. Cytoplasmic mRNA transcripts of the HLA-5.4 gene are

smaller than HLA-A, -B, and -C transcripts. Cytoplasmic RNA wasisolated from LCL 721 (lane 1), .221 (lane 2), a control .221transferent containing the pHeBo vector (lane 3), and independentpHeBo (5.4) --+.221 transferents Trl (lane 4), Tr24 (lane 5), and Tr28(lane 6) containing the transferred HLA-5.4 gene. The RNA sampleswere subjected to electrophoresis and RNA transfer blotting analysiswith the HLA-B7 cDNA general class I probe.

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pHeBo or pHPT vectors and transferred into .221 wereexpressed at levels seen in parental LCL 721 chromosomalcounterparts (Figs. 1B, 2, and 3; also data not shown). Withthe assurance that known class I genes could be expressedafter transfer into .221, we sought evidence concerningexpression of transferred non-HLA-A,B,C class I genes.

Expression of the HLA-5.4, -6.0, and -6.2 Genes. The threenon-HLA-A,B,C genes were individually inserted intopHeBo vectors and transferred into .221 by means ofelectroporation, and hygromycin-resistant transferents wereisolated. Initially, binding of mAb BBM.1 was quantified byflow cytometry in order to detect the de novo appearance ofclass I antigens on the cell surface. Increased binding ofBBM.1 to 12 of 14 independent pHeBo (6.0) -- .221 trans-ferents analyzed was unmistakable (Fig. 1C). The meanamount of BBM.1 bound by the pHeBo (6.0) -+ .221transferents was 60%o of that bound to HLA-C on .184 TGO3cells. None of 43 pHeBo (5.4) -* .221 (Fig. 1B) and 24 pHeBo(6.2) -* .221 (Fig. 1C) transferents bound significantly moreBBM.1 to their surfaces than did control .221 transferentscontaining the pHeBo vector alone (not shown).The binding of mAb W6/32 to transferents paralleled that

of BBM.1-i.e., W6/32 distinctly bound to pHeBo (6.0) -+

.221 transferents but not to the pHeBo (5.4) -* .221 and (6.2)-+ .221 transferents (Fig. 1E). However, one-dimensionalNaDodSO4 gel electrophoresis of BBM.1 immunoprecipi-tates (Fig. 2) and W6/32 immunoprecipitates (Fig. 3) from[35S]methionine-labeled cell extracts revealed that 5.4, 6.2,and 6.0 gene-containing transferents expressed a chains thatcoprecipitated with 82m (Fig. 2 Left) and were smaller thanHLA-A, -B, and -C a chains (Fig. 2 Right, lanes 2 and 6). Themolecular weights of the a-chain products of the 5.4, 6.2, and6.0 genes (Fig. 2 Left, lane 4) were judged to be about 40, 41,and 38 kDa, respectively. These results show that the 5.4,6.0, and 6.2 class I genes are expressible and encode a chainsthat associate with j32m. All three chains are closely enoughrelated to the HLA-A, -B, and -C a chains to share with themdeterminants that are recognized by mAb W6/32.The abundance of each non-HLA-A,B,C a chain was

judged to be <1% of the summed abundances of the HLA-A,-B, and -C a chains in mutant .114, which has just one copyof the MHC (Fig. 2 Right, lane 2). The relation of a-chainabundance to the 5.4, 6.0, and 6.2 DNA and RNA content ofthe transferents will be described in detail elsewhere. How-ever, one observation about the 5.4 mRNA is presented herebecause it may be useful in determining the relation betweenthe 5.4 gene and non-HLA-A,B,C genes studied by others.Fig. 4 shows that all three pHeBo (5.4) -- .221 transferentsthat were analyzed contained a class I-related mRNA thatwas distinctly smaller and far less abundant than the HLA-A,-B, and -C mRNAs detectable in LCL 721 with the HLA-B7cDNA probe.The absence of HLA-A, -B, and -C a chains in .221 cells

has allowed us to make another unusual observation bymeans of immunoprecipitation with BBM.1. In Fig. 2 Right,lane 4 clearly shows the presence of two j82m-associatedmolecules in a control transferent into which only the pHeBovector was transferred. The sizes of these two moleculescorresponded to those of the a chains encoded by 5.4 and 6.2,and both are apparently class I a chains: W6/32 immuno-precipitated two molecules of apparently the same sizes froma pHeBo -* .221 transferent (Fig. 3, lane 2). Therefore, thereis at least slight expression of two non-HLA-A,B,C f82m-associated molecules in the lymphoblastoid B cells westudied.

