Genuine CD7 expression in acute leukemia and lymphoblastic lymphoma

Leukemia Research VoL 14, No. 10, pp. 869--877, 1990. Printed in Great Britain.

0145-2126/90 $3.00 + .00 Pergamon Press plc

G E N U I N E CD7 EXPRESSION IN A C U T E L E U K E M I A AND LYMPHOBLASTIC LYMPHOMA H1ROTAKA OSADA,* NOBUHIKO EMI,* RYUZO UEDA,'I" MASAO SETO,t KOICHI KOIKE,~: TAIZAN SUCHI,:~ SEIJI KOJIMA,§ YUICHI OBATA* and TOSHITADA TAKAHASHI* * Laboratories of Immunology, ? Chemotherapy; and ~t Clinical Laboratory, Aichi Cancer Center, Chikusa-ku, Nagoya 464; and § Division of Hematology/Oncology, Children's Medical Center, The Japanese Red Cross Nagoya First Hospital, Nakamura-ku, Nagoya 453, Japan

(Received 13 January 1990. Revision accepted 28 April 1990) Abstract--CD7 has been used as a valuable marker for normal and malignant T cells and also for a proportion of acute nonlymphocytic leukemia (ANLL) cells. Difference in reactivity was noticed among CD7 antibodies, however, when tested against ANLL cells and myeloid/monocytoid cell lines; Tp40 antibody produced in our laboratories was not reactive with the HL-60 promyelocytic line, whereas 4A antibody was reactive, even though both detected a quite similar or an identical epitope on CD7 molecule. Preincubation of HL-60 cells with human immunoglobulin preparation clearly negated the reactivity by 4A, suggesting that 4A antibody is not reactive to CD7 itself, but it probably binds with immunoglobulin G Fc receptors expressed on HL-60 cells. Five cases of ANLL which were positive with 4A antibody were selected and tested with Tp40 antibody, and only two were found to be positive. Expression of CD7 mRNA in these two cases (but not in other cases) was also demonstrated by Northern blotting with a cDNA probe for CD7 recently cloned in our laboratories, indicating that CD7 is expressed on a certain fraction of ANLL, although the positive cases may be smaller than the reports so far appeared. A Northern blot study was also conducted with two acute lymphocytic leukemia cases and one lymphoblastic lymphoma case with CD7 +, CD2 -/-+, CD5 -/+- phenotype and germline T cell receptor fl genes. CD7 mRNA is expressed in all three cases and CD3 mRNA is also observed in two cases, suggesting that these tumor cells are of T precursor origin.

Key words: CD7, CD7 transcript, immunoglobulin Fc receptor, acute leukemia, lymphoblastic lymphoma.

In addition to the reactivity to T cells, CD7 antibodies were ~eported by us and others to react with a proportion of acute nonlymphocytic leukemia (ANLL) [9-13]. Occasional reactivity with A N L L cases was first suggested to be due to the binding of monoclonal antibodies via Fc receptors on the target cells, and not to the antigen on the surface, although the experiments were not conducted to prove it [10]. Sutherland et al., however, showed the genuine expression of CD7 on leukemia cells from one A N L L case by immunoprecipitation technique [11]. In our laboratories, we performed a survey of various types of leukemia and lymphoma cases with Tp40 and 4A antibodies detecting CD7 and found a difference in the reactivity between these two antibodies, when tested against myeloid/monocytoid cells, such as HL60 cells. Accordingly, a detailed serological study was conducted, together with a Northern blot study. The difference in serological reactivity is not due to the epitopes detected, but probably due to the isotype, i.e. y2a shows higher affinity for a Fcy receptor, FcyRI, than y1, resulting in a nonspecific positive

INTRODUCTION CD7 IS ONE of the major T cell antigens and is expressed predominantly on T cells [1-5]. We previously showed that CD7 is expressed on T precursor cells with C D 2 - phenotype separated from fetal thymocytes and also on acute lymphocytic leukemia ( A L L ) cells with germline T cell receptor (TCR) fl genes [2, 6]. Later we established mouse L cell transfectants stably expressing CD7 [7]. Recently, we assigned the gene for CD7 to chromosome 17 by somatic cell hybrids [8].

