RESEARCH ARTICLE | JANUARY 01 1982 Biochemical and immunologic heterogeneity of Ia glycoproteins isolated from a chronic lymphocytic leukemia.  M Letarte; ... et. al https://doi.org/10.4049/jimmunol.128.1.217 Related Content Speci c induction of syngeneic cytotoxic T lymphocytes by solubilized tumor antigen: fractionation of the speci c R-MuLVinduced leukemia antigen. J Immunol (July,1980) Production of interleukin 1 by human endothelial cells. J Immunol (April,1986) Partial puri cation and biochemical characterization of a T cell suppressor factor produced by human glioblastoma cells. J Immunol (February,1985) Downloaded from http://journals.aai.org/jimmunol/article-pdf/128/1/217/1016459/217.pdf by guest on 01 January 2023 J Immunol (1982) 128 (1): 217–223. 0022-1767/82/1281-0217$02.00/0 THE JOURNALOF bM4UNOLOGY Copyright 0 1982 by The American Association Of lmmunologists Vol. 128, No. 1. January 1982 Prmted ~n U S A BIOCHEMICAL AND IMMUNOLOGIC HETEROGENEITY OF la GLYCOPROTEINS ISOLATED FROM A CHRONIC LYMPHOCYTIC LEUKEMIA’ MICHELLE LETARTE2 AND JUDY FALK From the Research Institute, Hospital for Sick Children, and Toronto Western Hospital, Toronto, Canada The HLA-D/DR region of the human majorhistocompatibility complex ( ~ v ~ H and C ) ~the homologous I region of murine MHC control the expression of polymorphic antigens that play a crucial role in immunologic reactions (1 -6).The relationship between HLA-D determinants, defined by their ability to stimulate the mixed lymphocyte reaction, HLA-DR determinants, defined serologically as a series of polymorphic allospecificities, and the human la-like molecules, defined biochemically with monoclonal and hetero-antibodies, remains to be elucidated. HLA-DR determinants are carried on la-like molecules that are composed of 2 polypeptide chains of apparent m.w. 34.000 (a-chain) and 28,000 (&chain) (3, 4). By analogy to murine la antigens that are encoded for by at least 2 loci, I-A and I-E (5,61, it is expected that more than one locus within the HLA complex will code for la-like molecules. Partial amino acid sequence data have suggested that human la antigens, immunoprecipitated by xenoantisera, were related to murine I-E subregion products (3, 7, 8). Furthermore, reactivity of the murine A.TH anti-A.TL serum with human cells was shown to be due mostly to a cross-reaction between human la and mouse I-€ subregion product (9-1 2). More recentty, monoclonal antibody 21w4, produced in our laboratory against 19). Lampson and Levy (20) have described 2 monoclonal antibodies, L203 and L227, that in immunodepletion experiments recognize 2 different subpopulations of la molecules. It has been proposed that these 2 monoclonal antibodies define HLA-DR epitopes (21). Separation of subpopulations of la molecules should be feasible with monoclonal antibodies specifically recognizing determinants characteristic of a given subpopulation. In a recent paper it has suggested that subpopulations of la molecules could be fractionated by their susceptibility to papain digestion (22). Klareskog and co-workers (23) have suggested that human la molecules could be separated into 2 fractions by gel filtration; however, no antigenic differences were observed between the 2 fractions. In the present paper, we report the fractionation by gel filtration of la molecules obtained from chronic lymphocytic leukemia (CLL) cells into 2 antigenically distinct fractions. MATERIALS ANDMETHODS CeNs. Peripheral blood lymphocytes were obtained from patients with CLL undergoing leukopheresis inthe course of their treatment. The patient studiedhere may express an atypical form of the disease because no response was obtained with chemotherapeutic agents. An average of 5 x 10” cells could be recovered from a daily collection. Samples were often obtained 3 to 4 times before attempted chemotherapy. Erythrocytes were sedimented at unit gravity and lymphoblasts were washed in phosphatebuffered saline (PBS). Viability assessed by dye exclusion was usually greater than 98%. The leukemic cells of the patient studied here were of Received for publication May 13. 1981. the 8 cell type (with a white blood cell count of the order of 400.000/pl) Accepted for publication September 24, 1981. with less than 2% of the cells forming AET-rosettes (24) and 98% being The costs of publication of this article were defrayed in part by the payment positive for surface immunoglobulin (u and X) identified by fluorescence of page charges. This article must therefore be hereby marked advertisementin microscopy (25). The cells expressed la antigens detectable with rabbit accordance with 18 U.S.C. Section 1734 solely to indicate this fact. heterosera and with the species cross-reacting murine A.TH anti-A.TL ’ Thiswork was supportedbythe Medical ResearchCouncilandbythe serum. The histocompatibility phenotype of the cells was identified by the National Cancer Institute of Canada. standard cytotoxicity assays with the use of reagents from the 7th and 8th M. Letarte is a ResearchAssociate of theNationalCancer Institute of Canada. Address correspondenceto M. Letarte, Division of Immunology, HospitalInternational Workshop, the 1978 and 1979 American Workshop, the Canadian Red Cross, the National Institutes of Health, Dr. P. Terasaki, and for Sick Children, 555 University Avenue, Toronto, Ontario, Canada MSG 1X8. Abbreviations used in this paper: MHC, major histocompatibility complex; from our own laboratory. The cells from which la was purified inthe present CLL. chronic lymphocytic leukemia; RAM-Fc. F(ab’)* pepsin fragment of rabbit study expressed the antigens HLA-AP, AW32. 840, 844, CW5. DR4. MTIgG anti-mouse IgG-Fc; IAA. iodoacetamide; DTT. dithiothreitol. 3. 