Document gbVzV3GwnGn9YX5k71Mrk6n0e

A. E., am! J, AI. Allen. 1964. The AKR thymic antigen and its distribution leukemias and nervous tissues. J. Exf>. M ed. 120:413. A. IT, arid J. M. Allen. 1966. Mouse thymic iso-aritigens. Nature (Loud.) 3:521. !>, F.. S. l c>72. The- distribution of brain-associated 0 antigen cross-reactive ih mouse in the brain of other species. J . Immunol. 109:168. i-it, J., II. II. l'eter, J. D. Feldman, and \V. 0 . Wcigle. 1973. R abbit antiserum brain-aw toiaied thymus antigens of mouse and rat, XI. Analysis.of species1`cific and cress-reacting antibodies. J . Immunol. 110:1085. r, H. I [., J. Clagctt, J. D. Feldman, and W. 6 . Weigle. 1973. R abbit antiserum bra in-associated thymus antigens of mouse and rat. I. Demonstration of i bodies cross-reacting to T cells of both species. J . Immunol. 110:1077. port, M. M., and L. Graf. 1969. Immunochemical reactions of lipids. Progr. ilergy. 13:273. iqui, 11., and S. I. Hakomori. 1971. A revised structure for the . orssman ycolipid hapten. J . Biol. Chcm. 246:5766. g. $, J., W. J. Essclman, and C. C. Swcelev. 1973. Structure of a penlahexosyl ramide ( Forssman hapten) from canine intestine and kidney. J . Biol. Chcm. 18:0 ;-'N. :i>ni"ri. 5.. and G, D. Strychara. 1968. Investigations on cellular blood-group I. Isolation and chemical composition of blood-group ABH and " i-i'-an:igens of sphingoglycoiipid nature. Biochemistry. 7:1279. pori. ,\l .M., L. Graf, and R, Ledeen. 1968. Specificity of antiserum to brain' .mgiV'side Fed. Proc. 27:463. ca). T. A., and A. Sailer. 1969. Immunochemical studies of iso!.: human iram canuliuside components. J . Xciiroehcm. 16:301. lub. h. ' 1971. Brain-associated 0atuigen: reactivity of rabbit anti-mou" tai:. 'lilt mouse lymphoid cells. Cell. I minimal. 2:353. fu-n. and .M. Schlesingcr. 1970. Absorption of guinea pig serum with agaf Ii,;n ;>liint.i:um. 10:139 -i-lman, IV, J .. R. A. Lainc, and C. C. Sweeley. 1972. Isolation and characierixa* :un of ul\co-pliingolipids. Methods Enzymol. 28:140. Uii. J.. M. Lee.-, anil G. II. Sloane Stanely. 1957. A simple method for the lation and purification of total lipids from animal tissues. J . Biol. Chcm. 497. ennerholm. L. 1963. Chromatographic separation of human brain :tghosiT ./, Natrochcm. 10:613, idin, X. S. 1965. Blueprint records of thin layer chromatograms. J- Fipid^ 6:4-12. iettinen. T., and I-T. Takki-Luukkainen. 1959. Use of butyl acetate interm ination of sialic acid. Acta Chcm. Scond. 13:856. ,,fieri.. K. 1970. Xew developments in the study of gangliosidc struct^ Chan. Phys. Lipids. 5:205. neeli. J. L., R. E. Cone, and J. J. Marchalonis. 1973. Isolation of 0 antigen, tin* suriace of thymus lymphocytes. Nat. New Biol. 241:251. it vita, E. S., E. A. Boyse, and j . W. Uhr. 1973. Isolation and c!v:racte/ of a molecular complex containing Thy-1 antigen from the sur.acc thvmocvtes and T cells, Enr. J. Immunol. 3:446. p K /f/S - OCCURRENCE OF SURFACE IgM, IgD, ANp FREE LIGHT CHAINS ON HUMAN LYMPHOCYTES* By SHU MAN FU, ROBERT J. WINCHESTER, and HENRY G. KUNKEL (From The Rockefeller University, Neiv York 0021) (Received for publication 5 December 1973) Considerable evidence has accumulated indicating that the leukemic lympho cytes of patients with chronic lymphatic leukemia (CLL) carry surface im munoglobulin (Ig) of a single light- and heavy-chain class and that the heavy chain is usually IgM (1-3). This has been taken as evidence of the monorclonality of the proliferating cells. Recently it has been reported that IgD represents the surface-Ig of a significant number of norma! human peripheral blood lymphocytes (4, 5). Of special significance has been the study of Rowe [et al. (6) demonstrating that most normal lymphocytes bearing IgM also [have IgD on their surface. It appeared of special interest to study IgD in the monoclonal CLL situation and determine its relationship to the usual surface [IgM that is present. The findings indicate that the majority of CLL cases have gleukemic cells with IgD as well as IgM on their surface. In addition, free light [chains were frequently found. Materials and Methods r Anti-Intmunogiobtdin Anitscra.