JUNO, the receptor of sperm IZUMO1, is expressed by the human oocyte and is essential for human fertilisation.

STUDY QUESTION: Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process? SUMMARY ANSWER: JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion. WHAT IS KNOWN ALREADY: Fusion of gamete membranes is the culminating event of the fertilisation process, but its molecular mechanisms are poorly understood. Until now, three molecules have been shown to be essential: CD9 tetraspanin in the oocyte, Izumo1 protein on the sperm and Juno, its corresponding receptor on the oocyte. Oocyte CD9 and sperm IZUMO1 have been identified in human gametes and their interaction is also well-conserved among several mammalian species. The presence of JUNO on human oocytes, however, has not yet been reported, nor has its role in fertilisation been investigated. STUDY DESIGN, SIZE, DURATION: We selected an anti-human JUNO antibody in order to investigate the presence of JUNO on the oocyte membrane surface and studied its potential involvement in gamete membrane interaction during fertilisation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Monoclonal antibodies against human JUNO (anti-hJUNO mAb) were produced by immunisation of mice with HEK cells transfected with the putative human JUNO sequence (HEK-hJUNO). These antibodies were used for immunostaining experiments and in vitro fertilisation assays with human gametes (GERMETHEQUE Biobank). MAIN RESULTS AND THE ROLE OF CHANCE: Three hybridoma supernatants, verified by immunostaining, revealed specifically HEK-hJUNO cells. The three purified monoclonal antibodies, FJ2E4 (IgG1), FJ8E8 (IgG1) and FJ4F5 (IgG2a), recognised the soluble recombinant human JUNO protein and, in a western blot of HEK-hJUNO extracts, a protein with an expected MW of 25 kDa. In addition, soluble recombinant human IZUMO protein inhibited the binding of anti-hJUNO mAbs to cells expressing hJUNO. Using these anti-hJUNO mAbs in immunostaining, we identified the presence of JUNO protein at the plasma membrane of human oocytes. Furthermore, we revealed a progressive expression of JUNO according to oocyte maturity. Finally, we showed that human zona-free oocytes, inseminated in the presence of anti-hJUNO mAb, were not fertilised by human sperm. These results suggest that, as seen in the mouse, JUNO is indeed involved in human gamete membrane fusion during fertilisation. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In accordance with French bioethics laws, functional tests were performed using zona-free oocytes, which of course does not fully encompass all normal in vivo physiological conditions. However, these in vitro tests do provide direct information regarding sperm-oocyte membrane interactions. WIDER IMPLICATIONS OF THE FINDINGS: Mechanisms of gamete fusion appear to be homologous between mice and humans. However, some differences do exist and analysing the human mechanisms is essential. In fact, this is the first report describing the presence of JUNO on human oocytes and its involvement in human fertilisation. This discovery allows further examination of the understanding of molecular mechanisms that drive gamete fusion: a crucial challenge at a time when infertility affects 16% of reproductively active couples. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Agence Nationale pour la Recherche, Grant no. ANR-13-BVS5-0004, and by Association Institut du Cancer et d'Immunogénétique (ICIG). There are no competing interests.

Severely reduced female fertility in CD9-deficient mice.

CD9 is a widely expressed cell surface molecule that belongs to the tetraspanin superfamily of proteins. The tetraspanins CD9, KAI-1/CD82, and CD63 are involved in metastasis suppression, an effect that may be related to their association with beta1 integrins. Knockout mice lacking CD9 were created to evaluate the physiological importance of CD9. CD9-/- females displayed a severe reduction of fertility. Oocytes were ovulated but were not successfully fertilized because sperm did not fuse with the oocytes from CD9-/- females. Thus, CD9 appears to be essential for sperm-egg fusion, a process involving the CD9-associated integrin alpha6beta1.

Outcome of treatment after first relapse in adults with acute lymphoblastic leukemia initially treated by the LALA-94 trial.