21 7 Downloaded from http://journals.aai.org/jimmunol/article-pdf/128/1/217/1016459/217.pdf by guest on 01 January 2023 human la, was shown to react with murine la.7-like specificity la glycoproteins have been isolated from human that maps to the I-E subregion (1 3). chronic lymphocytic leukemic cells (CLL) by Lens culiEvidence for the presence of human I-A-like molecules is still naris chromatography and by filtration on ACA-34 Ultrogel. la antigenic activity, measured byinhibitionof the weak. We have shownthat there was a cross-reaction between cellular radioimmunoassay, was separated by gel filtralab antigens and human la antigens that cannot be readily tioninto 2 fractions,peak I andpeak II. Monoclonal accounted for by an I-E cross-reaction because mice of hapantibodies, produced against peak II glycoproteins, aplotype b do not express I-E antigens (12). An anti-rat la monpear to recognize different antigenic determinants of la oclonal antibody. MRC-0x3, cross-reactive with murine la.9 molecules.Monoclonalantibody 18a4 reacted with la specificity of I-A subregion, was shownto be reactive with cells moleculesof peaks I and II, whereasmonoclonalantiof individuals expressing HLADR1,2, and w6 specificities bodies 18c2 and 18d5 reacted almost exclusively with (14). Goyert and Silver (15) have described a monoclonal peak II molecules both in the cellular radioimmunoassay antibody SG 171 that precipitated la molecules bearing 2 and by immunoprecipitation. In addition to antigenic difdistinct a-chains, demonstrated by peptide map analysis. One ferences, minor variations in the apparent m.w. of the la of the a-chains identified by SG 171 antibody showed amino polypeptide chains were observed between peaks I and acid sequence homology with the a-chain encoded for by the II. These results indicate the existence of antigenically distinct subsets ofla molecules that are separable by gelmurine I-A subregion. Reports by other investigators who used human allosera filtration. have also suggested the heterogeneity of la molecules (16- FALK 21 8 JUDY AND [VOL.128 LETARTE MICHELLE RAM-Fc ('251-F(ab')2pepsin fragment of rabbit IgG anti-mouse IgG-Fc) was added 125 ng and approximately 100.000 cpm). After 2 hr, cells were washed, transferred, and counted. All assays were done in triplicate in microtitre plates. lmrnunoprecipitation andpolyacrylamidegel electrophoresis (PAGE). '251-labeledfractions were incubated with Proteln A-Sepharose 48 for 1 hr at 4°C before incubationwith specific antibody. In a typicalexperiment, 25 pl of precleared radiolabeled sample (1 x 1O6 cpm) were incubated with 25 pI of specific antibody for 1 hr. The following concentrations of antibodies were added for optimal immunoprecipitation: A.TH anti-A.TL serum, 1 / 5 dilution; DA/2 hybridoma lgG, 0.3 mg/ml; 18a4, 18c2. and 18d5 hybridoma culture supernatants. undiluted. Protein A-Sepharose 4 8 (25 pl) beads were added to each sample. The beads were washed twice and eluted by boiling for 5 min in 20/0sodium dodecyl sulfate (SDS) in 0.1 M Tris. pH 6.8.Eluates were counted and appropriatealiquots were analyzed by PAGE. Samples were prepared by boiling in reducing (DTT) or nonreducing conditions andby alkylation with IAA. One-dlmensional PAGE analysis was performed accordingto Maize1(40)by using 10% acrylamide slab gels. Molecular weight markers were run with all gels (Pharmacia, Piscataway, NJ). Autoradiography was done with Kodak XR-1 x-ray film and Dupont Cronex Hi-Plus intensifying screens (41) with exposure varying from 8 to 96 hr. RESULTS Preparation of glycoproteins from human CLL cells. Efficient solubilization of la antigens from CLL cells derived from several patients has been obtained with the conjugated bile salt taurocholate. In several cases, 100% of the la antigens measured on intact cells was recovered in the soluble extract assessed by inhibition of the cellular radioimmunoassay. In some cases, the detergent decreased the ability of the leukemic cells to inhibit the binding of anti-la antibodies to glutaraldehyde-fixed target cells. Even in those cases, however, the amount of la measured in the soluble extract was comparable to that of the leukemic cells incubated with taurocholate. The soluble extract of CLL cells was fractionated on Lens culinaris to obtain a glycoprotein fraction. With the material described in the present study, as small an amount as 20% of the la activity and 2% of the proteins present in the extract were recovered in the specifically bound and eluted fraction. Repeated chromatography of the unbound fraction, containing 70 to 80% of the la activity did not yield significantly more la glycoproteins, eliminating the possibility that saturation of the column was responsible for the low percentage binding. These results suggest that the la glycoproteinsstudiedhere may represent a fractionof a heterogeneously glycosylated mixture. Fractionation of human la antigens by gel fiftration. The glycoprotein fraction prepared from the taurocholate extract of CLL cells was fractionated by ACA-34 Ultrogel chromatography. la antigenic activity, measured by inhibition of the cellular radioimmunoassay, was found in 2 fractions. When detected with the murine alloserum A.TH anti-A.TL, shown previously to react strongly with la of human 8 cells and 6-CLL (1 1, 121, most of the activity was eluted between 250 ml and 280 ml, whichwerepooledandlabeledpeak II (Fig. la). Antigenic activity measured with DA/2 hybridoma. shown tobinda monomorphic determinant of human la molecules (24). was distributed in 2 fractions (Fig. 1a). The first fraction, 190 to 240 ml, was rechromatographed after incubation in 1% cholate and 0.005 M DTT. It did not shift significantly under these conditions and remained ahead of peak II; it was labeled peak I (Fig. lb). Stokes radii of 6.3 nm and 5.2 nm were calculated for peaks I and II, respectively, by using the markers citedin Materials and Methods (26-28). Similar gel chromatography profile of human la antigens has been reported (23). laantigens were separated on Sepharose 6B into 2 fractions with Stokes radii of 5.5 nm and 4.0 nm; however, no antigenic differences were