Abbreviations: ANLL, acute nonlymphocytic leukemia; ALL, acute lymphocytic leukemia; TCR, T cell receptor; LbL, lymphoblastic lymphoma; Ig, immunoglobulin; FITC, fluorescein isothiocyanate; TdT, terminal deoxynucleotidyltransferase; mCD3, membrane CD3; cyCD3, cytoplasmic CD3; MPO, myeloperoxidase. Correspondence to: Dr H. Osada, Laboratory of Immunology, Aichi Cancer Center Research Institute, Chikusaku, Nagoya 464, Japan. 869

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reaction [14, 15]. Expression of CD7 m R N A was always observed in Tp40 positive cases, but not in 4A ÷ Tp40- cases, suggesting that Tp40 reactivity shows genuine expression of CD7 molecule. We showed here that a certain fraction of A N L L cases express CD7 antigen and that a part of A L L and lymphoblastic lymphoma (LbL) cases with germ line T cell receptor fl gene express CD7 antigen as well.

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MATERIALS AND METHODS Cells and cell lines HL-60 and HPB-ALL lines were maintained with RPMI1640 supplemented with 10% fetal bovine serum (FBS). Leukemia and lymphoma cells were cryopreserved in liquid nitrogen with RPMI-1640 containing 10% FBS and 2% dimethyl sulfoxide (DMSO). Diagnosis of leukemia was essentially based on Fab classification [16]. Monoclonal antibodies and flow cytometric analysis Tp40 (IgG1) antibody detecting CD7 was produced in our laboratory [2], while 4A (IgG2a) antibody was kindly provided by Dr Morishima, Nagoya University School of Medicine, Nagoya, Japan [17]. Ta60a to CD25 was produced in our laboratory [18], while Tac to CD25 was given by Dr Uchiyama, Kyoto University School of Medicine, Kyoto, Japan. Other monoclonal antibodies to CD7(Leu9), CD2(OKTll), CD3(Leu4, OKT3), CD5(Leul), CD8(Leu2, OKT8), CD13(MCS2), CD14 (My4), CD20(Leul6, B1) and DR(OKDR) were purchased. Surface antigens were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ig A + G + M F(ab')2 (Zymed, San Francisco, CA) after reacting with monoclonal antibodies, and then studied by FACStar (Becton-Dickinson, Sunnyvale, CA) as previously described [2]. The blocking of CD7 reactivity to HL-60 and leukemia samples by immunoglobulin (Ig) preparation was carried out as follows; the target cells were incubated with 10% aggregated human IgG preparation (1 mg/ml, Sigma 14506, St Louis, MO) for 30 min at room temperature and then stained with CD7 antibodies. 125I-labeling of Tp40 and 4A antibodies for antibody binding inhibition was carried out as previously described [18]. In the case of CD3, cytoplasmic staining was also carried out: the cells to be stained were pelleted and fixed for 15 min at -20°C with 1% paraformaldehyde in 50% ethanol, and then reacted with Leu4 antibody. After washing, FITC-conjugated anti-mouse Ig A + G + M F(ab')2 was added, washed again and then assayed by FACStar. OKT3 was not appropriate for cytoplasmic staining, as reported by Janossy et al. [19]. Southern and Northern blotting analyses Both analyses were carried out under the experimental conditions as described previously [6]. A partial cDNA clone for CD7 was cloned in our laboratories: the 40 mer oligonucleotide probe corresponding to 569--608 nucleotides (nt) of the 1.7kb cDNA clone described by Aruffo & Seed [20] was synthesized and used to screen the ~.gt-ll cDNA library prepared from a Jurkat T cell leukemia line (purchased from Clonotech Inc., Palo Alto, CA). Consequently, a J61 clone (0.6 kb) was isolated. A 1.3kb

