Rearrangement and expression of immunoglobulin genes and expression of Tac antigen in hairy cell-leukemia. (recombinant DNA/B-cell differentiation/T-cell ...
Proc. Nati Acad. Sci. USA Vol. 80, pp. 4522-4526, July 1983
Medical Sciences
Rearrangement and expression of immunoglobulin genes and expression of Tac antigen in hairy cell -leukemia (recombinant DNA/B-cell differentiation/T-cell growth factor/interleukin 2/monoclonal antibody)
STANLEY J. KORSMEYER*, WARNER C. GREENE*, JEFFREY COSSMANt, SU-MING HSUt, JANE P. JENSEN*, LEONARD M. NECKERSt, SANDRA L. MARSHALL*, AJAY BAKHSHI*, JOEL M. DEPPER*, WARREN J. LEONARD*, ELAINE S. JAFFEt, AND THOMAS A. WALDMANN* *Metabolism Branch and tLaboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Marvland 20205 Communicated by DeWitt Stetten, Jr., April 22, 1983
ABSTRACT The origin and exact stage of differentiation of the neoplastic cells that comprise hairy cell leukemia have remained uncertain. As Ig heavy and light chain genes must both undergo a DNA rearrangement during B-cell development but rarely do so within other hematopoietic lineages, we examined these genes in this leukemia. The neoplastic cells of all eight cases demonstrated rearranged heavy and light chain genes and, in two cases examined, contained the corresponding mRNA for heavy and light chain Ig. Consistent with this B-cell genotype, all cases displayed cell surface HLA-DR and B-cell-associated antigens. Unexpectedly, all cases demonstrated cell surface Tac antigen, which previously had been restricted predominantly to select T-cell malignancies and activated T cells. Prior studies suggested that the antiTac monoclonal antibody recognized a peptide associated with the binding of interleukin 2 (T-cell growth factor) in such T cells. Immunoprecipitation with anti-Tac and NaDodSO4/polyacrylamide gel electrophoresis revealed an antigen on leukemic hairy cells with a Mr of 53,000-57,000, identical in size to the receptor on activated T cells. This apparent biphenotypic status might reflect a transformation-associated expression of the Tac antigen in this leukemia. Alternatively, hairy cell leukemia may be a malignancy of a unique stage of normal B-cell differentiation in which the Tac antigen is expressed.
Considerable controversy has surrounded the cellular origin and the stage of differentiation of the neoplastic cells comprising hairy cell leukemia (HCL), leukemic reticuloendotheliosis (14). These neoplastic mononuclear cells characteristically have a ruffled cell surface and contain acid phosphatase resistant to tartaric acid (3). However, no fully equivalent cell within normal cellular differentiation has been identified. Several investigations have found the presence of B-cell-associated surface antigens (5-7), whereas others have reported T-cell-associated surface antigens (8-10) upon these leukemic cells. The vast majority of cases do bear surface Ig, but the presence of avid Fc fragment receptors (11) and reports of multiple Ig isotypes (5, 12) have raised questions as to whether the Ig in all cases was actually synthesized by the neoplastic cells. In this study we determined the patterns of Ig gene arrangement and the expression of Ig genes within HCL. Ig genes must undergo a prerequisite reorganization at the DNA level during the differentiation of a B cell (13-15). This occurs in a sequential order in which heavy (H) chain gene rearrangement precedes that of the light (L) chain genes, of which the K-chain genes generally rearrange before the A chain genes (16-18). In
contrast, leukemic T cells do not show L chain gene rearrangements and usually (18 of 20 cases) also retain their H chain genes
in the germ-line form (19, 20). We show here that the HCL cells examined possess Ig gene rearrangements and mRNA responsible for Ig production, thus placing these leukemic expansions within the B-cell lineage. In addition, the observed patterns of H and L chain gene rearrangements suggest a rather mature stage of B-cell differentiation. Surprisingly, the leukemia cells of all eight cases reacted with the anti-Tac antibody (21, 22), which appears to recognize, at least in part, the membrane receptor for interleukin 2 (IL-2; or T-cell growth factor) on activated T cells (23). The unexpected presence of Tac antigen upon the malignant B cells of HCL raises the possibility that an IL-2 receptor or a similar antigen might be expressed at certain stages of normal B-cell development.