observed between the 2 fractions (23). Downloaded from http://journals.aai.org/jimmunol/article-pdf/128/1/217/1016459/217.pdf by guest on 01 January 2023 T-CLL cells were also obtained from a patient (250,000 white blood cells/pl) undergoing leukopheresis in the course of his treatment. Ninetyeight percent of the cells formed AET-rosettes and nosurface immunoglobulin could be detected. Antisera and cell lines. DA/2 monoclonal antibody, shown tobinda monomorphic determinant of la molecules (26). was a generous gift from Dr. Michael Crumpton, ICRF, London, England. A.TH anti-A.TL serum, which also reacts with a monomorphic determinant of human la molecules (1 1, 121,was obtained from Cedar Lane, Hornby, Ontario, Canada, P3/ X63-Ag8 myeloma cells were kindly given by Dr. Cesar Milstein, Cambridge, England. HSC-3, a B lymphoblastoid cell line, was a gift from Dr. Erwin Gelfand. Hospital for Sick Children. Solubilization of la antigens from human CLL cells. CLL cells were resuspended at 1 X l o 9 cells/ml In 0.05 M tris-(hydroxymethy1)aminomethane (Tris), pH 7.0, 0.15 M NaCI, 0.01 M iodoacetamide (IAA). Alkylation of free disulfide groups was carried out for 1 hr at 4OC. It has been reported that the formation of disulfide bonds can occur during purification and can be prevented by alkylation with IAA before and during detergent solubilization (27). Extraction was initiated by adding a solution of 2% taurocholate (Calbiochem. LaJolla. CA) in the above buffer supplemented with 0,002 M phenylmethyl sulfonyl fluoride to the cell suspension. After gentle stirring tor 9 0 min at 4"C, the mixture was centrifuged at 5,000 x G for 3 0 min. and a soluble fraction was obtained by centrifugation at 6,000,000 x g/ min with a Beckman Type 45 Ti rotor. The soluble extract was frozen at -70°C. The presence of la was monitored by the inhibition of cellular radioimmunoassay and the yield was estimated. Preparation of glycoprotein fractions from CLL cells. Soluble taurocholate extract from CLL cells was fractionated by Lens culinanslectinchromatography (28). In a typical experiment, 1 gram of extract proteins was applied to a 25-ml column of lectin-Sepharose 4 8 coupled at a ratio of 2 mg/ml, and the column was equilibrated in 0.05 M Tris, pH 7.0, 0.1 5 M NaCI, 0.001M CaCI2, 0.001 M M n C h and 19/0 cholate buffer. After extensive washing, the specifically boundfractionwas eluted from the column with 0.5 M a-methylmannose inthe same buffer. Approximately 20 mg of glycoprotein was recovered. Protein concentration was estimated by the Hartree method (29). Gel filtration on ACA-34 Ultrogel. The glycoprotein fractlon obtained by Lens culinaris chromatography of the CLL cell extract was concentrated and fractionatedon ACA-34 Ultrogel (LKB. Stockholm, Sweden). The column (2.6 cm x 87 cm) was equilibrated in 0.01 M Tris, pH 7.0, 0.1 5 M NaCI containing either 0.6% or 1% cholate. In most cases, fractionation was performed in 0.001 M DTT.3 la antigenic activity was monitored by inhibition of cellular radioimmunoassay. Fractions containing la were pooled and referred to as peaks I and II. The Stokes radius of peaks I and II was calculated according to the method of Siege1 and Monty (30). Two independent determinatlons were done by using the following markers: myoglobin 1.7 nm. bovine serum albumin @SA) 3.5 nm, transferrin 3.85 nm, aldolase 4.6 nm, and IgG 5.1 nm (30-32). '251-/abelingof proteins. Tracelabeling of la-containing fractions, namely peaks I and 11, obtained after ACA-34 chromatography was done at a chloramine T to protein molar ratio of 30:l In 0.6% cholate, in 0.05 M phosphate buffer, pH 7.0, and in the presence of dimethyl sulfoxide at a ratio of 50,000 mol/mol of protein (33. 34). The reaction was stopped by the addition of excess tyrosine. and free '251 was removed by Sephadex G50 chromatography. In a typical experiment, 25 pg protein was iodinated by 0.3 mCI and aspecific activity of 2 X 10' cpm/pgprotein was obtained. Preparation of monoclonal antibodies to human la antigens. A.TH mice (I")were immunized 1.p. on days 0 and 18 with 25 Kg of ACA-34 peak II proteins (see Fig. 1 ) emulsified in complete Freund's adjuvant; 10 pg of ACA-34 peak II proteins in PBS were given on day 28. On day 32, spleen cells prepared from 2 mice were fused with P3/X63-Ag8 myeloma cells (35,36) at aratio of 5:l with 50% polyethylene glycol1000(Baker, Phillipsburg, NJ). Cells were then plated at 2.5 X lo5viable myeloma cells per milliliter in H21 medium (GIBCO. Grand Island. NY). supplemented with 15% fetal calf serum (FCS) and containing 2 X 10-6 M hypoxanthine, 2 X 10.' M aminopterine, and 0.8 x 10"M thymidine. After 12 days, the supernatants of 24 fast-growing wells (out of 280 wells) were tested for anti-la reactivity by cellular radioimmunoassay. Four wells were selected on the basis of high binding to B-CLL and low binding to T-CLL or to the T lymphoblastoid line, Molt-3. These selected hybrids were cloned in methyl cellulose (37). Three positive clones, 18a4, 18c2, and 18d5, were grown in large quantities. The antibodies secreted by the 3 clonesused in the present studies were found to be IgGl and K. Cellular radioimmunoassay of l a antigens. The amount of la present in extracts and in partially purified la fractionswas measured by the inhibition of cellular radioimmunoassay (1 2, 38. 39). Soluble fractions were diluted directly in microplates in 0.5% BSA/PBS (25 pl) and incubated for 1 6 hr with the appropriate dilutionof specific anti-la antibody. Residual binding of absorbed antibody was measured by the addition of 1 x 1O6 glutaraldehydefixed B-CLL cells (25 pl). After 1 hr, cells were washed 3 times and 1251- 21 9 HETEROGENEITY OF HUMAN la FROM CLL CELLS 19821 .