--Antisera against isolated IgM, IgG, IgA, IgD, Fab of Fraction II human 7 -globulin (Fr II), and kappa and lambda Bence Jones proteins were Raised in New Zealand Red rabbits. For immunofluorescence, all antisera conjugated with let- nelhylrhodamine isothiocyanate according to Amante et al. (7) were absorbed with an [gammaglobulinemic scrum bound to the Sepharose 4B gel activated by cyanogen bromide, ctbe anti./i antiserum (Rho-anti-i) was further absorbed with Sepharosc-bound Fr II, the dvho-unli-7 ) with Sepharose-bound IgM, IgA, Bence Jones kappa and lambda pro&, the anii-cr (Rho-anti-or) with Sepharose-bound Fr II, IgM, Bence Jones kappa and ntbda proteins, and the anti-5 (Rho-anli-5) with Sepharose-bound Fr II, IgM lambda and ftA . lambda myeloma proteins. The anti (Fab)-7 -serum (Rho-anti-Fab) was not absorbed Antiserum specificities were ascertained by double diffusion and direct hemagglulinaThe Rho-anti-/:, Rho-anti-7 , Rho-anli-a, and Rho-anti-S gave a single precipitin line normal human sera. The Rho-anti-/i agglutinated only IgM-coated red cells, the Rho- only IgG-coated red cells, th? Rho-anti-a only IgA-coated red cells, and the RhoS TM only IgD-coatcd red cells. The Rho-anli Fab had activities against both kappa and iO 1 ^elerm*nants- When the Rho-anti-Fab was absorbed with Sepharose-bound IgG 0r IgG kappa, it was specific for kappa or lambda determinants, ^hemagglutination inhibition, the anti-ju antiserum after absorption with Fr II aggluti|>M -coated reci ce]|s ancj this agglutination inhibited only by either. IgM kappa or 1investigation has been supported by U. S. Public Health Service Grants No. RR-102, and AM 04761. Sti. ^JOURNAL OP EXPERIMENTAL MEDICINE VOLUME 139, 1974 451 1 -152 PU, WINCHESTER, KUNKEL URIEP DEFINITIVE REPORT IgM lambda proteins. The anti-5 aniiserum was absorbed with Fr II and a normal semm wiih low levels of IgD. After absorption, it was specific for 6-determinants only. Similar absorptions and specificity experiments were done with the anti-7 and anli-a antisera. Anti-kappa lienee Jones antisera were absorbed with purified pooled whole human 7 -globulin or human sera. Alter this absorption, the antisera were specific for free kappa chain only. Similar absorptions were done with the anti-lambda antisera. An anli-Fr II light-chain antiserum (anti-Fr II-L) was a generous gift from Dr. \V. Epstein of the University of California at San Francisco. Tliis antiserum was used after absorption with whole myeloma proteins and a normal human Lymphocyte Separation.--Mononuclear cells were isolated from the peripheral blood of chronic lymphocytic leukemia patients by Ficoll-Hypaque discontinuous gradients. The cells were washed six times with 50 ml phosphate-buffered saline (PUS). For hemag .itioij inhibition, cells were suspended in the concentration of 1-2 X 108 cells/ml. Fc. .mtnunofiunrcsccnce, 0.5-1 X 10" cclls/ml suspensions were made. Viability of all lymphocyte prepa rations was greater than 95% b> trypan blue exclusion. Iimunofiuorcscenec.--lmmunofluoresccnL staining of lymphocyte surface Ig was per formed as described by Pernis ct al. (8). For each preparation, 100-400 cells were counted. Antisera were routinely centrifuged to remove agegrgates before use. Absorption of the Rho-:~ anti-/i by Scpharose-linked IgM, the Rho-anti-7 by Sepharose-bound IgG (purified from Fr II by DEAE column chromatography), the Rho-anti-a by Sepharose-bound IgA, theRho-anti-Fab by Sepharose-bound IgG and the Rho-anti-6 by an isolated IgD myeloma pro-" tein removed the staining activities of these antisera. In these experiments, the blocking pnL teins used were different from the immunizing proteins. I Hemagglutination and Hemagglutination Inhibition.