Fifty-four percent of adults with acute lymphoblastic leukemia (ALL) who entered the LALA-94 trial experienced a first relapse. We examined the outcome of these 421 adult patients. One hundred and eighty-seven patients (44%) achieved a second complete remission (CR). The median disease-free survival (DFS) was 5.2 months with a 5-year DFS at 12%. Factors predicting a better outcome after relapse were any transplant performed in second CR (P<0.0001), a first CR duration >1 year (P=0.04) and platelet level >100 x 10(9)/l at relapse (P=0.04). Risk groups defined at diagnosis and treatment received in first CR did not influence the outcome after relapse. The best results were obtained in a subset of patients who were eligible for allogeneic stem cell transplantation (SCT). Geno-identical allogeneic SCT was performed in 55 patients, and 3 patients received donor lymphocyte infusions. Forty-four transplantations were performed from an unrelated donor (of which four were cord blood). We conclude that most adult patients with recurring ALL could not be rescued using current available therapies, although allogeneic SCT remains the best therapeutic option.

Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma.

There is much debate about the way in which epithelial tumors metastasize. It has been proposed that the bone marrow (BM) acts as a tumor cell reservoir. We injected human hepatocellular carcinoma (HCC) cells (Mahlavu cell line) into the livers, circulation or BM of NOD/SCID mice and circulating tumor cells were quantified. When injected under the Glisson capsule, a primary tumor developed and continuously yielded circulating tumor cells. Liver tumor removal led to a very low level of Mahlavu cells both in blood and BM 30 days later. When Mahlavu cells (cultured or from BM of primary mice femurs) were intravenously injected into mice, the number of cells in the bloodstream (BS) steadily decreased, whereas the BM was not significantly colonized. When Mahlavu cells were directly injected into one femur, the controlateral femur was not colonized. Microscopic analysis and a sensitive PCR assay (<1 Mahlavu cell/nuclear cells) both failed to detect human tumor cells in other organs regardless of injection route. In conclusion, our model strongly supports the hypothesis that HCCs continuously release cells into the BS. However, in sharp contrast with the current hypothesis, the BM is not specifically colonized by tumor cells but could store them at a very low level.

Autologous stem cell transplantation in adults with acute lymphoblastic leukemia in first complete remission: analysis of the LALA-85, -87 and -94 trials.

To evaluate the results of autologous stem cell transplantation (ASCT) in a large population of adults with acute lymphoblastic leukemia (ALL) in first complete remission (CR), we performed an individual data-based overview of the last three trials from the LALA group. Overall, 349 patients with ALL prospectively randomized in the consecutive LALA-85, -87, and -94 trials to receive either ASCT or chemotherapy as post-CR treatment were analyzed. Eligibility criteria were 15-50-year-old patients without sibling donors in both LALA-85/87 trials and 15-55-year-old patients with high-risk ALL and no sibling donors in the LALA-94 trial. Intent-to-treat analysis, which compared 175 patients from the ASCT arm to 174 patients from the chemotherapy arm, showed that ASCT was associated with a lower cumulative incidence of relapse (66 vs 78% at 10 years; P=0.05), without significant gain in disease-free or overall survival. Despite a possible lack of statistical power, a nested case-control analysis performed in 85 patient pairs adjusted for time to transplant and prognostic covariates confirmed these intent-to-treat results in patients actually transplanted. Of interest, the reduced relapse risk after ASCT translated in better disease-free survival in the 300 rapid responders who reached CR after the first induction course.

Allogeneic stem cell transplantation improves the outcome of adults with t(1;19)/E2A-PBX1 and t(4;11)/MLL-AF4 positive B-cell acute lymphoblastic leukemia: results of the prospective multicenter LALA-94 study.

Adult patients with acute lymphoblastic leukemia (ALL) and t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4 have a poor outcome. We have evaluated the impact of an intensified post-remission therapy using a high-dose chemotherapy course followed by allogeneic or autologous SCT on the outcome of 58 patients with t(1;19)/E2A-PBX1 (E2A group, n=24) or t(4;11)/MLL-AF4 (MLL group, n=34) treated in the LALA-94 multicenter prospective study. Patients in the MLL group had higher WBC counts and more frequent DIC. CR rates achieved by MLL and E2A groups were similar to other B-cell ALL (87, 82 and 86% respectively). While in CR, patients with a donor were assigned to alloSCT (n=22), the remaining patients with were randomized between autoSCT (n=15) or chemotherapy (n=8). Five-year overall survival was 31 and 45% for E2A and MLL groups, respectively. In both groups, DFS was higher in the alloSCT arm as compared to autoSCT and chemotherapy arms. The results of this study show that chemotherapy intensification did not overcome the poor prognosis of adults with t(1;19)/E2A-PBX1. Allogeneic SCT should thus be offered in first CR to patients with t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4. New therapeutic approaches are needed for patients without donor.