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FIG. 1. The reactivity of CD7 antibodies (Tp40, 4A and Leu9) to HL-60 and HPB-ALL cells. HL-60 promyelocytic leukemia line (A, B) or HPB-ALL T ALL line (C, D) was stained with each CD7 antibody after preincubation with (B, D) or without (A, C) human IgG preparation and analyzed by FACStar. (A) Leu9 (. . . . ) and 4A ( .... ) antibodies bind to HL-60, while Tp40 (o • • e) does not. - - , negative control. (B) The binding of 4A and Leu9 to HL-60 is completely blocked by Ig preincubation. (C) Tp40, 4A, and Leu9 antibodies show similar reactivities to HPB-ALL. (D) Preincubation with Ig shows no influence on the binding.

mRNA band was detected in the L cell transfectant expressing CD7 [7], while no band was observed in parental L cells. The dideoxy method showed that the J61 clone consists of 98-139 nt and 572-1136 nt of the CD7 cDNA clone of Aruffo & Seed [20]. The probes for CD3 (pDJ4, pPGBC9), TCR fl (Jurfl2), TCR ,/, TCR i5 (pMI19), and terminal deoxynucleotidyl transferase (TdT) genes were kindly provided by Dr Cox Terhorst, Dr Tak Mak, Dr Carlo Croce and Dr Osamu Koiwai [21-25].

RESULTS Reactivity o f C D 7 antibodies to H L - 6 0 cells The reactivity of CD7 antibodies (Tp40, 4A, and Leu9) to HL-60 and H P B - A L L was shown in Fig. 1. Both 4A and Leu9 were positive with HL-60 cells (which do not express CD7 m R N A as described below), whereas Tp40 was negative, although they all belong to CD7 (Fig. 1A). With H P B - A L L cells (which express 1.3kb CD7 m R N A as described below), all three antibodies showed positive reactions (Fig. 1C). .. It is possible that the difference in reactivity may be explained by the epitope detected by each antibody. Thus, epitope analysis was carried out by antibody

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FIG. 2. Analysis of the epitopes detected by Tp40 and 4A antibodies. (A) The binding of FITC-labeled Tp40 to HPBALL cells is inhibited by preincubation of 4A ( .... ) as completely as by Tp40 (e • • •) itself. Leul to CD5 ( ) shows no effect on the Tp40 binding. (B) The binding of 125I-labeled 4A is blocked not only by 4A, but also by Tp40. The binding of 125I-labeled Tp40 is also blocked by both antibodies. Ta60b to CD25 was used as a negative control. Control, preincubation without antibody.

binding inhibition assay. As shown in Fig. 2A, the binding of FITC-conjugated Tp40 to HPB-ALL cells was competitively inhibited by 4A as completely as by Tp40 itself, while Leul, as a negative control, showed no inhibition. Fig. 2B showed reciprocal inhibition of Tp40 and 4A binding to HPB-ALL cells. Antibody 4A inhibited the binding of ~25I-Tp40 to HPB-ALL as completely as Tp40 itself, and vice versa. These results implied that these two antibodies detect a quite similar or an identical epitope.

Blocking of 4A antibody reactivity to HL-60 by human immunoglobulin Since HL-60 cells are known to express IgG Fc receptors, Fcy RI and FcyRII [15], the reactivity of 4A and Leu9 antibodies may be explained by the higher affinity to these receptors. Thus, the experiments were conducted to see whether human IgG preparation is able to block this reactivity. Fig. 1B showed that 4A and Leu9 antibodies no longer bind

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to HL-60 cells after preincubation with human Ig. On the other hand, binding of 4A and Leu9 to HPBALL was not blocked by human Ig treatment, as that of Tp40 (Fig. 1D). These results suggested that 4A and Leu9 bind to the ligand on HL-60 different from CD7 molecule, probably to Fc3'R. The isotype of Tp40 is IgG1, while that of 4A and Leu9 is IgG2a, suggesting that IgG2a antibody may bind to FcyR on HL-60 cells, resulting in positive reactions, even though HL-60 cells do not express CD7 molecule. To study further the relation between murine IgG2a antibodies and FcyR, the binding of monoclonal antibodies with two different subclasses, IgG2a and IgG1, against the same CD was examined. All IgG2a antibodies studied (OKT3 for CD3, OKT8 for CD8, B1 for CD20, and Tac for CD25) showed binding to HL-60 cells, whereas IgG1 antibodies (Leu4 for CD3, Leu2 for CD8, Leul6 for CD20, and Ta60a for CD25) did not. The binding of IgG2a antibodies to HL-60 cells was completely inhibited by the addition of aggregated human Ig (data not shown).