--Hemagglutination and i- cglutir lion inhibition experiments were carried out as described previously (9). Pu , , protdnf were coated onto human red cells by the bisdiazolized benzidine method (BDB). A lymphocy suspension was incubated with an equal volume of appropriately diluted antisera for 45 !_ and after centrifugation, 0.1 ml of the supernate was used to test its ability to agglutinili indicator red cells. More than 90-95% lymphocytes were viable after absorption. By parison of the inhibition efficiency of the cell suspensions in serial dilutions and known amof inhibitory proteins, the gross amount of surface Ig was estimated, Initially, considerable difficulty was encountered in applying hemagglutination techn to estimate' lymphccyte surface IgM. This was overcome by applying antisera of b limited specificities for certain regions of the fi chain. Details of this method will be desc.^ elsewhere. RESULTS Surface 1g of C LL Lymphocytes.-- Both IgM and IgD were found on surface of CLL lymphocytes as shown by representative cases A, B,C, a , (Table I). The Rho-anti-Fab antiserum measured the total percentage ol* I'atirni TABLE I l.ymphocyte Surface Ig of Representative CLL Cases -- .. ---- -- -- ----------- 1 Immunofluorescence leM IgD Fab Kappa A XS 89 94 6 B 75 67 74 70 C 12 87 93 6 D XO 3 86 S3 FU, WINCHESTER, KUNKEL BRIEF DEFINITIVE REPORT 453 bearing cells in these patients since the antiserum had both anti-kappa and anti-lambda activities. In the first twe instances (A and B) the majority of the Ig-bcaring ljuiphocytes were stained with both the Rho-anti-^ and the Rhoanti-6. Since these antisera were highly specific for fx or 6-determinants it was apparent from the percentages stained that the vast majority of cells bore both jgM and IgD on their surface. In the third instance (C) IgM was not detectable .by fluorescence except in a small number of cells which repxesmtecLat-1east-in- normiiPBr'cells in the sample. No significant number of y - and cr-bearing lymphocytes were found in these cases. A total of 15 CLL cases were studied and in 10 a high percentage of the cells showed IgD; in most instances IgM was found as well on the same high percentage of cells. Four cases showed IgM and not IgD; three showed IgD and not IgM; one showed low percentages of IgM, ), and Fab. In all the cases studied there was predominantly one class of light chains on lymphocytes of an individual CLL patient. The pattern of stain- by these three rhodamine-conjugated antisera was finely granular. Howr, the intensities of lymphocyte staining by these antisera varied among different patients. In general, when IgM and IgD were both present, Rhoti-ju and Rho-anti-5 stained with similar intensities. iConsiderable experience has been gained recently in the use of hemaggltitina?on inhibition techniques for obtaining semiquantitative information on sur:Igs on lymphocytes. This method has the advantage of applying antisera of early defined specificity as determined by experiments such as those shown in le II. In three cases in Table I, IgD was readily detected on the lymphosurface in quantities that clearly related the IgD to the leukemic cells, was detectable similarly in cases A and B but barely so in case C. No IgG IgA were detected in any of the cases. In case C, lambda determinants were d and no kappa determinants were detected. In all respects the results similar to those obtained by fluorescence. detection of Free Light Chains on C L L Lymphocytes.