METHODS HCL Cases Examined. Leukemic cells from the eight patients examined here displayed the typical morphological characteristics of HCL cells and had tartrate-resistant acid phosphatase detected within the neoplastic hairy cells of their spleens or blood (1-4). The neoplastic cells examined were obtained either from the peripheral blood of patients with high leukemic cell counts after splenectomy or were teased free from splenic tissue and then enriched by Ficoll/Hypaque gradient centrifugation. In several cases splenic tissue blocks that were diffusely infiltrated by uniform, monotonous-appearing hairy cells were studied directly. Monoclonal Antibody Studies and Ig Characterization. When available, leukemia cells in suspension were allowed to react with monoclonal antibodies and were analyzed by flow microfluorometry as described (20; 24). Prior to staining, cells were incubated at 370C in 5% C02/95% air for 30 min to allow shedding of cytophilic Ig. In addition, controls were performed in which a purified mouse myeloma protein of the appropriate IgG subclass (identical to the antibody utilized) was incubated with the hairy cells to assess nonspecific Fc fragment binding. Fluorescein-conjugated anti-Tac antibody was utilized in dual fluorescence studies together with an appropriate, biotin-conjugated anti-L chain antibody, which was then treated with avidin-linked Texas Red (25). Such dual staining was performed on cells with and without prior incubation with a nonreactive mouse myeloma protein of the IgG2a subclass (identical to antiTac). Such cells were interrogated simultaneously with both an argon and a krypton laser with suppression filters. In addition to staining with monoclonal anti-Ig reagents, cells were also examined with fluorescein isothiocyanate-conjugated F(ab')2 rabAbbreviations: HCL, hairy cell leukemia; IL-2, interleukin 2; H chain, heavy chain; L chain, light chain; VH, DH, andJH, the variable, diversity, and joining segments of the H chain genes; CH, the constant region of H chain; kb, kilobase(s).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. NatL Acad. Sci. USA 80 (1983)
bit antibodies to human ,u, f, 'y, a, K, and A chains. When no suspension cells were available (cases 5, 6, 7, and 8), preserved splenic tissue blocks were stained with antibodies utilizing a sensitive avidin-biotin immunohistochemical procedure as described (26). DNA and RNA Analysis of Ig Genes. High molecular weight DNA was extracted from leukemic cells in suspension or directly from cryo-preserved splenic tissue blocks. This genomic DNA was digested to completion with the appropriate restriction endonuclease (BamHI or EcoRI), size-fractionated by agarose electrophoresis, and transferred to nitrocellulose paper (20). Such nitrocellulose blots were hybridized to nick-translated [32P]DNA probes of the Ig genes shown to be capable of recognizing germ-line and rearranged alleles (Fig. LA) (27-29), washed at the appropriate stringencies, and then visualized on autoradiograms. Total RNA was extracted from the leukemic cells of two cases by resuspending the cells in 4 M guanidine thiocyanate, subjecting them to disruption with a Polytron homogenizer (Brinkmann), and centrifuging the RNA through a 5.7 M CsC12 gradient to form a pellet. These total RNA preparations were electrophoresed through a formaldehyde gel, transferred to nitrocellulose paper (30), and then hybridized with the aforementioned probes and a 7.5-kilobase (kb) germ-line BamHIHindIII fragment containing the H chain constant (CH) region gene Cy4 Receptor Characterization. Purified hairy cells were obtained by depleting sheep erythrocyte-binding T cells from the leukemic lymphocytes of case 2. Purified cells (5 x 106 cells) were washed and surface-labeled with 1"I as described (23). Labeled cells were extracted, and the supernatants were cleared with a monoclonal Ig (RPC5; IgG2a-K) and 10% formaldehydefixed Cowan I strain staphylococci as described (23). This supernatant was immunoprecipitated with purified anti-Tac or control UPC10 (another IgG2a-K). The final, washed pellet was resuspended in 0.1 M dithiothreitol/1% NaDodSO4, boiled, and then analyzed by 7.5% discontinuous NaDodSO4/polyacrylamide gel electrophoresis (23).