No expansion of the pre-B and B-cell compartments in the bone marrow of patients with multiple myeloma.

We have recently demonstrated that the CD24 antigen density of bone marrow (BM) lymphoid cells discriminates between pre-B cells and mature B-cells. Using this new method, we evaluated the B-cell lineage in the BM and peripheral blood (PB) of 18 patients with multiple myeloma (MM). First, the percentage of pre-B cells was significantly reduced by 40% in the BM of patients with MM: 2.3% +/- 2.2% versus 5.7% +/- 2.8% of normal BM lymphoid cells (P less than 0.01). This finding was associated with a significant reduction (50%) of the percentage of mature B-cells in both BM and PB, especially in patients with progressive disease (P less than 0.05). In contrast to what has been reported previously, we have not found any pre-B cells in the PB of these patients with MM. Secondly, BM pre-B and B-cell patients with MM did not express any activation markers (CD23, CD25, or CD71 antigens) and no CD5+ B-cells were found in the BM unlike in PB (8% CD5+ B-cells). Taken together, these data do not support the concept of a direct involvement (i.e, expansion or activation) of pre-B cells in MM without excluding the possibility of an early oncogenic event at the pre-B cell stage. Furthermore, our data emphasize this important reduction of the B-cell compartment (including that of pre-B cell) as a major cause of the humoral immunodeficiency in MM.

Transcriptional regulation of the human CD9 gene: characterization of the 5'-flanking region.

The CD9 antigen, initially discovered on B lineage leukemic cells, belongs to the tetraspan superfamily of surface molecules. If no precise function has been assigned to any of these molecules, there are some indications that they could be involved in cell adhesion and cell migration, as well as malignant progression. The CD9 antigen is associated with surface proteins such as VLA integrins or HB-EGF precursor. Transfection of CD9 in melanoma cells reduces tumor growth and metastasis. The heterogenous distribution of the CD9 antigen suggests a complex regulation of its expression. We have previously characterized the CD9 gene and shown that transcription could be initiated at several sites in the TATA-less 5'-flanking region. We show here, using as a model two human leukemic cell lines with erythromegakaryocytic potential, HEL and K562, that the [-205, -154] region supports a promoter activity when cloned ahead of a CAT reporter gene. Mutagenesis analysis suggested the presence of a positive element located within the [-170, -154] region. Gel shift experiments using HEL extracts were compatible with the binding of the transcriptional factor Sp1 to the [-237, -205] region and indicated that a non-identified protein binds to the 3' end of the [-205, -154] region.

A new cytokine (IK) down-regulating HLA class II: monoclonal antibodies, cloning and chromosome localization.

The role of HLA class II Antigens in the control of the immune response is determined not only by the genetic polymorphism of these molecules, but also by their density on the cell surface. It is therefore essential to identify the signals that modulate HLA Class II gene activity in normal and neoplastic cells. We have purified a cytokine (IK factor, 19 kDa) secreted by the leukemic cell line K562 and several cancer cells, which inhibits HLA Class II antigen induction by IFN-gamma. We produced specific mAbs which antagonize the biological effect of IK in colon carcinoma Colo 205 cells induced to express HLA-DR molecules by IFN-gamma. Moreover, in Colo 205, HLA-DR can also be induced by the protein synthesis inhibitor Cycloheximide (0.1 micrograms ml-1); and addition of IK factor almost completely abolishes HLA class II expression. We have also performed the cloning and the sequencing of a specific cDNA. This probe recognizes a 2.1 Kb mRNA in different cell types. The nucleotide sequence exhibits no homologies with known cytokines. IK gene localization shows that it maps on chromosome 2p15-p14. The transient transfection of the cDNA in COS cells induces the secretion of a biologically active 19 kDa protein which is recognized in Western blot by 1C5B11 blocking mAb. This paper reports the characterization of a new cytokine down-regulating HLA class II Antigens, whose analysis will help to better understand HLA class II gene regulation and the mechanism of escape from immunorecognition in cancer cells.

Sequence and expression of seven new tetraspans.

