A novel human T cell antigen preferentially expressed on mature T cells and shared by both well and poorly differentiated B cell leukemias and lymphomas

A new lymphocyte differentiation antigen shared by all normal T cells and some malignant B cells was defined by a monoclonal antibody designated 12.1. This antibody reacted with all peripheral blood T cells but not with normal B cells and B cell lines. Analysis with a fluorescence activated cell sorter showed that the expression of 12.1 antigen changes during T cell maturation. Most thymocytes, blasts of acute T cell leukemia, and cells from established leukemic T cell lines bear a small amount of 12.1 antigen. In contrast the majority of peripheral blood T cells, activated T cells, and leukemic T cells of the Sezary syndrome bear a large amount of 12.1 antigen. Unexpectedly, antibody 12.1 reacted with leukemic cells from most patients with B-type chronic lymphocytic leukemia (CLL) and some patients with lymphosarcoma cell leukemia (LSCL). Among these leukemias, expression of the 12.1 antigen was not correlated with the stage of B cell maturation, with the amount of surface immunoglobulin on the cells, or with the presence or absence of monoclonal gammapathy. In a comparative serologic analysis the antigen defined by antibody 12.1 was distinct from the p67 T cell antigen (defined by monoclonal antibody 10.2) that is also known to be expressed by B-type CLL cells.     

Characteristics of a monoclonal antibody (WT-31) that recognizes a common epitope on the human T cell receptor for antigen

We describe a monoclonal antibody, WT-31, that reacted with all human T lymphocytes. Electrophoretic analysis of the material reacting with WT-31 revealed that it precipitated predominantly an 80-kD disulfide-linked heterodimer from the cell surface-labeled T leukemic cell line HPB-ALL. This heterodimer was identical to the one precipitated with a recently described monoclonal reagent, T40/25, which recognizes a clonotypic structure on HPB-ALL. The target antigen of WT-31 comodulated with T3 after incubation of T cells with excess anti-T3 antibody, indicating that the WT-31 target antigen is associated with T3. We also found that anti-T3 reagents, but not the clonotypic reagent T40/25, blocked binding of FITC-labeled WT-31 to HPB-ALL cells. This indicates that the T cell receptor epitope recognized by WT-31 is located close to the epitopes recognized by the anti-T3 reagents anti-Leu-4 and SPV-T3b but distal from the clonotypic T40/25 epitope. Functional studies showed that WT-31 reacts similar to anti-T3 antibodies. It is mitogenic for resting T cells, blocks cytolysis mediated by alloantigen-specific CTL clones, and induces antigen-nonspecific cytolysis by CTL clones against Daudi target cells. WT-31 did not inhibit the formation of conjugates, but it blocked cytolysis just before or during the Ca2++-dependent programming for lysis. We conclude that WT-31 is an antibody that recognizes a common determinant on the T cell receptor for antigen. The present results support the notion that the two chains of the T cell receptor (alpha and beta) form a functional protein ensemble with the three invariable T3 polypeptide chains (T3-gamma-, delta-, epsilon).     

Germline antibody V regions as determinants of clonal persistence and malignant growth in the B cell compartment.

Antibody V gene expression was studied in a subpopulation of murine B cells (Ly1 B) which was enriched by cell transfer and had earlier been shown to persist in the immune system over long periods of time. Among 17 hybridomas derived from Ly1 B cells of two different mice, eight were progeny of only three different B cell precursors which apparently had expanded to clones of large size, in the absence of detectable somatic mutation of their antibody V regions. Furthermore, several clonally independent cells expressed identical, unmutated V genes. These data define a novel pathway of B cell development in which cells expressing a selected set of germline antibodies are continuously propagated in the organism. A Ly1 B cell leukemia derived from a similar transfer experiment expressed a VH gene that had been isolated in three independent Ly1 B cell hybridomas, suggesting that the leukemic cells had been equally selected in this pathway.     

Growth of single B cell clones is enhanced by products of a T cell line

In this paper we show the presence of a B cell growth-promoting activity in T cell replacing factor (TRF) supernatants from a monoclonal T cell line and polyclonally activated splenic T cells. The target cell of this activity is indisputably shown to be the B cell, which indicates that T cell-derived factors can act directly on B cells. The effect of monoclonal TRF-containing supernatant from the C.C3.11.75 Dennert cell line, (DL)TRF, which demonstrates B cell growth-promoting activity, is to increase the frequency of B cell clones stimulated by mitogens as opposed to increasing B cell clone sizes. (DL)TRF B cell growth enhancement is observed when B cells are activated by fetal calf serum mitogens, lipopolysaccharide (LPS), dextran sulfate (DXS), or LPS + DXS. The growth-promoting activity of (DL)TRF appears to be that of a costimulator rather than a classical growth factor because (DL)TRF alone is not sufficient to maintain clonal growth of activated B lymphoblasts.     