genes when their DNAs were examined with the joining (JH) gene region probe (Table 1). Previous studies have indicated
that cells of the B-cell lineage from the earliest recognizable Bcell precursors through terminally differentiated plasma cells display H chain gene rearrangements (13-18). In contrast, the vast majority of hematopoietic cells that pursue other than the B-cell pathway of differentiation (T cells, granulocytes, and monocytes) retain germ-line H chain genes (refs. 19 and 20; unpublished data). Thus, at the H chain gene level, the HCL cells had rearrangements suggesting their commitment to the B-cell pathway of development. This included cases 6 and 7 in which surface H chain Ig was not demonstrated (Table 1). Cells of all cases showed at least one JH rearrangement, presumably corresponding to the productive allele bearing an effective recombination of variable, diversity, and joining (VH, DH, and JH) gene segments capable of producing a complete H chain Ig molecule. The other nonproductive or "excluded" H chain gene allele within malignant B cells has been shown to be in the germline form, also rearranged, or in some cases the JH gene elements have been deleted (15, 16, 19, 20). Similar patterns were seen within these leukemic hairy cells (Table 1), and the specific H chain gene patterns of cases 1 and 2 are shown in Fig. 1B. Case 1 had both of the H chain genes rearranged as detected by hybridizing theJH region probe with an EcoRI digest. In addition, both C, regions were apparently deleted, probably reflecting a deletional H chain class switch event to a more 3'located CH region on both alleles (31). Consistent with these gene rearrangements, case 1 had cell surface IgG present (Table 1) and y chain mRNA was detectable (Fig. 1B). Case 2 also had both H chain alleles in a rearranged state as demonstrated with the JH region probe and EcoRI-digested DNA (Fig. 1B). However, the H chain gene rearrangements appear to have created similarly sized BamHI fragments, as but a single yet diffuse band was detected in the BamHI digests when probed with the same IH region gene (Fig. 1B). Examination of these BamHI digests revealed that the C. region was retained on at least one of these alleles. Although this case is predominantly a cell surface IgG-positive leukemia, approximately 15% of the leukemic cells of case 2 bore surface IgM (Table 1), and ,u chain mRNA was indeed detectable within these cells (Fig. 1B). The precise mechanism by which such a leukemia can produce two
RESULTS Ig H Chain Gene Rearrangement and Expression. All eight cases of HCL studied demonstrated recombinations of H chain
Table 1. Ig gene patterns and cell surface markers of eight HCL patients Cell surface markers Monoclonal antibodies Case idenHLA/ tificaDR CALLA Leu 4/ Leu 3/ Leu 2/ Antition B1 BA-1 BA-2 SC2 J5 OKM1 OKT3 OKT4 OKT8 3A-1 Tac 1
+
+
-
2
+
+
3
+
+
+
-
+
-
+
-
+
-
+
-
+ _
44 + + + + + 5 + +
-
-
-
_
-
-
-
-
+
_
_
-
+
_
_
-
+
-
+
-
+
-
4523
-
,u
Surface Ig 8 y a K
-
-
+
-
(15%)-
+
-
+ _ +
-
+
+ -
-
+
+
Ig gene patterns Heavy A chain K A - JH:2 Re 1 Re Germ C 2 Del JH:2 Re Del 1 Re Re - JH 1Re 1 Re Germ
CA:i1
Re ~~~~~~~~~~~~~~~~~~~~~~~~~CA:l +JHAlReDel iRe -ND ND JH:lRe Del 2Re
-
-
C,:1 Re + + ND ND ND ND - + JH:iRe NoRe* 2Re + + ND ND ND ND - + JH:2 Re NoRe* 1 Re + 8 + + + + - - - + JH:1Re NoRe* lRe The presence of surface markers was determined by fluorescence-activated cell sorting (cases 1-4; +, >30% reactivity). Leukemic cells within splenic tissue sections from cases 5-8 were examined directly with an avidin-biotin complex assay. ND, nondetectable; Re, rearranged; Del, deleted; Germ, germ line. * Cells examined from cases 6, 7, and 8 contained some nonleukemic cells that contributed germ-line K chain genes. Thus, K chain deletions could not be assessed in these instances. 6
+
7
+
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.