The tetraspans are components of large molecular complexes that include also non-tetraspan molecules, in particular integrins. We have identified and sequenced several new members of the tetraspan superfamily, called NET-1 to NET-7 (new EST tetraspan). Sequence analysis of the NET reveals a structure typical for tetraspans, with the presence of four transmembrane domains delimiting two extracellular regions as well as conserved amino acid residues. The NET are differentially expressed in human cell lines.

Adult acute lymphoblastic leukemia: a multicentric randomized trial testing bone marrow transplantation as postremission therapy. The French Group on Therapy for Adult Acute Lymphoblastic Leukemia.

PURPOSE: In a prospective multicenter study, we analyzed the benefits of allogeneic bone marrow transplantation (BMT) in a nonselected group of adult patients with acute lymphoblastic leukemia (ALL) and, by a randomized trial, evaluated the effectiveness of autologous BMT over chemotherapy as postremission therapy in patients younger than 50 years who were not candidates for allogeneic BMT. PATIENTS AND METHODS: After induction therapy that randomized patients to receive one of two anthracycline-containing regimens, either daunorubicin (DNR) or zorubicin (ZRB), patients were assigned to postremission treatment according to age and results of HLA typing. Patients younger than 40 years with an HLA-identical sibling (group 1) were scheduled to receive cyclophosphamide 60 mg/kg on days 1 and 2, total-body irradiation (TBI), and allogeneic BMT. Patients older than 50 years (group 2) received the chemotherapy arm composed of three monthly consolidation courses (DNR or ZRB, cytarabine, and asparaginase) followed by maintenance chemotherapy (modified L10 regimen). The remaining population (group 3) was randomly assigned to receive, after the three 1-month consolidation courses, either the chemotherapy arm or autologous BMT following a conditioning regimen similar to that of group 1. RESULTS: Of the 572 assessable patients, 436 achieved complete remission (78% +/- 2% for DNR v 74% +/- 3% for ZRB; P = .3). The estimated 3-year disease-free survival (DFS) rate for the 116 patients included in group 1 was 43% +/- 5%. Both autologous BMT (95 patients) and chemotherapy (96 patients) produced comparable 3-year DFS rates (39% +/- 5% v 32% +/- 5%) and survival durations (49% +/- 5% v 42% +/- 5%). However, late relapses after 36 months were mainly observed in the chemotherapy arm. CONCLUSION: This first interim analysis did not demonstrate a benefit of this autologous BMT procedure over classical maintenance chemotherapy in patients with ALL who received consolidation chemotherapy.

Upregulation of CD9 expression during TPA treatment of K562 cells.

The CD9 antigen, a major platelet glycoprotein, is a member of the tetraspan superfamily. We show that treatment of K562 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) which induces megakaryocytic differentiation, leads to a seven-fold increase in CD9 expression, which becomes associated with the integrin beta1, suggesting that it is functionally relevant. The upregulation of CD9 expression precedes the appearance of the megakaryocytic-specific marker GPIIb (CD41) as well as integrins beta3 (GPIIIa/CD61), alpha v (CD51) and VLA-2 (CD49b). Both GPIIb/IIIa expression and CD9 upregulation are dependent on protein kinase C (PKC) activation since they are blocked by the specific inhibitor GF109203X. Steady-state levels of CD9 and GPIIb mRNA were also measured by quantitative RT-PCR. Both messengers were detected on resting cells and were shown to accumulate during TPA treatment. However, the increase of the CD9 mRNA was detected much earlier than the increase of GPIIb mRNA (1-2 h vs 24-48 h). Using different constructs of the 5'-flanking domain of the CD9 gene cloned ahead of the CAT reporter gene, we could demonstrate that a responsive element was located in a 52 bp fragment of the promoter of the CD9 gene. Altogether, these data suggest that CD9 upregulation in the megakaryocytic lineage could occur at early stages of differentiation.

Mouse monoclonal antibodies to the human C3b receptor.