DISCUSSION

The isolation of HLA-A,B,C-null lymphoblastoid B-cellmutant .221 and the demonstration that cloned HLA-A, -B,

and -C genes are expressed normally after their transfer into.221 defines a system for assaying the expressibility andcharacterizing the products ofnormal and altered HLA-A, -B,and -C class I genes and of non-HLA-AB,C class I genes inhuman cells. DNA sequencing has shown that the class Isegments on 5.4-kb, 6.0-kb, and 6.2-kb HindIII-generatedDNA fragments from LCL 721 are clearly candidates forbeing such genes (refs. 11 and 14; also unpublished data),whereas mapping with deletion mutants has shown that theyare located distal to the HLA-C locus in the MHC (ref. 12;also unpublished data). Transfer ofthe HLA-5.4, -6.0, and 6.2genes into .221 is followed, in each case, by the appearanceof a molecule that is coprecipitated with f32m (Fig. 2). Eachof these molecules is related to the HLA-A, -B, and -C achains because each is precipitable by mAb W6/32 (Fig. 3).The three non-HLA-A,B,C a chains are not fusion proteinsresulting from recombination of the transferred class I seg-ments with chromosomal DNA because the 5.4, 6.0, and 6.2genes were introduced into .221 in physical association withthe pHeBo vector: such plasmids replicate as extrachromo-somal elements in mutant derivatives ofLCL 721 (10), and wedirectly confirmed the presence of the three non-HLA-ABCgenes on extrachromosomal plasmids in the transferentsstudied here (not shown). The observation that the threenon-HLA-A,B,C a chains that are precipitable by anti-classI mAbs differ in size from each other and from HLA-A, -B,and -C a chains is additional strong evidence that they areencoded by the transferred genes.While the HLA-5.4, -6.0, and -6.2 genes can be expressed

in lymphoblastoid B cells when they are part of extrachro-mosomal plasmids, some evidence for expression of theirchromosomal counterparts also exists. The presence ofRNAtranscripts of the 6.2 gene in diverse normal and abnormalcells including LCL 721 has prompted its designation ashuman class I gene HLA-E (unpublished data). The absenceof the HLA-A, -B, and -C antigens on mutant .221 has nowallowed us to use the allo-indifferent anti-class I mAbsBBM.1 and W6/32 to demonstrate the existence of an HLA-Ea chain in association with f32m in pHeBo(6.2) -- .221transferents. Furthermore, Fig. 2 Right (lane 4) and Fig. 3(lane 2) show small amounts of an endogenously producedclass I a chain that corresponds in size to the HLA-6.2 achain. Very little information about expression of the chro-mosomal HLA-5.4 and -6.0 genes in any type of cell exists atpresent except for the presence of a small amount of whatmay be the HLA-5.4 a chain in .221 (Fig. 2 Right, lane 4 andFig. 3, lane 2).The existence of a chains encoded by the HLA-5.4, 6.0,