Northern blot analysis of CD7 in HL-60 To confirm the absence of the CD7 molecule on HL-60 cells, Northern blot analysis was conducted with the CD7 probe J61, which was cloned in our laboratory as described in Materials and Methods. Authentic 1.3kb mRNA was detected in RNA extracted from HPB-ALL cells and peripheral hlood lymphocytes (PBL), but it was absent from HL-60 cells (Fig. 3A). This result confirmed that the ligand on HL-60 to which 4A and Leu9 bind was not a CD7 molecule.

Tp40 reactivity and CD7 mRNA expression on A N L L cells Some of the CD7 antibodies commonly used for serological typing of PBL and leukemia cells are known to belong to an IgG2 isotype, e.g. 4A, Leu9, WT-1, 3A1-4G6 and OKT16. Thus, some of the ANLL cases positive with these IgG2 antibodies may not be true CD7 positive ones. Accordingly, 11 leukemia cases with 4A +, membrane CD3 (mCD3)phenotype were selected and further examined (Table 1). All five cases in group A and B had myeloid phenotype, myeloperoxidase (MPO)-positive, DR +, CD13 +, CD14 -~/-. In respect to Tp40 reactivity, three cases (group A) were negative, while two cases (group B) were positive, although all five cases were 4A +. Interestingly, the 4A reactivity to group A cases was completely blocked by human IgG preparation, whereas the reactivity to group B, C and D cases remained. To confirm the presence of CD7 on two ANLL cases in group B, CD7 mRNA

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t2D7 FIG. 3. Northern blot analysis with J61 CD7 probe. (A) Ten ~tg total R N A extracted from H P B - A L L (HPB), HL60, and PBL was analysed with a partial CD7 c D N A probe, J61 after electrophoresis. Authentic 1.3 kb band (shown by t>) is detected in H P B - A L L and PBL. No transcript of CD7 is recognized in HL-60. (B) Ten micrograms of total R N A from 4A ÷, m C D 3 - acute leukemia cases was hybridized with a partial CD7 c D N A probe, J61. The numbers over the lanes represent the case numbers in Table 1. The group to which each case belongs is also indicated with brackets. Among A N L L cases, none in group A have CD7 transcripts, while abundant CD7 transcripts are recognized in both cases of group B. Two cases in group C have small amounts of CD7 transcripts, while two cases of group D have large amounts. The amount of applied R N A is assessed by nonspecific binding to 28S ribosomal R N A . Two bars in the left show the size of 28S and 18S ribosomal RNAs. (See Table 1.)

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CD7 antigen and mRNA expression was studied by Northern blotting (Fig. 3B). Authentic 1.3 kb CD7 m R N A was clearly recognized in R N A extracted from these Tp40 ÷ cases, but was absent from three Tp40- cases in group A. TdT expression was also studied, and two cases in group B were positive for 2. lkb transcripts, whereas three cases in group A were negative. We studied 26 additional ANLL cases (M1, M2, or M3, 10 cases; M4 or M5, 7 cases; M6, 1 case; and M7, 8 cases) with flow cytometry (which were not included in Table 1). Seven cases showing the 4A+/ Tp40 ÷ phenotype consisted of one M1 case (10% of M1, M2, or M3) and six M7 cases (75% of M7). Three cases showed 4A+/Tp40 - phenotype, and all were M4 or M5 cases (43% of M4 or M5), whereas no M1, M2, or M3 case showed this phenotype. Expression o f CD7 m R N A in A L L cases with immature T cell phenotype, CD7 ÷, C D 5 - and m C D 3 -