* 9467100 200 300 rnl eluent A00 Figure I . Fractionation of human la antigens by gel filtration. A glycoprotein fraction (8 mg) obtained by Lens culinaris chromatography of human E-CLL taurocholate extract was applied to a column of ACA-34 Ultrogel equilibrated in 0.6% cholate, 0.01 M Tris. pH 7.0, 0.15 M NaCl with 0.001 M DTT (panel a). The sample was treated with 0.005 M DTT for 1 hr before application. Markers used to standardize the column were: blue dextran, BD: human transferrin, J . 76.000: bovine serum albumin, A. 67.000, and myoglobin, M. 17.000. la activity of the fractions was measured in the la cellular radioimmunoassay by inhibition ofbindingofDA/2 IgG (0)and A.TH anti-A.TL serum (A)to glutaraldehyde-fixed B-CLL cells.Fractions 190 to 240 ml of panel a were pooled, concentrated, incubated in 0.005 M DTT for 1 hr and rerun on the same ACA-34 column equilibrated in1 % cholate, 0.001 M DTT. The percentages of la activity recovered ineach fraction were calculated as an average of 3 independent assays, each done in triplicates. The 2 peaks of activity are referred to as peak I (eluent 225 ml to 245 ml of panel b ) and peak I1(eluent 250 ml to 280 ml of panel a). 43- 30- 20To eliminate the possibility that the resolution of la antigens into 2 fractions was due to the presence of residual lectin shed during the affinity chromatography step, a crude plasma membrane fraction was prepared by disruption of CLL cells according to Snary et al. (4). The membrane fraction was solubilized in cholate, reduced with 0.005 M DTT, and chromatographed as in Figure 1b. Again, the la activity was distributed into 2 peaks with most A.TH anti-A.TL reactivity in peak II and with DA/2 reactivity in both peaks I and II. Production of monoclonal antibodies to human la antigens. In an attempt to identify possible structural differences between the la molecules resolved by gel filtration into peaks I and 11, monoclonal antibodies were produced against ACA-34 peak II proteins. An aliquot of the latter, labeled with lZ5l,was visualized by PAGE (Fig. 2). Four to 5 major polypeptide bands were visible under nonreduced conditions (estimated m.w. 26.000, 32.000. 57,000, and 100,000) and reduced conditions (estimated m.w. of29,000.34.000.67,000.90,0,00, and 100.000). 14- 1 2 Figure 2. PAGE of radioiodinated ACA-34 Peak II. A radioiodinated aliquot of ACA-34 peak 11, after fractionation onSephadex G-50. was analyzed by PAGE with 10% acrylamide. The sample was run either reduced (track 1) or unreduced (track 2). The speclfic activity of the radiolabeled sample was 3 x 10' cpm/pg protein; approximately 14 ng and 50.000 cpm were applied per track. Autoradiography was done with Kodak XR-1 x-ray film and Dupont Cronex Hi-Plus intensifying screens with exposure of 24 hr. The positions of m.w. markers are indicated on the left. Downloaded from http://journals.aai.org/jimmunol/article-pdf/128/1/217/1016459/217.pdf by guest on 01 January 2023 The major la-like bands thus shift from 26,000 to 29,000 (pchain) and from 32.000 to 34.000 (a-chain) upon reduction. Peak I. when radiolabeled, gave a pattern similar to peak I1 except that the 100,000 polypeptide band was less intense. Three hybridoma clones derived from the same fusion and producing antibodies selected for their reactivity with B-CLL and B lymphoblastoid cell lines, but negative with T leukemias and T cell lines, have been used in the present study. Figure 3 illustrates strong and specific binding of culture supernatants of clones 18a4. 18c2. and 18d5 with B-CLL cells, with minimal binding to T-CLL cells. The 3 monoclonal antibodies were of IgG1 subclass and showed complement-dependent cytotoxicity only in the presence of rabbit anti-mouse IgG serum (1:lo). All normal B cells and B-CLL cells tested were lysed whereas T cells were not, suggesting that the antibodies were recognizing nonpolymorphic determinants of la molecules. Immunoprecipitation of human la antigens by monoclonal antibodies. Immunoprecipitation experiments were performed to confirm that the reactivity of clones 18a4. 18c2. and 18d5 was la-specific. Figure 4 demonstrates that la-like polypeptide bands are precipitated with A.TH anti-A.TL murine alloserum, monoclonal antibody DA/2, and culture supernatants from clones 18a4.18~2.and 18d5. Nonimrnune A.TH serumor P3/ X63-Ag8 culture supernatant gave low background values. The 220 JUDY AND LETARTE MICHELLE 18151296- 18c2 \ 1 2- 18d5 oclonal antibodies 18c2 and 18d5. The determinants could be absent, suggesting different subpopulations of la molecules in peaks I and II. This hypothesis would be reinforced by them.w. differences observed for the la chains in peaks I and II. The antigenic determinants could also be inaccessible in peak I molecules as aresult of differential glycosylation, altered folding, or aggregation of la chains. In the latter case, the physicochemical differences observed between peaks I and II may not reflect antigenically different la chains,but may reflect chains in a different conformational state. Differential reactivity in the cellular radioimmunoassay of la molecules of peak I and peak I/. The ability of la molecules of peaks I and IIto block the reactivity of various anti-la antibodies to B-CLL target cells was measured by inhibition of the cellular radioimmunoassay. Equivalent dilutions of peak I and peak II proteins (in the preparation shown in Fig. 1, 1 mgof protein was recovered from both peak I andpeak II) wereequally efficient at absorbing the reactivity of either DA/2 or 18a4 monoclonal antibodies for B-CLL cells (Fig. 6a and b). However, peak II contained approximately 5 times more A.TH antiA.TL-reactive determinants and 25 times more 18d5 reactive determinants than peak I (Fig. 6c and d). It should be noted that although most of the A.TH anti-A.TL reactive material is in peak II (Figs. 1 and 61, la can still be precipitated from peak I with this antiserum (Fig. 5). In the case of 18d5-reactive maif any was found in peak l both by cellular terial, a small amount radioimmunoassay and by immunoprecipitation(Figs. 5 and 6). Theantigenicdeterminants reactive with 18c2 monoclonal .' " _ " " " 2 LOG, ANTIBODY DILUTION 1 Figure 3. Reactivity of anti-human la monoclonal antibodies. Culture supernatants of hybridoma 18a4. 