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Proc. Natl. Acad. Sci. USA 80 (1983)
H chain isotypes is uncertain but suggests that a process other than the classic deletional H chain class switch might be involved. L Chain Gene Rearrangement and Expression. We have previously noted an apparent sequence in L chain gene recombinations within other malignancies of the human B-cell series and even within normal B cells (16-18, 20). This hierarchy of
finitive cell surface L chain isotype was demonstrated (case 5) had a pattern of rearrangement consistent with a A chain-producing cell (Table 1). The two cases (cases 1 and 2) in which RNA was examined displayed the expected messages (K and A chains, respectively) corresponding to their L chain gene rearrangements and apparent surface Ig (Fig. 1B). Cell Surface Antigen Expression as Detected by Monoclonal Antibodies. Consistent with the findings of Ig gene rearrangements, the HCL cases frequently displayed a number of B-cell-associated antigens. Cells from all eight cases examined reacted with the monoclonal antibody B1 that detects a B-cellassociated antigen (32) and with the SC2 monoclonal antibody that recognizes a nonpolymorphic HLA-DR determinant (33) (Table 1). Four of eight cases demonstrated the p30 B-cell-associated antigen recognized by the BA-i monoclonal antibody (34). Two of eight cases displayed some p24 antigen as recognized by the BA-2 antibody (35), whereas three showed a reaction with OKM1, which recognizes monocytes and certain other cells (36). None of the cases displayed the common acute lymphoblastic leukemia antigen (called CALLA), which is frequently found on B-cell precursor leukemias and is detected by the J5 antibody (37) (Table 1). Correspondingly, none of eight cases reacted with the 3A-1 (38), OKT6, Leu 4/OKT3, Leu 3/OKT4, or Leu 2/OKT8 (39, 40) monoclonal antibodies recognizing various T-cell-associated
L chain gene utilization appears to begin with attempts at K chain and, if unsuccessful, proceeds to the A chain all 20 human T-cell lines or leukemia cells examined have had germ-line L chain genes (19, 20). Of note, all cases of HCL suspected of K chain production based on detection of surface Ig had rearranged K chain genes with germline A chain genes (Table 1). Similarly, all HCL cases with demonstrable cell surface A chain had corresponding A chain gene recombinations (Table 1). In the instances in which highly pure leukemic cells with A chain gene rearrangements were available for study (cases 2, 4, and 5) the K chain genes had been deleted. This finding of K chain gene deletion is characteristic of A chaingene recombination genes. In contrast,
producing B-cell malignancies (17). The remaining three cases with A chain gene rearrangements (cases 6, 7, and 8) had no K chain rearrangements present; but nonleukemic cells were present in these particular preparations and contributed their germ-line K chain genes, so that the extent of K chain deletion could not be assessed. In addition, the case in which no deA
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FIG. 1. (A) HumanIggene probes. The JH probe was a 2.4-kb Sau3A fragment and recognized a germ-line 17-kb BamHI fragment or a 16-kb EcoRI fragment in non-B-cell sources ofDNA (27). The 1.3-kb germ-line EcoRI fragment used as the C,, probe uniformly identified a 17-kb-sized BamHI fragment in germ-line DNA (27). The 2.5kb EcoRI CK probe recognized a 12.0kb-sized germ-line BamHI fragment in all individuals (28). The combined CA probe consisted of a 0.8-kb BamHIEcoRI fragment containing the CA1 gene and a 1.2-kbBamHl-EcoRI fragment containing the CA2 gene. This probe could discern the several polymorphic patterns of A chain genes in man (29). (B) Case 1: DNA genomic blots revealed two rearranged (arrows) H chain genes in the leukemic hairy cells (7C) as compared to the germline genes (indicated by dash marks and kb size markers) in the control cells (IC) when probed with the JH region. Both C, genes were deleted consistent with a H chain class switch, and the hairy cells had an appropriately sized y chain RNA present when compared to an IgG-producing B-cell line (yB). A K chain gene was rearranged and K chain RNA was present, whereas the A chain genes were germ line and no A chain RNA was evident. Case 2: Two rearranged JH regions were detected in an EcoRI digest but were not clearly separable in theBamHI digest. At least one C, region was retained and ,u chain RNA was also detected within these hairy cells (MC). The rearranged A gene produced detectable RNA, whereas the K chain genes were deleted and, thus, were not transcribed.
Medical Sciences: Korsmeyer et al.