We previously studied CD7 ÷, CD2-, mCD3-, D R - ALL, and reported that a fraction of CD7 ÷, CD5 ÷, CD2- leukemia cases were pre-T cell leukemia, according to Southern blot study with Ig and TCR probes. CD7 ÷, CD5- and CD2- leukemia, however, showed no rearrangements of Ig and TCR genes [6]. In this study, six cases with Tp40 ÷, mCD3phenotypes were studied (Table 1 and Fig. 3B). Three cases (group C) with CD7 ÷, CD5 -/-+, CD2 -/-+, D R - phenotype expressed moderate amount of CD7 mRNA. CD3 expression [mRNA and cytoplasmic CD3 (cyCD3)] was also observed in two out of three. One of them (case 8) expressed 1.1 kb TCR [3 mRNA, without TCR fl rearrangement. This case, however, showed rearrangement of both TCR "¢ and 8 genes. Three cases (group D) expressed CD2 (case 9) or CD5 (cases 10 and 11) in addition to CD7. CD7 mRNA, CD3 mRNA and cyCD3 were detected in all three cases, but the amounts of CD7 and CD3 mRNA and cyCD3 in case 9 were smaller than those in cases 10 and 11.

DISCUSSION The previous reports by us and others demonstrated the antigens detected by Tp40, 4A, Leu9, 3A1 and WT-1 antibodies to be the same molecule using various techniques such as sequential immunoprecipitation (with CD7 positive cells and also with CD7 positive mouse L cell transfectant), and twodimensional electrophoresis [2, 7, 10, 11, 17, 26, 27]. The present study, combined with the reports described above, further demonstrated that the epitope recognized by Tp40 is identical to or quite similar to that by 4A, Leu9, 3A1 and WT-1. The

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present study suggested that the difference in reactivity between Tp40 and two other CD7 antibodies (4A and Leu9) is due to Fc receptors for IgG expressed on the target cells, i.e. the reactivity by 4A and Leu9 antibodies (IgG2a) to CD7 m R N A negative HL-60 cells is probably due to F w R I , because monomeric murine IgG2a was reported to bind with high affinity to F w R I , rather than to F w R I I or F w R I I I [14, 15]. In contrast, the reactivity by Tp40 (IgG1) antibody showed a good correlation to CD7 mRNA expression, suggesting the genuine specificity to CD7 molecule. In addition, the present study showed that ~,2a antibodies in the other CDs such as CD3 and CD8 tend to show non-specific binding to HL-60 cells. Thus, it is advisable to use antibodies with isotypes other than IgG2a for testing cells of myeloid/monocytoid origin. Interestingly, three out of 26 ANLL cases showed 4A+/Tp40 phenotype, and all were M4 or M5 cases, suggesting that 4A÷/Tp40 - phenotype is associated with M4 and M5 cases, which tends to express high density and/or affinity FW receptors (such as F w R I ) on the leukemic cells. This finding also supports the contribution of FW receptors to the binding of the IgG2a antibody to such target cells. However, such a reaction to Fc receptors was not always seen, when antibodies with ~,2a isotype were used. Even among IgG2a antibodies, different binding activities to HL-60 cells were shown; OKT3 and Tac showed weaker binding than 4A, Leu9, OKT8, and B1 (data not shown), although all were IgG2a antibodies. It is possible that the factors other than Fc receptors may cause non-specific binding of antibody molecule to myeloid/monocytoid cells. Thus, it is important to study the serological specificity of each monoclonal antibody before using it for leukemia phenotyping. Another important finding obtained in this study is that certain TdT ÷ ANLL cases express CD7, which is in accordance with the previous report by Sutherland et al. [11]. Using WT-1 (IgG2a) antibody to CD7, Vodinelich et al. [10] reported that 6% of ANLL cases and 13% of blastic crisis in chronic myelocytic leukemia cases are CD7 positive. Later, Seremetis et al. [12] reported that 5 of 9 TdT ÷ ANLL cases express CD7 using Leu9 (IgG2a) and/or 3A1 antibodies. Thus, it may be possible that some of the CD7 positive cases reported in their studies may not be genuine CD7 positive cases. Thus, it is likely that the CD7 positive ANLL cases are smaller than the reports so far appeared. In retrospect, both 4A+/ Tp40 ÷ ANLL cases in Table i are considered to have developed following other hematological diseases (myelodysplastic syndrome in case 4 and polycythaemia vera in case 5). One 4A+/Tp40 ÷ M1 (out of additional 26 ANLL cases) also developed several