18c2. and 18d5 were titrated by using the cellular radioimmunoassay. 1 x 10' glutaraldehyde-fixed B-CLLcells (0,A,D or T-CLL cells (0.A, 0)were incubated for 2 hr with the dilutions of culture supernatant indicated on the abscissa. Cells were washed and incubated for 2 hr with lZ5lRAM-Fc (25 ng per assay, 108,000 cpm).Cellswerewashedandcounted. Assays were done in microculture trays in triplicate. Background binding with 0.5% BSA/PBS has been subtracted. 9467- 43- m.w. calculated for the la-like bands precipitated from radio30II were 25,000 (weak), 27,000, and labeledACA-34peak 33,000 under nonreducingconditions. The nature of the higher m.w. bands is undetermined. The band of m.w. 25.000 may not be la-like because it is precipitated by the control culture supernatant (track 7, Fig. 4); however, it is not reactive with 20A.TH nonimmune serum (track 1, Fig. 4). The monoclonal antibodies produced against ACA-34 peak II were tested forreactivity against ACA-34 peakI. Immunoprecipitation of la-like bands from peaks I and II, respectively, is 14shown in Figure 5. The first striking observation is that monoclonal antibodies 18d5 and 18c2, although reacting with radiolabeledpeak II. react poorly with radiolabeled peak I; A.TH 1 2 3 4 5 6 7 anti-A.TL serum, DA/2, and 18a4 monoclonal antibodies react Figure 4. Immunoprecipitationof la antigensfrompeak II bymonoclonal wellwith both peaks I and II. Secondly,the m.w.of the la antibodies. A radioiodinated aliquot of ACA-34 peak II (visualized in Figure 2) polypeptide chains precipitated from peak I (25,000 (strong), was immunoprecipitated with anti-la antibodies and protein-A Sepharose. The following antibodies were used which precipitated. respectively. the cpm indi29,000 (weak), and 31.000) are slightly different from those cated (100 pl eluant): 1) A.TH nonimmune serum, 5400 cpm; 2) A.TH anti-A.TL precipitated from peak II (25,000 (weak), 27,000 (strong), and serum, 162,000 cpm:3) DA/2 IgG.81,000cpm:4) 18a4 hybridoma culture supernatant. 135,000 cpm; 5 ) 18d5 hybridoma culture supernatant. 52,000 33,000). Control culture supernatantand A.THnonimmune cpm; 6) 18c2 hybridoma culture supernatant. 79.000 cpm;7) P3/X63-Ag8 serum precipitated minimal cpm and no la-like bands from peak culture supernatant. 18,000 cpm. Forty-microliteraliquotsofsamples 1 to 7were run undernonreducing I. conditions in tracks 1 to 7, respectively. Polyacrylamide slab gel electrophoresis These results suggestedthatpeak I la molecules do not was performed with 10% acrylamide. and autoradiograms were exposed for24 express accessible antigenic determinants recognized by mon-hr. Downloaded from http://journals.aai.org/jimmunol/article-pdf/128/1/217/1016459/217.pdf by guest on 01 January 2023 \ [VOL. 128 HETEROGENEITY OF HUMIAN la FROM CLL CELLS 19821 *, . .I 1 i- ,I!- 1 .' . . " ., * :., 4 1, Figure 5. Comparison of immunoprecipitates of human la antigens obtained from peak I and peak II. Radioiodinated aliquots of peaks I and II were immunoprecipitated with anti-la antibodies and protein A-Sepharose. The following antibodies were used and precipitated the cpm indicated from the respective peaks I and 11: A.TH anti-A.TL. tracks 1 and 2. 103.000 cpm and 162,000 cpm; DA/2 hybridoma, tracks 3 and 4 . 7 4 . 0 0 0 cpm and 81,000 cpm: 18a4 hybridoma, tracks 5 and 6, 128.000 cpm and 135.000 cpm; 18c2 hybridoma, tracks 7 and 8 . 2 0 . 0 0 0 cpm and 79,000 cpm: 18d5 hybridoma, tracks 9 and 10. 10,000 cpm and 52.000 cpm. Aliquots (40 81) of the eluants were run under nonreducing conditions Tracks 1, 3. 5. 7, and 9 are immunoprecipitates of peak I whereas tracks 2, 4 , 6, 8. and 10 are immunoprecipitates of peak 11. In the same experiment, control A.TH nonimmune serum precipitated 5200 cpm and 5 4 0 0 cpm. and control P3/X63-Ag8 culture supernatant precipitated 12.000 cpm and 18,000 cpm from peaks I and 11, respectively. PAGE analysls In 10% acrylamide was performed for 16 hr. and autoradiograms were developed for 24 hr. Controls for peak II were identical to those visualized in tracks 1 and 7 of Figure 4. The A.TH nonimmune serum control for peak I showed no detectable band; the control P3/X63-Ag8 immunoprecipitate was identical to the 18d5 immunoprecipitate (track 9). antibody were also found by cellular radioimmunoassay to be more abundant in peak I I than in peak I, confirming the results of Fig. 5. DISCUSSION A procedure was established for the rapid and effective solubilization of la antigens from human B-CLL leukemic cells. Extraction with the conjugated bilesalt taurocholate from intact cells was selected as the method of choice. Taurocholate has been shown tobe relatively mild in itseffects on biologic membranes. At low concentrations (0.2%), it can release externally-orientated proteins of the plasma membranes of erythrocytes and lymphocytes without cell breakage(42).In the present study, selective solubilization of la did not occur with low concentrations of taurocholate; a concentration of 1% was needed to ensure maximum recovery of la antigenic activity in the extract. However, taurocholate does not lyse nuclei easily, and unlike deoxycholate, it can be used with intact cells. It also has minimal effects on la antigens, and often all la activity measured at the cell surface can be recovered in the soluble extract. In contrast to nonionic detergents, taurocholate has a small monomeric micellar weight that does not affect substantially the apparent molecular weight (m.w.1 of isolated membrane proteins. Taurocholate, being a trihydroxy bile salt, forms very small micelles with an aggregation number less than 10, and their size is not affected by ionic strength, temperature, or concentration of detergent (43). Taurocholate thus constitutes a detergent of choice for rapid extraction of biologically intact la