-- It is known that antit be made to light chains that detect determinants that are hidden when t chains exist in combination with heavy chains in whole proteins (11). _antisera were prepared to both kappa and lambda Bence-Jones proteins after absorption showed such "free chain" specificity. Table II illustrates li. TABLE II , Specificity of Anti-Kappa Chain Scrum Absorbed with Normal Human Scrum Inhibitor protein concentration (mc/ml) 1 0.1 0.0! U.001 0.0001 0.000 Kappa Bence Jones '.lambda lienee Jones ..IgG K IgM K formal human serum (diluted 1/ 10) Uni protein; Kappa Bence Jones protein. 0 000 2 222 tr 2 2 2 2 222 2 222 0 2 2 2 2 2 2 2 2 2 45-1 FU, WINCHESTER, KUNKEL BRIEF DEFINITIVE REPORT this point. Only kappa Hence-Jones proteins inhibited the agglutination of the kappa protein coated red cells by the antiserum whereas whole l : rj k..ppa Ig.M kappa, and normal human sera did not inhibit the agglutination. Similar specificity only for free lambda chains was also obtained from anti-lambda Hence-Jones antisera. The anti-Fr II-L had specificities for both free kappa and lambda chains. When these hemagglutination inhibition systems were applied to the quanti tation of free light chains on CLL lymphocytes, lymphocytes of seven CLL patients were found to have readily delectable amounts of free light chain on their surface (Table III). The range of free light chains was between 125-1 000 ng per H's cells Free light chains were found on both kappa- and lambda- I'alimi TABLE III Detection of Free Light Chains on CLL Lymphocyte Surface Class of ligia ch a in F ree light ch a in ' Kappa l- Kappa G Kappa I I Kappa 1 Kappa J L a m l da K Lambda n g / / 0 * ctllt 1 ,0 0 0 500 250 125 125 2 2 * A j u i m a i e values from the semiquantitative hemagglutination inhibition lechniq bearin'.' CLL lymphocytes. Determination of the total light chains on the say ( 'LL < - b tie- usual anti-kappa and anti-lambda antisera with specificit! aganM light i Lain determinants shared by both free chains and those in binaii' ii with heavy chains, indicated that the free light chains represented, signifu ant portion of the total. In only one of eight cases studied were the light chains undetectable. DISCUSSION In view of the monoclonal nature of the proliferating CLL cells with on single <lass of light chains, these cells are proving extremely useful for stud the characteiistics of a homogeneous cell population. Their H-ccll nature high percentages of Ig-bearing cells is now firmly established in aim cases (5). The findings that the majority of CLL lymphocytes carry bot and IgM on their surface offers strong supportive evidence for the concl. of Rowe et al. (6) from their studies on normal human lymphocytes. PcrT Rowe have also observed IgD on leukemic: lymphocytes recently- ^ was obtained in the present study as well as the previous one (6) Personal communication. FU, WINCHESTER, KUNKEL BRIEF DEFINITIVE REPORT 455 and IgD 'ire formed by the cells themselves and are not taken up from the serum. The single light-chain type for-each leukemia, with both kappa and lambda represented, offers strong evidence on this point. It also indicates that both the IgD and IgM have similar light chains. It appears probable that they also possess identical V regions and should share the same idiotypic sped ftri tv. This is currently under investigation. The significance of the free light chains on the leukemia cells remains to be determined. The use of antisera specific for the free chains determinants utTered a simple procedure for their detection. It remains to be determined whether similar amounts are found on normal If cells. More quantitative procedures for the light chains as well as for IgM and IgD are required to answer questions such as this. Evidence for some free surface light chains has been obtained in single instances previously by other methods (11, 12). 