Proc. Nati Acad. Sci. USA 80 (1983)
antigens. In unexpected contrast, however, all eight cases of HCL did display binding of the anti-Tac monoclonal antibody (21, 22). This antibody recognizes an activation antigen on T cells and blocks the binding of IL-2 to such cells (23). Although an occasional B-cell malignancy has been noted to bear some cell surface Tac antigen, we have not noted such consistent reactivity in B-cell chronic lymphocytic leukemia, Burkitt lymphoma, Epstein-Barr virus-transformed B-cell lines, B-cell precursor leukemias, or normal B cells (ref. 21; unpublished observations). The staining of cells from four cases of HCL with a fluorescein-conjugated anti-Tac antibody after the cells were incubated with an irrelevant nonlabeled mouse IgG2a myeloma protein confirmed that the reactivity was due to the presence of Tac antigen and not to Fc binding of the monoclonal antibody. Dual fluorescence studies were performed utilizing this fluorescein-conjugated anti-Tac antibody and the appropriate anti-L chain antibody, which was coupled to biotin and allowed to react with avidin-linked Texas Red. These examinations, performed on cells from cases 1-4, confirmed that the very same
AI
a
b
c
4525
d
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97.592.5 /
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_
43-
43
-
,,
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FIG. 3. NaDodSO4/polyacrylamide gel comparison of immunoprecipitations with anti-Tac and a control Ig, UPC10, of the surface-iodinated cells from an IL-2-dependent T-cell line and the HCL cells of case 2. A broad Mr 53,000-57,000 band was present in the anti-Tac immunoprecipitations (lanes a and c) of the hairy cell as well as the T-cell line but was absent with the control Ig (lanes b and d). A smaller contaminating band was present in all gel lanes, including the Mr marker lane M.
II
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Fluorescence (log scale)
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FIG. 2. (A) The fluorescence profile of cells of case 4 when stained with a biotin-conjugated anti-A chain monoclonal antibody and then allowed to react with avidin-linked Texas Red ( ) as compared to background fluorescence (---). (B) A fluorescence profile revealing binding of a fluorescein-conjugated anti-Tac antibody to cells of case 4. (C) A linear contour plot of the two-color, dual fluorescence comparison of anti-Tac and anti-Ig. The same cells that bore surface Ig (sIg) were also positive for Tac (-87% of the total population). A population of cells that was negative for both markers (-8% of the total) was clearly separable from the stained HCL cells.
leukemic cells that expressed cell surface Ig expressed Tac antigen as well (Fig. 2). Initial Characterization of the Cell Surface Tac Antigen in HCL Cells. The HCL cells of case 1 were purified by removing any residual sheep erythrocyte-binding T cells, leaving 1% or less of contaminating T cells as assessed by Leu 1, Leu 4, or OKT1L monoclonal antibodies. The surface membrane antigen recognized by anti-Tac was then characterized on these purified leukemic cells. The HCL cells and, as a control, an IL-2dependent T-cell line established from the same patient were surface-iodinated and immunoprecipitated with either anti-Tac or UPC10. A broad Mr 53,000-57,000 band was immunopre-cipitated by anti-Tac but not by the control monoclonal UPC1O (Fig. 3). The membrane antigen identified on these hairy cells was essentially identical in size to that of the Tac receptor present on the IL-2-dependent T-cell line. To further assess whether reactivity with anti-Tac indicated the presence of a functional IL-2 receptor, aliquots of the purified hairy cells were incubated in the presence of a lectin-depleted IL-2 preparation, and proliferation was assessed by [3H]thymidine incorporation. In the presence of IL-2, proliferation in these hairy cell cultures was very modest, ranging from 1.5- to 3.0-fold greater than that obtained in cultures without IL-2. DISCUSSION The demonstration of appropriately rearranged H and L chain Ig genes and their corresponding mRNA within HCL cells indicates that these leukemic lymphocytes are committed B cells at the Ig gene level. Consistent with this B-cell classification, all cases displayed cell surface HLA-DR and reactivity with the B1 antibody, and some cases also showed reactivity with the BA1 or BA-2 monoclonal antibodies. In addition, other investigators have demonstrated the incorporation of radiolabeled amino acids into Ig within selected cases of HCL (41, 42). In keeping with these findings, none of the HCL cells reacted with any of a number of monoclonal antibodies-3A-1, OKT6, Leu 4/OKT3,
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Proc. Natl. Acad. Sci. USA 80 (1983)
Medical Sciences: Korsmeyer et al.