molecules from large volumes of cells. Effective solubilization of murine la antigens can also be achieved with taurocholate (44). A glycoprotein fraction, representing only 20% of the la of the B-CLL taurocholate extract, was fractionated by ACA-34 gel filtration into 2 antigenically distinct peaks, referred to as peaks I and II. The sample had been reduced with 0.005 M DTT before fractionation, and the column was pre-equilibrated and run in cholate with 0.001 M DTT to prevent disulfide bond formation, which could lead to dimer formation (27). We have shown previously that murine la antigenic activity, fractionated in cholate, was distributed over a wide volume, unless it was The reduced with 0.005 M DTT beforegelfiltration(44). reduced murine la sample, in cholate, had a Stokes radius of 4.3 nm, which was analogous to that reported for unreduced rat la in deoxycholate (45). In the present report, Stokes radii of 6.3 nm and 5.2 nm were estimated for peaks I and I I , respectively, in the presence of cholate and DTT (Fig. 1). Klareskog and co-workers (23) reported that human HLA-DR antigens, solubilized from leukemic and spleen cell crude membrane preparations with deoxycholate, could be resolved by gel filtration in deoxycholate, but without DTT, into 2 fractions with calculated Stokes radii of 5.5 nm and 4.0 nm. They estimated by sedimentation analysis that their fractions I and II corresponded to m.w. of 147,000 and 76.000, respectively; upon storage in detergent, fraction I displayed a tendency to become of a size identical to fraction I I (23). It was suggested therefore that fraction I may represent a dimer of fraction I I that could have arisen during the solubilization procedure. No antigenic difference could be found between fractions I and II by Klareskog and co-workers. Our results are different. Peak I does not convert to peak I I by storage in cholate, and antigenic differences are observed between peaks I and II. CLL cells were alkylated with 0.01 M IAA before solubilization; IAA was also present throughout the extraction procedure to prevent artefactual disulfide bond formation (25). Furthermore, samples were reduced with 0.005 M DTT before gel filtration, and columns were always equilibrated in 0.001 M DTT. Thus, the high Stokes radii observed for peaks I and II do not appear tobe due to formation of disulfide bonds between la chains or between la chains and other polypeptide chains. We would suggest that la chains, possibly due to the mild procedures used for solubilization, are joined together by noncovalent interactions and/or are highly asymmetric. The interactions observed could either occur within the plasma membrane of the leukemic cells or arise as aconsequence of thedisruption of the hydrophobic bonds normally responsible for membrane integrity. We have not attempted to calculate the m.w. of la molecules from fractions I and I I ; sedimentation analysis of la purified by affinity to monoclonal antibody 18a4, before gel filtration, should give a better estimate of the m.w.of peaks I and II. It should be mentioned that the determination of apparent Stokes radius of asymmetric molecules by gel filtration in the presence of detergents, with the use of globular proteins for calibration,needs to be interpreted with caution (26-28). It is used here only as an indication of the size heterogeneity of la antigenic activity. Ourresults demonstrate that human la molecules canbe separated into antigenically different fractions by gel filtration. This agrees with results of previous studies suggesting the existence of families of la molecules. Monoclonal antibodies L203, L227, Q2/80, and 0 5 / 1 6 , which recognize nonpolymorphic determinants of la molecules, were shown by immu- Downloaded from http://journals.aai.org/jimmunol/article-pdf/128/1/217/1016459/217.pdf by guest on 01 January 2023 - ( 1 22 1 222 MICHELLE LETARTE AND JUDY FALK 01 D A / 2 Afroction I froction I I hybridoma [VOL.128 c) A.TH anti-A.TL serum o hybridomo b] 1804 2 IOO- hybridoma d) 18d5 i BO- 40- 20I 1 I 1000 100 I I IO 1 I 1000 100 1 IO Dilution of fraction Figure 6. DIXerential reactivity of la molecules of peaks I and 1 I with monoclonal antibodies. Peaks I (A)and 1 I (0)obtained afler ACA-34 chromatography of BCLL glycoproteins (Figure 1) were assayed for their ability to block the reactivity of DA/2 hybridoma (a). 18a4 hybridoma(b), A.TH anti-A.TL serum (c) and 18d5 hybridoma (d) to B-CLL cells. FractionsI and 11, obtained as shown in Figure 1 and each containing 1 mg of protein, were diluted as indicated on the abscissa. A 25amount of each dilution was incubated for 1 6 hr with 25 pl of antibody at the following dilutions: DA/2 IgG, 1.2 pg/ml: 18a4 culturesupernatant, 40-fold dilution; A.TH anti-A.TL serum, 1000-fold dilution; 18d5 culturesupernatant, 20-fold dilution. Residual binding activity of the antibodies was tested by adding glutaraldehydefixed B-CLL (25 +I).Afler 2 hr incubation and 3 washes, '251-RAM-Fc (25 ng, 66,000 cpmper assay) was added. The 100% values are the binding values observed with the unabsorbed antibodies: 15,780 cpm for DA/2; 14.873 cpm for 18a4; 1 1,240 cpmfor A.TH anti-A.TL, and 7060 cpm for 1865.The ordinate represents the percentage of anti-la binding activity remaining after absorption of the various antibodies with peaks I and 11, respectively. All assays were done in triplicate. nodepletion experiments to react with subpopulations of la molecules (20, 46). Several interpretations can be given to explain the heterogeneity of la molecules. By analogy to the murine system, it is likely that more than one locuscodesfor la molecules. No recombinant family studies have clearly established the existence of a locus, separate from the HLA-DR locus, and coding for serologically detected la-like specificities. However, population studies and immunochemical data support the existence of more than one locus coding for la-like molecules (1, 