Mouse monoclonal antibodies were raised against the human C3b receptor (CR1) molecule that had been purified from solubilized erythrocytes membranes. Four hybridomas were selected, cloned and expanded because their supernatants reacted strongly with insolubilized CR1 by ELISA and intensely stained B-dependent areas of the spleen and glomerular podocytes by indirect immunofluorescence. The four monoclonal antibodies, named J3D3, J8B10, J3B11 and J7C2, were IgG1 immunoglobulins. J3D3 immunoprecipitated two protein bands of apparent mol. wts 200,000 and 220,000 from 125I-surface-labeled human erythrocytes, which correspond to the two major allotypic forms of CR1. By indirect immunofluorescence, monoclonal antibodies stained polymorphonuclear leucocytes (PMN), most peripheral blood B-cells and a small subset of peripheral blood T-cells. J3D3 bound to CR1 on erythrocytes, PMN and lymphocytes with an affinity of 1-3 X 10(9) M-1 and recognized 170-1330 antigenic CR1 sites with an average of 740 sites/erythrocyte in 100 healthy individuals, approx. 50,000 sites/PMN and 15,000 sites/lymphocyte. There was a bimodal distribution of CR1 numbers on erythrocyte in the normal population. The four monoclonal antibodies similarly inhibited CR1-mediated decay of preformed cell-bound alternative- and classical-pathway C3 convertase sites. Two antibodies, J3D3 and J3B11, inhibited C3b-dependent rosette formation with lymphocytes, although much less efficiently than F(ab')2 polyclonal anti-CR1 antibody. Differences that were observed in the relative capacity of the antibodies to inhibit some of the functions of CR1 and in their ability to compete for binding of 125I-J3D3 to CR1 on erythrocytes, suggested that they are directed against different epitopes on CR1. Monoclonal antibodies provide useful means to assess and analyze the biological and immunoregulatory functions of the C3b receptor.

Possible involvement of two proteins (phosphoprotein and CD9 (p24)) in regulation of platelet calcium fluxes.

The monoclonal antibody ALB6 directed against the leukocyte differentiation antigen CD9 (p24) increases the calcium incorporation into isolated platelet membrane vesicles enriched in internal membranes. The similarities of the effects of both the monoclonal antibody and the catalytic subunit of the cAMP-dependent protein kinase (C, subunit), which phosphorylates a protein of an apparent molecular mass of 23 kDa, led us to investigate the relationship between CD9 (p24) and the 23-kDa phosphoprotein (p23). ALB6IgG does not inhibit the C.subunit-induced phosphorylation of p23 and the immunoadsorption by ALB6IgG of p24 associated to membrane vesicles does not alter the phosphorylation pattern. Thus, proteins of similar molecular mass appear to be involved in calcium fluxes: one is recognized by the ALB6 antibody while the other can be phosphorylated by the C-subunit.

Characteristics of platelet aggregation induced by the monoclonal antibody ALB6 (acute lymphoblastic leukemia antigen p 24). Inhibition of aggregation by ALB6Fab.

The leukemia-associated cell surface antigen p 24 is found on normal platelets as well as on Bernard Soulier syndrome and thrombasthenia type I platelets. ALB6 IgG (a monoclonal antibody against p 24) induces the aggregation of platelets from normal donors but not from thrombasthenia. In contrast, ALB6 Fab inhibits platelet aggregation induced by collagen, ADP, thrombin, ionophore A 23187 and ALB6 IgG. The results suggest that ALB6 interferes with a mechanism common to all aggregation pathways; the possible mechanisms are discussed.

Retinal S-antigen epitopes in vertebrate and invertebrate photoreceptors.

Monoclonal antibodies specific for the retinal S-antigen were obtained by hybridization of spleen cells from a BALB/c mouse immunized with bovine S-antigen and NS-1 myeloma cells. Five selected antibodies specifically labeled the photoreceptor cells of the retina by immunofluorescence. Whereas antibody S9E2 only reacted with bovine S-antigen, the other antibodies showed interspecies cross-reactivity. They were used for the characterization of specific epitopes of S-antigen in photoreceptors from a wide range of species representative of various classes of vertebrates and invertebrates. The presence of S-antigen in distant species (vertebrates, Amphioxus, nemerteans, annelids, molluscs) indicates a high phylogenetic stability and suggests an important role for this protein in photoreceptor function.

CD9 gene deficiency does not affect smooth muscle cell migration and neointima formation after vascular injury in mice.