Leu 3/OKT4, and Leu 2/OKT8-that recognize various accepted T-cell-associated antigens. Thus, the reactivity of all tested HCL cells with the anti-Tac antibody was in surprising contrast with all of the other findings. The T-cell activation antigen recognized by the anti-Tac monoclonal antibody has been observed primarily on some T-cell malignancies and activated T cells. This finding no doubt reflects the fact that at least a component of the p53-57 antigen recognized by anti-Tac is associated with the binding of IL-2 in activated T cells (23). The presence of the Tac antigen upon cells that by all other criteria are malignant B cells raises several interesting possibilities. First, the simultaneous expression of both B-cell- and T-cell-associated genes could be interpreted as evidence for a biphenotypic leukemia. It is conceivable that a biphenotypic status might occasionally result from a transformation-associated activation of a gene-in this particular case, the gene encoding the Tac antigen. It is of note that, within adult T-cell leukemias, there is a strong correlation between the expression of cell surface Tac antigen and the presence of the human Tcell leukemia virus (HTLV) (43). Perhaps the presence of Tac antigen upon B-cell-type HCL cells suggests that a similar retrovirus might be involved in the pathogenesis of this leukemia. In this regard, Kalyanaraman et al. (44) have found a new subtype of the human retrovirus (designated HTLV-II) within a variant T-cell line (Mo) established from a patient with HCL. Alternatively, HCL may be a malignancy of a unique stage of normal B-cell differentiation in which an IL-2 receptor or a closely related protein is expressed. Recently, the precise factors required for the growth and subsequent differentiation of normal B cells have come under close scrutiny, and several factors unique to B cells have been identified (45). However, other data have suggested the possibility that IL-2 itself is utilized in some B-cell expansions (46). It is intriguing that the Tac antigen on HCL cells is immunoprecipitable and has a Mr of 53,00057,000, identical in size to the receptor present on activated normal human T cells. However, the short-term proliferation of purified HCL cells in the presence of IL-2 was not dramatic, suggesting the possibility that the Tac antigen identified on these malignant B cells may not be fully identical with that found on activated T cells. Alternatively, additional growth factors may be required for the successful expansion of these cells. As there may be a normal equivalent cell of HCL that expresses a Tac antigen, this leukemia is a useful model in further examining the role of this antigen in the B-cell lineage. We wish to thank Dr. Geraldine Schechter, Dr. Curtis Ries, and Dr. William Lee for allowing us to study their patients, and Janet Shelton for her assistance in preparation of this manuscript. 1. Bouroncle, B. A. (1979) Blood 53, 412-436. 2. Catovsky, D. (1977) Clin. Haematol. 6, 245-268. 3. Braylan, R. C. & Burke, J. S. (1979) Annu. Rev. Med. 30, 17-24. 4. Golomb, H. M., Catovsky, D. & Golde, D. W. (1978) Ann. Intern. Med. 89, 677-683. 5. Jansen, J., Schuit, H. R. E., Meijer, C. J. L. M., van Nieuwkoop, J. A. & Hijmans, W. (1982) Blood 59, 52-60. 6. Posnett, D. N., Chiorazzi, N. & Kunkel, H. G. (1982)J. Clin. Invest. 70, 254-261. 7. Jansen, J., LeBien, T. W. & Kersey, J. D. (1982) Blood 59, 609614. 8. Saxon, A., Stevens, R. H. & Golde, D. W. (1978) Ann. Intern. Med. 88, 323-326. 9. Cawley, J. C., Burns, G. F., Nash, T. A., Higgy, K. E., Child, J. A. & Roberts, B. E. (1978) Blood 51, 61-69. 10. Guglielmi, P., Preud'homme, J. L. & Flandrin, G. (1980) Nature (London) 286, 166-168. 11. Jaffe, E. S., Shevach, E. M., Frank, M. M. & Green, I. (1974) Am. J. Med. 57, 108-114. 12. Burns, G. F., Cawley, J. C., Worman, C. P., Karpas, A., Barker, C. R., Goldstone, A. H. & Hayhoe, F. G. J. (1978) Blood 52, 11321147.
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