2, 1619). Sequential immunoprecipitation with the use of alloantisera suggested that DR, MB, and also MT determinants were found on different la molecules (16, 19, 47). It has also been argued that MB and MT specificities were expressed on the same molecules as those carrying the DR specificities (48). More recently, preliminary sequence data supports the view that a subpopulation of human la molecules, homologous to murine I-A molecules, does exist (1 5). DR molecules have been demonstrated to be homologous to murine I-E molecules (3, 7, 8). The heterogeneity observed here could also be due to allelic variation because the patient could be heterozygote, although only DR4 could be identifiedon his leukemic cells. Studies with several leukemic patients suggested that the reactivity of 18a4 and 18d5 monoclonal antibodies did not correlate with the DR allotype (49). Results have also indicated that 18d5 determinants are usually less abundant than 18a4 determinants on the surface of leukemic cells (49), further substantiating the idea that 18d5 representsasubpopulation of la molecules. The presence of subpopulations of la molecules, identifiable by serologic and physicochemical criteria, could be a reflection of the extent of processing of la polypeptide chains. Peak I, of higher Stokes radius than peak 11, may represent a cytoplasmic configuration of la molecules for which incomplete processing of the oligosaccharide moieties would be expected. The conformational arrangement of the chains in the complex of larger size (peak I) could render the antigenic sites 18d5 and 18c2 inaccessible for binding. The possibility that the antigenic determinants 18d5 and 18c2 are found on the oligosaccharide chains of certain la polypeptide chains, only present in peak II, has not been excluded. Differential extent of glycosylation of la polypeptide chains might explain the variations in apparent m.w. observed for both the major a- and /3-like bands of peaks I and II (Fig. 5). It is also possible that the polypeptide chainof m.w. 29,000, present in peak I but absent from peak II (Fig. 51,represents an invariant chain of la molecules (50). This invariant chain could be associated with only certain la polypeptide chains, namely the incompletely glycosylated or cytoplasmic ones. Further studies should establish if the heterogeneity observed here is due tothe existence of subsets of la molecules, to the association of certain la determinants with glycosylated subpopulations of molecules, or to the masking of antigenic sites by formation of complexes between la polypeptide chains. Acknowledgments. We thank Ms. Jane Addis and Ms. Christina Messinger for their excellent technical work. We also thank Dr. S. Sutton of the Toronto Western Hospital forproviding us with CLL Cells. Downloaded from http://journals.aai.org/jimmunol/article-pdf/128/1/217/1016459/217.pdf by guest on 01 January 2023 60- HETEROGENEITY OF HUMAN CELLS CLL la FROM 19821 223 to HLA-DRw determinants. Tissue Antigens 16:30. 27. Springer, T. A.. R. J. Robb. C. Terhorst. and J. L. Strominger. 1977. Subunit and disulfide structureof monomeric and dimeric forms ofdetergent-soluble 1. Mann. D. L., L. Abelson, S. Harris, and D. 8 . Amos. 1976. Second genetic HLA antigens. J. Biol. Chem. 252:4694. locus in the HLA region for human B-cell alloantigens. Nature 259:145. 28. Letarte-Muirhead, M., A. N. Barclay. and A F. Williams. 1975. Puriflcation 2 Van Rood, J. J..A. van Leeuwen, M. Jonker. A. Termijtelen, and B.A. of the THY-1 molecule, a major cell-surface glycoprotein of ratthymocytes. Bradley. 1977. Polymorphic B-cetl determinants in man. Cold Spring Harbor Biochem. J. 151:685. Symp. Quant. Biol. 11:417. 29. Hartree, E. F. 1972. Determination of protein. a modification of the Lowry 3. Springer, T. A,. J. F. Kaufman. C. Terhost. and J. L. Strominger. 1977. method that gives a linear photometric response. Anal. Biochem. 48:422. Purification and structural characterization of human HLA-linked B-cell an30. Siegel, L. M.. and K. J. Monty. 1966. Determination of molecular weights tigens. Nature 268:213. and frictional ratiosof proteins in impure systems by use of gelfiltratlon and 4. Snary. D.. G. J. Barnstable, W. F. Bodmer. P. N. Goodfellow, and M. J. density gradient centrifugation. Biochim. Biophys. Acta 112:346. Crumpton. 1977. Cellular distribution, purification and molecular nature of 31. Tanford, C.. Y. Nozaki, J. A. Reynolds, and S. Makino. 1974. Molecular human la antigens. Scand. J. Immunol. 6:439. characterization of proteins in detergent solutions. Biochemistry 13:2369. 5. Uhr, J. W., J. D. Capra. E. S. Vitetta. and R. G .Cook. 1979. Organization of 32. Nozaki. Y., N. M. Schechter. J. A. Reynolds, and C. Tanford. 1976. Use of the immune response genes. Both subunits of murine I-A and I-E/C molegel chromatography for the detection of the Stokes radii of proteins in the cules are encoded within the I reglon. Science 206:292. presence and absence of detergents. A re-examination. Biochemistry 15: 6. Klein, J.. L. Flaherty, J. L. vande Berg. and D. C. Shreffler. 1978. H-2 3884. haplotypes, genes, regions and antigens: first listing. Immunogenetics 6: 33. Sonoda, S.. and M. Schlamowitz. 1970. Studies of lZ5l trace labeling of 489. immunoglobulin G by chloramine T. Immunochemistry 7:885. 7. McMillan. M.. J. M. Cecka. D. B. Murphy, H. 0. McDevitt. and L. Hood. 34. Stanley, E. R.. G. Hansen, J. Woodcock,and D. Metcalf.1975. Colony 1978. Partial amino acid sequence of murine lad antigens of the I-E/Cd stimulating factor and the regulation of granulopoiesis and macrophage subregion. Immunogenetics 6:137. production. Fed. Proc. 34:2272. 8. Allison. J. P.. L. E. Walker, W. A. Russell, M. A. Pellegrino. S. Ferrone, R. A. 35. Kohler. G.. and C. Milstein. 1976. Derivation of specific antibody-producing human DR Reisfeld, J. A. Frelinger, and J. Silver. 