The hypothesis that CD9, a member of the tetraspanin family, plays a role in smooth muscle cell (SMC) migration was tested with the use of a vascular injury model in wild-type (CD9+/+) and CD9-deficient (CD9-/-) mice. Neointima formation 3 weeks after electric injury of the femoral artery was not significantly different in CD9+/+ and CD9-/- mice (area of 0.019 +/- 0.0034 mm2 versus 0.013 +/- 0.0036 mm2; mean +/- SEM, n = 6). The medial areas were also comparable, resulting in intima/media ratio's of 1.3 +/- 0.15 and 0.90 +/- 0.22, respectively. Nuclear cell counts in cross-sectional areas of the injured region were comparable in media (33 +/- 5 versus 27 +/- 2) and neointima (135 +/- 16 versus 97 +/- 17) of CD9+/+ and CD9-/- arteries. Immunocytochemical analysis revealed expression of CD9 in the endothelium, by SMC in the media and by some fibroblasts in the adventitia of non-injured femoral arteries. Three weeks after injury, there appeared to be a gradient of increased CD9 expression from the adventitia to the neointima, in which SMC are abundantly present. Immunogold labeling and electron microscopy with non-injured femoral arteries of CD9+/+ mice confirmed the presence of CD9 at the surface of adventitial fibroblasts and in SMC or pericytes, as well as in the endothelium. Thus, in this model CD9 is highly expressed by migrating SMC, but deficiency of CD9 does not affect SMC migration or neointima formation after perivascular injury.

Effects of monoclonal antibodies raised against the common acute lymphoblastic leukemia antigen on endopeptidase-24.11 activity.

The common acute lymphoblastic leukemia antigen (CALLA, CD10) has been identified as neutral endopeptidase-24.11 (NEP), a mammalian ectoenzyme involved in the inactivation of regulatory peptides, such as the enkephalins and atrial natriuretic peptide. Twenty monoclonal antibodies directed against the human antigen, were tested for their ability to inhibit the enzymatic activity of the human and rat peptidases expressed by cell lines. Six anti-CALLA antibodies were found to inhibit 50% or more of the hydrolysis of D-Ala2-leucine enkephalin by the neutral endopeptidase present on the human leukemic cell line Reh6 and, to a lesser extent, the hydrolysis of atrial natriuretic peptide. This may indicate that their binding may affect regions of the active site more important for the dipeptidylcarboxypeptidase activity of the enzyme. Only four antibodies cross-reacted with the peptidase from the rat epithelial cell line Rat2, as shown by membrane immunofluorescence, and these also partially inhibited enzyme activity. No antibody was able to inhibit completely the activity of the human and rat enzymes and all the active antibodies appeared to behave as non-competitive inhibitors of substrate cleavage. These monoclonal antibodies could be used in mapping studies of NEP.

A new set of monoclonal antibodies against acute lymphoblastic leukemia.

Six monoclonal antibodies produced by immunization of Balb/c mice with common acute lymphoblastic leukemia (cALL) cells were tested against various types of normal and malignant tissues. ALB1 and ALB2 are directed to the cALL antigen (CALLA gp100); ALB6 recognizes a determinant of p24; ALB7, ALB8 and ALB9 have a pattern of reactivity similar to Ba1. None of these antibodies specifically identify cALL but they should be useful tools for diagnosis or depletion of bone marrow in autologous therapy in transplantation. In addition, the example of ALB6 which acts as a platelet aggregating agent, suggests that the study of other cell systems expressing the antigens associated with cALL may shed light on the function of these antigens and subsequently on the physiopathology of the leukemic cells.

Detection of binding sites for spiroperidol on leukemic cells: its value for the phenotype characterization of lymphoid leukemias.

The specific binding of the dopamine antagonist spiroperidol was studied in leukemic cell samples of various phenotypes. Among these only B-cell samples from chronic lymphocytic leukemias (7/7) and some "null" cell samples from acute lymphoblastic leukemias (2/7) showed specific binding. B cells from a prolymphocytic leukemia were negative as were also T-lymphoïd and non-lymphoïd leukemic cells at different stages of maturation. This pattern can be clearly correlated with the previous results obtained with normal blood cells and on cell lines. Moreover, it suggests that the detection of spiroperidol binding sites could provide a new means of distinguishing different phenotypes among B cells and early lymphoïd cells. Our results open the way to further studies which might show a correlation between spiroperidol binding sites and the new immunological markers defining subsets among non-T lymphoïd cells, as well as defining their physiological meaning.