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years after pure red cell aplasia. Furthermore, six (75%) of eight M7 cases studied showed 4A+/Tp40 + phenotype; four were patients with Down's syndrome, in which trisomy 21 may play a yet unidentified role in the development of M7 leukemia [28] ~.Therefore, A N L L with a 4A÷/Tp40 + phenotype seems to be a particular group of leukemias, associating with some hematological diseases or Down's syndrome. From early studies of T cell malignancy, Haynes et al. [1, 5] found CD7 to be a good marker for T cell A L L and proposed it as a marker of early T cell lineage development. In the previous study with Tp40 ÷, CD2-, m C D 3 - A L L , we demonstrated that a fraction of Tp40 ÷, CD5 ÷ leukemia cases are of T cell origin based on T C R 13 gene rearrangement analysis [6]. We also found that Tp40 is positive in certain leukemia cases with CD5- phenotype and germline T C R 13genes [6]. The present study clearly showed that CD7 m R N A is expressed in all Tp40 ÷, CD5 -/± A L L or LbL cases (group C) and that CD3 m R N A was expressed in two of three group C cases. These results suggested that a fraction of CD7 ÷, CD5-/±, CD2 -/± , m C D 3 - A L L or LbL are of immature T cell origin, and also that CD7 expression precedes CD3 expression during T cell differentiation. In addition, the results in group D cases suggested CD7 expression increases together with CD5 expression. Recently, Haynes' group proposed that CD7 ÷, CD4-, CD8- acute leukemia is a new category of malignant pluripotent lymphohematopoietic disease [29]. Three of nine cases in their report may belong to group C in this study, with regard to antigen phenotype. Thiel et al. reported that pre-T A L L (CD7 ÷, CD2-) differed significantly from CD2 ÷ TA L L in some clinical features and had poorer prognosis [30]. Further study is, however, still needed to determine the cell lineage and the stage of differentiation of CD7 positive acute leukemia. The present study showed the importance of measuring m R N A expression to obtain accurate information for the antigen expression; m R N A expression is negative, when antibody reactivity is non-specific as observed with 4A antibody to HL-60, while m R N A expression is positive, even when the antigen expression is restricted to cytoplasm as in the case of CD3 in T cell precursors, i.e. these cells are generally scored as CD3 negative, because the flow cytometric study conducted at present can detect only surface expression, (but not cytoplasmic expression), of the antigen. In this study, we developed the condition for cytoplasmic staining of CD3 antigen, which should be applicable for other antigens. In conclusion, flow cytometric study on surface and cytoplasmic

expression of antigen molecules, together with Northern blot study on m R N A expression, will provide detailed information for antigen expression.

Acknowledgements--This work was supported in part by Grant-in-Aid for the Comprehensive Ten-Year Strategy for Cancer Control from the Ministry of Health and Welfare; Grant-in-Aids for Cancer Research from the Ministries of Education, Science, and Culture and of Health and Welfare in Japan; and by grants from the Cancer Research Institute, Inc., New York, NY.