1978.Murinelaand tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6:51 1. antigens: homology of amino terminal sequences. Proc. Natl. Acad. Sci. 75: 36. Galfre, G.. S. C. Howe, C. Milstein. G. W. Butcher. and J. C. Howard. 1977. 3953. Antibodies to major histocompatibility antigens produced by hybridcell 9. Delovitch, T. L., and J. Falk. 1979. Immunochemical evidence for structural lines. Nature 266:550. homology between murine and human la antigens. Immunogenetics 8:405. 37. Worton, R. G.. E. A. McCulloch. and J. E. Till. 1969. Physical separation of 10. Lunney. J. K., D. L. Mann, and D. H. Sachs. 1979. Sharing of la antigens hemopoietic stem cells from cells forming colonies in culture.J. Cell Physiol. between species 111. la specificitiesshared between mice and human beings. 74:171. Scand. J. Immunol. 10:403. 38. Letarte-Muirhead, M.. R. T. Acton. and A. F. Williams. 1974. Preliminary 11.Ing. P. M., J. A. Falk, M. Letarte. T. L. Delovitch, and R . E. Falk. 1979. characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in Serologic cross-reaction ofmurine and human la antigens. Transplant. Proc. detergents. Biochem. J. 14351. 1 1 :1745. 39. Letarte. M.. H.-S. Teh. and G. Meghji. 1980. Increased expression of la and 12. Letarte. M.. and J. Falk. 1980.Analysis of serologic cross-reactions between Thy-1 antigens on mitogen-activated murine spleen lymphocytes. J. Immuhuman and mouse la antigens. J. Immunol. 125:1210. 13. Falk, J.,J. B. L. Addis, and M. Letarte. 1981. Species cross-reactive la nol. 125370. determinants: detection by monoclonal antibody to human la. Abstracts of 40. Maizel, J. V. 1971. Polyacrylamide gel electrophoresis of viral proteins. the American Association for Clinical Histocompatibility Testing, Orlando, Methods Virol. 5 1 7 9 . FL. P. 32. 41. Swanstrom. R.. and P. R . Schank. 1978. X-ray intensifying screens greatly 14.McMaster, W. R.. and A. F. Williams. 1979. Monoclonal antibodies to la enhance the detection by autoradiography of the radioactive isotopes 32P antigens from rat thymus: cross-reactions with mouse and human and use and lZ5l.Anal. Biochem. 86:184. in purification of rat la glycoproteins. Immunol. Rev. 47:117. 42. Holdsworth. G.. and R. Coleman. 1976. Plasma-membrane components can be removed from isolated lymphocytes by the bile salts glycocholate and 15. Goyert. S. M., and J. Silver. 1981. Isolation of I-A subregion-like molecules taurocholate without cell lysis. Biochem. J. 158:493. from subhuman primates and men. Nature. In press. 43. Small, D. M. 1967. Size and structure of bile salt micelles in molecular 16. Tosi. R.. N. Tanigaki. D. Centis. G. B. Ferrara. and D. Pressman. 1978. association in biological and related systems. Advances in Chemistry Series, Immunological dissection of human la molecules. J. Exp. Med. 148:1592. Washington. Pp. 31 -52. 17. Duquesnoy. R. J., M. Marrari, and K. Annen. 1979. Identification of anHLA44. Cruz, L., and M. Letarte. 1979. Glycoproteins of murine LPS blasts: resoDR-associated system of B c e l l alloantigens. Transplant. Proc. 1 1:1757. 18. Katagiri. M., H. lkeda, N. Maruyama, J. Moriuchi. A. Wakisaka, S. Kimura. lution and interactions. In The Molecular Basis of Immune Cell Function, M. Aizawa, and K. Itakura. 1979. Evidence for two €3-cell alloantigen loci in edited by J. G. Kaplan. Elsevier/North Holland Biomedical Press, Amsterthe HLA-D region. Immunogenetics 9:335. dam. Pp. 99-107. 19. Tanigaki, N., R. Tosi. D. Pressman, and G. 8. Ferrara. 1980. Molecular 45. McMaster, W. R., and A. F. Williams. 1979. Identificationof la glycoproteins a gene locus closely linked identification of human la antigens coded for by in rat thymus and purification from ratspleen. Eur. J. Immunol. 9:426. to HLA-DR locus. Immunogenetics 10:151. 46. Quaranta, V., M. A. Pellegrino. and S. Ferrone. 1981. Serologic and immu20. Lampson, L. A,. and R. Levy. 1980. Two populations of la-like molecules on nochemical characterization of the specificity of four monoclonal antibodies a human B-cell line. J. Immunol. 125:293 to distinct antigenic determinants expressed in subpopulations of human la21. Grumet, F. C.. D. J. Charron. B. M. Fendley. R. Levy, and D. 8. Ness. 1980. like antigens. J. Immunol. 126:548. HLA-DR epitope region definition byuse of monoclonal antibody probes. J. 47.Markert. M. L., and P. Cresswell. 1980. Polymorphism of human B-cell Immunol. 1252785. alloantigens: Evidence for three loci within the HLA system. Proc. Natl. Acad. Sci. 77:6101. 22. Tanigaki. N.. R. Tosi. K. Koyama. and D. Pressman. 1980. Purification and separation of subsets of human la molecules by papaindigestion. Immunol48. Karr. R. M.. D. L. Mann. T. C. Fuller, G. E. Rodey, and B. D. Schwartz. ogy 39:615. 1981. HLA-DR molecules bear the MB and MT antigens. Abstract of the 23. Klareskog. L.. L. Tragardh, L. Rask. and P. A. Peterson. 1979. Isolation and American Association for Clinical Histocompatibility Testing, Orlando, FL. P. characterization of detergent-solubilized human HLA-DR transplantation an40. tigens. Biochemistry 18:1481. 49. Addis, J. B. L., J. Falk. and M. Letarte. 1981. Quantitation with monoclonal 24. Kaplan, M. E., andC. Clark. 1974. An improved rosetting assay for detection antibodies of la antigenic determinants on B-lymphocytic leukemic cells. of human T lymphocytes. J. Immunol. Methods 5:131 Abstract of the American Association for Clinical Histocompatibility Testing, Orlando, FL. P. 20. 25. Alexander, E. L., and S. K. Sanders. 1977. F(ab’)* reagents are not requlred if goat, rather than rabbit, antibodies are used to detect human surface 50. Shackelford. D. A., and J. L. Strominger. 1980. Demonstration of structural immunoglobulin. J. Immunol. 1 19: 1084. polymorphism among HLA-DR light chains by two-dimensional gel electro26. Brodsky. F. M.. P. Parham, and W. F. Bodmer. 1980. Monoclonalantibodies phoresis. J. Exp. Med. 151 :144. REFERENCES Downloaded from http://journals.aai.org/jimmunol/article-pdf/128/1/217/1016459/217.pdf by guest on 01 January 2023