and -6.2 genes and the paucity of their expression must nowbe reconciled with the apparent presence of all three of thegenes in .221 (not shown). Our results with .221 and itstransferents show that expression of the genes is regulated inat least two ways in lymphoblastoid B cells. First, while thegenes clearly are transcribable, transcripts of the chromo-somal genes are rare (5.4 and 6.2) or absent (6.0) (not shown).Second, presence of the HLA-5.4 and -6.2 a chains intransferents was not manifested as increased binding ofmAbsBBM.1 and W6/32 to cell surfaces. This failure probably didnot result from inadequate quantity; the quantities of thesechains were similar to that of the HLA-6.0 a chains (Figs. 2Left and 3), which strikingly increased surface binding ofBBM.1 and W6/32. Therefore, the HLA-5.4: and -6.2 achain-f2m dimers (i) may be secreted, (ii) may be present inthe cell membrane but inaccessible to external antibody, or(iii) may not be transported to the cell surface. Appropriatecell fractionations will resolve this issue, but it is worth notingevidence for large differences in transport of murine H-2Kk:and H-2Dk a chain f32m dimers to the cell surface, resultingfrom differential efficiency of processes that occur in theendoplasmic reticulum (27). This suggests the interesting

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possibility that the HLA-5.4, -6.0, and -6.2 a chains might bedisplayed on the surfaces of cells in which such processes aremore efficient for these non-HLA-A,B,C molecules than theyare in lymphoblastoid B cells. Alternatively, the normalfunctions of the HLA-5.4, -6.0, and -6.2 a chains may notinclude cell surface display of the kind involving HLA-A, -B,and -C a chains.A report of expression of a sequenced non-HLA-A,B,C

gene after its transfer into mouse L cells recently appeared(28). While the reported gene resembles the HLA-6.2 gene insome respects, several differences in their sequences andbehavior prompt hesitation before concluding they are inde-pendent isolates of the same gene. Previous studies ofnon-HLA-A,B,C antigens have emphasized antigen expres-sions of human T lymphocytes because of possible analogieswith mouse Qa and Tla antigens. According to this guide, theHTA1/T6 molecule can be excluded from considerationbecause it is encoded by a non-class-I gene that is not locatedon chromosome 6 (29, 30). Available information (e.g., refs.31-45) about other f2m-associated human T-cell antigensdoes not permit their definite identification with the HLA-5.4, -6.0, or -6.2 molecules. Paul et al. (46) have described acloned, class I gene encoding a cell surface antigen that isrecognized on activated T lymphocytes by specific al-loantisera. Since the antigen described did not bind mAbW6/32, it probably is not the HLA-6.0 molecule describedherein. Vasilov et al. (47) noted at least two W6/32- andanti-j2m-precipitable T-cell molecules that were smaller thanthe HLA-A and -B a chains. It may be feasible to determinethe relationship between those molecules and the non-HLA-A,B,C molecules described here by means of coelectropho-resis of transferent and T-cell immunoprecipitates.So far, TCA antigens (40) correspond most closely, but not

yet conclusively, to the HLA-6.0 antigen: their a chains aresmaller than HLA-A, -B, and -C a chains, are encoded bygenes that are linked distally to the HLA-A locus, and are notnormally expressed on lymphoblastoid B cells. In general,since pHeBo (5.4) -- .221, pHeBo (6.0) -+ .221, and pHeBo(6.2) -* .221 transferents each prominently express just oneclass I a chain, the use of .221 and of the transferents asimmunogens, targets, and antibody-absorbing cells shouldallow unambiguous determination of which, if any, of thealloantiserum reagents used to define non-HLA-A,B,C classI-like antigens specifically recognize the three a chainsdescribed herein. This will be another way of obtainingevidence for the expression of the HLA-5.4, -6.0, and -6.2 achains in vivo.

We thank Dr. Sherman Weissman for the HLA-B7 probe, SandraGuthrie and Carol Kass for flow cytometry, Dr. Donna Paulnock forassistance with immunoprecipitations, and Lynn Tague for preparingthis manuscript. This research was supported by Grants AI-15486 (toR.D.) and AI-18124 (to H.T.O.) from the National Institutes ofHealth and Grant Im-379 from the American Cancer Society. Y.S.was a predoctoral trainee supported by Public Health Service Grant5T-32-GM07133; H.T.O. is a Scholar of the Leukemia Society ofAmerica. This is paper 2965 from the University of WisconsinLaboratory of Genetics.

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