REFERENCES 1. Haynes B. F. (1981) Human T lymphocyte antigens as defined by monoclonal antibodies. Immunol. Rev. 57, 127. 2. Tsuge I., Ueda R., Nishida K., Namikawa R., Seto M., Maruyama T., Takamoto S., Matsuoka H., Torii S., Ota K. & Takahashi T. (1984) Five antigens on human T cells detected by mouse monoclonal antibodies. Clin. exp. Immun. 58, 444. 3. Furley A. J., Mizutani S., Weilbaecher K., Dhaliwal H. S., Ford A. M., Chan L. C., Molgaard H. V., Toyonaga B., Mak T., van den Elsen P., Gold D., Terhorst C. & Greaves M. F. (1986) Developmentally regulated rearrangement and expression of genes encoding the T cell receptor-T3 complex. Cell 46, 75. 4. van Dongen J. J. M., Quertermous T., Bartram C. R., Gold D. P., Wolvers-Tettero I. L. M., Comans-Bitter W. M., Hooijkaas H., Adriaansen H. J., De Klein A., Raghavachar A., Ganser A., Duby A. D., Seidman J. G., van den Elsen P. & Terhorst C. (1987) T cell receptor-CD3 complex during early T cell differentiation. Analysis of immature T cell acute lymphoblastic leukemias (T-ALL) at DNA, RNA, and cell membrane level. J. lmmun. 138, 1260. 5. Haynes B. F., Denning S. M., Singer K. H., Kurtzberg J. (1989) Ontogeny of T-cell precursors: a model for the initial stages of human T-cell development. Immun. Today 10, 87. 6. Suzuki H., Ueda R., Obata Y., Seto M., Hirata R., Iwamura H., Ota K. & Takahashi T. (1986) T cell receptor 15 chain gene rearrangements in leukemias with immature T cell phenotype. Jpn J. Cancer Res. 77, 1069. 7. Emi N., Ueda R. & Takahashi T. (1985) Isolation of stable mouse L cell transfectant expressing human T cell differentiation antigen Tp40. J. Immun. 134, 2817. 8. Osada S., Utsumi K. R., Ueda R., Akao Y., Tsuge I., Nishida K., Okada J., Matsuoka H. & Takahashi T. (1988) Assignment of a gene coding for a human T-cell antigen with a molecular weight of 40,000 daltons to chromosome 17. Cytogenet. Cell Genet. 47, 8. 9. Ueda R., Namikawa R., Ota K., Suchi T. & Takahashi T. (1985) In Macrophage Biology, Progress in Leukocyte Biology. Vol. 4. (Reichard S. and Kojima M. Eds), p. 791. Alan R. Liss, Inc., New York. 10. Vodinelich L., Tax W., Bai Y., Pegram S., Capel P. & Greaves M. F. (1983) A monoclonal antibody (WT1) for detecting leukemias of T-cell precursors (T-ALL), Blood 62, 1108. 11. Sutherland D. R., Rudd C. E. & Greaves M. F. (1984)

CD7 antigen and mRNA expression Isolation and characterization of a human T lymphocyte-associated glycoprotein (gp40). J. lmmun. 133, 327. 12. Seremetis S. V., Pelicci P-G., Tabilio A., Ubriaco A., Grignani F., Cuttner J., Winchester R. J., Knowles II D. M. & Dalla-Favera R. (1987) High frequency of clonal immunoglobulin or T cell receptor gene rearrangements in acute myelogenous leukemia expressing terminal deoxyribonucleotidyl-transferase. J. exp. Med. 165, 1703. 13. Sobol R. E., Mick R., Royston I., Davey F. R., Ellison R. R., Newman R., Cuttner J., Griffin J. D., Collins H., Nelson D. A. & Bloomfield C. D. (1987) Clinical importance of myeloid antigen expression in adult acute lymphoblastic leukemia. New Engl. J. Med. 316, 1111. 14. Unkeless J. C. (1989) Function and heterogeneity of human Fc receptors for immunoglobulin G. J. clin. Invest. 83, 355. 15. Fanger M. W., Shen L., Graziano R. F. & Guyre P. M. (1989) Cytotoxicity mediated by human Fc receptors for IgG. Immun. Today 10, 92. 16. Bennett J. M., Catovsky D., Daniel M-T., Flandrin G., Gaiton D. A. G., Gralnoick H. R. & Sultan C. (1976) Proposals for the classification of the acute leukaemias. Br. J. Haemat. 33, 451. 17. Morishima Y., Kobayashi M., Yang S. Y., Collins N. H., Hoffmann M. K. & Dupont B. (1982) Functionally different T lymphocyte subpopulations determined by their sensitivity to complement-dependent cell lysis with the monoclonal antibody 4A. J. Immun. 129, 1091. 18. Ueda R., Nishida K., Koide Y., Tsuge I., Seto M., Yoshida M., Miyoshi I., Ota K. & Takahashi T. (1985) Two mouse monoclonal antibodies detecting two different epitopes of an activated lymphocyte antigen on adult T-cell leukemia cells. Cancer Res. 45, 1314. 19. Janossy G., Coustan-Smith E. & Campana D. (1989) The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases. Leukemia 3, 170. 20. Aruffo A. & Seed B. (1987) Molecular cloning of two CD7 (T-cell leukemia antigen) cDNAs by a COS cell expression system. E M B O J. 6, 3313. 21. Gold D. P., Puck J. M., Pettey C. L., Cho M., Coligan J., Woody J. N. & Terhorst C. (1986) Isolation of cDNA clones encoding the 20kD nonglycosylated polypeptide chain of the human T cell receptor/T3 complex. Nature 321, 431.