15 CLL cases were investigated in the present study and it was apparent both fluorescence and hemagglutination inhibition studies that IgD was nt on the surface of the leukemia cells in most but not all cases. In three instances the IgD was present in a high percentage of cells without detectable ;M. The major question concerning the presence of both Igs on these B tells their relationship with respect to receptor function. This question remains answered but it appears clear that IgD must play a key role. SUMMARY An analysis was made of the immunoglobulin surface markers of the t ells of tients with chronic lymphatic leukemia (CLL) in view of previous evidence their monoclonal B-cell character. The simultaneous presence of IgM and 1on the surface of the majority of lymphocytes was demonstrated b\ both lunofluorescence and hemagglutination inhibition in most cases. However, were observed with surface IgM without IgD a< well as cases with lcD >ut IgM. IgG and IgA were absent. Studies of the light chains in d ic a te d y a single class in a given case. In addition to bound light chains, free light ns were readily demonstrated in most cases through the use of antisera he for "free chain" determinants. It thus appeared that there arc- three `types of surface Ig on CLL lymphocytes, IgM, IgD, and free light chains thank Doctors B. Lee, L. Reich, H. Al'Mondhiry (Memorial Hospital, Neu York) -Doctors A. Sawiiski and K. Rai (Long Island Jewish Hospital, Long Island, N Y j itheir help in collecting the blood samples from the leukemic patients. We also thank Dr Epstein for his generous gift of the anti-Fr II-L antiserum. REFERENCES fey, H. M., E. Rabellino, and B. Pirofsky. 1971. Immunoglobulins on the surface j?* lymphocytes. IV. Distribution in hypogammaglobulinemia, cellular immune ^deficiency and chronic lymphocytic leukemia. J . Clin. Invest. 50:2368. upland, S. S., J. B. Natvig, and I*. Stavem. 1972. Immunological characteriza>n f lymphocytes in lymphoproliferative diseases. Restriction of classes, p a s s e s , and Cm allotypesof membrane-bound lg. Scand. J. Immunol 1:551. 456 FU, WINCHESTER, KUNKEL BRIEF DEFINITIVE REPORT 3. lentwich, Z., and H. G. Kunkel. 1973. Specific properties of human 11 and T lymphocytes and alterations in disease. Transplantation Rev. 16:29. 4. Van Boxel, J. A., \V. E. Paul, W. D. Terry, and I. Green. 1972. IgD bearing human lymphocytes. J . Iimntntol. 109:648. 5. Rowe, D. S., K. Hug, \V. P. Faulk, J. N. McCormick, and H. Gerber. 1973. IgD on the surface oi periferial blood lymphocytes of human newborn. Nat. New Biol. 242:155. 6. Rowe, I). S,, K. Hug, L. Forni, and II. Pernis. 1973. Immunoglobulin D as a lymphocyte receptor. J . Exp. Med. 138:965. 7. Amante, L., A. Ancona, and L. Forni. 1972. The conjugation of immui..,iobuHn with letramcthylrhodaminc isolhiocyanate. J . Immunol. Methods. 1:289, S. Pernis, Ji.. L. Forni, and L. Amante. 1970. Immunoglobulin spots on the surface of rabbit lymphocytes. J . Exp. Med. 132:1001. 9. Naivig, J. B., and H. G. Kunkel. 1967. Detection of genetic antigens utilizing gamma globulins coupled to red blood cells. Nature (Land.). 216:68. 10. Epstein, \Y. V., M. Tan, and D. Gross. 1964. Blocked antigenic sites on theLchain of human gamma globulin. Nature {Lond.). 202:1175. 11. Eskeland, T., E. Klein, M. Inoue, and B. Johansson. 1971. Characterization of immunoglobulin structures from the surface of chronic lymphocytic leukemia cells. J . Exp. Med. 134:265. 12. Moroz, E,, L. Shalmon, and J. Hahn. 1973. Synthesis of surface im;v..:::oglobulia by lymphocyte leukemia cell in vitro. Eur. J . Immunol. 3:16.