877

22. van den Elsen P., Shepley B-A., Borst J., Coligan J. E., Markham A. F., Orkin S. & Terhorst C. (1984) Isolation of cDNA clones encoding the 20K T3 glycoprotein of the human T-cell receptor complex. Nature 312, 413. 23. Yoshikai Y., Anatoniou D., Clark S. P., Yanagi Y., Sangster R., van den Elsen P., Terhorst C. & Mak T. W. (1984) Sequence and expression of transcripts of the human T-cell receptor 13-chain genes. Nature 312, 521. 24. Koiwai O., Kaneda T. & Morishita R. (1987) Analysis of human terminal deoxynucleotidyltransferase cDNA expressible in mammalian ceils. Biochem. Biophys. Res. Commun. 144, 185. 25. Isobe M., Russo G., Haluska F. G. & Croce C. M. (1988) Cloning of the gene encoding the ~ subunit of the human T-cell receptor reveals its physical organization within the ~-subunit locus and its involvement in chromosome translocations in T-cell malignancy. Proc. natn. Acad. Sci. U.S.A. 85, 3933. 26. Palker T. J., Scearce R. M., Hensley L. L., Ho W. & Haynes B. F. (1986) Comparison of the CD7 (3A1) group of T cell workshop antibodies in Leukocyte Typing H, Vol. 2. (Reinherz E. L., Haynes B. F., Nadler L. M., Bernstein I. D. Eds) p. 303. Springer, New York. 27. Mann D. L., Haynes B. F., Thomas C., Cole D., Fauci A. S. & Poplack D. G. (1983) Heterogeneity of acute lymphocytic leukemia cell surface markers as detected by monoclonal antibodies. J. natn. Cancer Inst. 71, 11. 28. Hayashi Y., Eguchi M., Sugita K., Nakazawa S., Sato T., Kojima S., Bessho F., Konishi S., Inaba T., Hanada R. & Yamamoto K. (1988) Cytogenetic findings and clinical features in acute leukemia and transient myeloproliferative disorder in Down's syndrome. Blood 72, 15. 29. Kurtzberg J., Waldmann T. A., Davey M. P., Bigner S. H., Moore J. O., Hershfield M. S. & Haynes B. F. (1989) CD7 ÷, CD4-, CD8- acute leukemia: a syndrome of malignant pluripotent lymphohematopoietic cells. Blood 73, 381. 30. Thiel E., Kranz B. R., Raghavachar A., Bartram C. R., LOftier H., Messerer D., Ganser A., Ludwig W-D., Biichner T. & Hoelzer D. (1989) Prethymic phenotype and genotype of pre-T ( C D 7 + / E - R ) - cell leukemia and its clinical significance within adult acute lymphoblastic leukemia. Blood 73, 1247.