Interleukin 2 acts directly on a Tac-positive cloned B cell line established from a patient with adult T cell leukemia

A Tac-positive B cell line termed K3B was established from a patient with adult T cell leukemia (ATL). This cell line had EBNA antigen and human T cell leukemic virus (HTLV) provirus besides B1 antigen and surface immunoglobulin. A cloned Tac-positive B cell line termed K3B01 was obtained from K3B by the limiting dilution method. The K3B01 cells were shown to absorb IL 2 activity in a tonsillar IL 2 preparation. By using this cloned cell line and a purified recombinant IL 2 preparation, it was shown that the proliferation of K3B01 cells was enhanced by the addition of recombinant IL 2. Moreover, this response was inhibited by anti-Tac antibody. These results demonstrate definitively that IL 2 acts directly on B cells through IL 2 receptors on them.     

Molecular cloning of interferon-gamma inducible genes from a murine pre-B cell leukemia.

In the present study, a pre-B cell leukemia L1210-C7, representing a very early stage of the B lineage, was used to characterize the molecular mechanisms exploited by IFN-gamma to modulate B cell activity. A cDNA library was prepared with poly (A)+ RNA from cells stimulated with IFN-gamma and three cDNAs clones complementary to IFN-gamma inducible mRNAs were isolated by differential screening. Of these, the 9.5 cDNA hybridized to a 2.4 kb mRNA not homologous with previously cloned IFN-gamma inducible mRNAs. Furthermore, when compared with RNAs obtained from cells of different origins (fibroblasts and T cells) the 9.5 mRNA appeared to be increased only in cells belonging to the B lineage. Taken as a whole, these results demonstrate that in leukemic pre-B cells IFN-gamma induces the expression of a gene that could be employed as specific cell activation marker.     

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.     

A human CD4- T cell leukemia subclone with contact-dependent helper function.

Helper T lymphocytes provide a contact dependent signal to resting B cells that is required for optimal differentiation into Ig-secreting cells. The surface structure(s) on T cells that mediate helper function have not been identified but are known to be induced by T cell activation. A CD4- subclone of the Jurkat leukemic T cell line (D1.1) was isolated and found to be distinct from CD4+ Jurkat clones and a variety of other T and non-T cell leukemic lines in that coculture of D1.1 with resting B cells induced B cells to specifically express surface CD23 molecules, a marker of B cell activation. Furthermore, Jurkat D1.1 induced B cell proliferation and terminal B cell differentiation into IgG-secreting cells in the presence of T cell-dependent B cell mitogens. Similar to the helper effector function of activated T cells, the effects of Jurkat D1.1 were neither Ag nor MHC restricted. Paraformaldehyde fixed Jurkat D1.1 cells remained competent to activate B cells while D1.1 supernatants and diffusible factors were inactive. The effect of Jurkat D1.1 on B cell activation was distinct from that of rIL-4 and was not inhibited by antibodies to IL-4. Together these observations suggested that surface structures on D1.1 and not secreted factors, mediated contact-dependent helper effector function.     

Characterization of murine T cell activation signals produced by B lymphoma cells

The activation requirements of alloreactive and antigen reactive murine T cells were examined by stimulating class II restricted T cell clones with monoclonal B lymphoma cells. One B lymphoma cell line (T27A) was found to stimulate IL 2 release from some alloreactive T cell clones without stimulating any significant T cell proliferation response. The same B lymphoma cells are capable of stimulating IL 2 release and proliferative responses from other T cell clones. Evidence is presented suggesting that B lymphoma cell stimulation of these T cell clones is largely IL 1 independent and that at least some T cell clones may require activation signals other than Ia, antigen, and IL 1. The addition of exogenous, purified IL 1 to the T cell activation assays was found to have a wide range of stimulatory effects on the proliferative responses of different T cell clones. The absence of comparable IL 1-induced stimulation of IL 2 secretion suggests that IL 1 primarily enhances antigen specific T cell proliferation through mechanisms other than acting as a co-stimulant for IL 2 release.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.     

Cellular immune response to human sarcomas: cytotoxic T cell clones reactive with autologous sarcomas. I. Development, phenotype, and specificity

Human T cell clones cytotoxic for autologous sarcoma cell lines have been developed from patient JM with an osteogenic sarcoma, and from patients EG and RM with malignant fibrohistiocytoma. These clones were derived from the cocultivation of peripheral blood lymphocytes (PBL) with the respective patient's autologous irradiated established tumor cell lines (AIT). After two cycles of stimulation for 5 days in bulk culture, these "educated" lymphocytes were seeded at a density of 1 X 10(6) cells/well in 24-well plates and were cultured in the presence of highly purified natural IL 2 and AIT, the latter serving as a feeder layer. Cell numbers were reduced from the initial seeding density by one log each week until reaching a density of 10(2) cells. These cells were found to be stable in viability and cytotoxic activity, after which limiting dilution was then performed. Within 4 to 6 wk, clones were isolated with unique specificities. These clones were capable of proliferating to a total density of 10(9) cells/ml and maintained their specific cytotoxicity for more than 6 mo. Testing with a panel of target cells of various histotypes, cold-target inhibition assays, and blocking of cytotoxicity with anti-HLA monoclonal antibodies showed that the T cell clones recognize a common sarcoma-associated antigen and that the lysis is HLA restricted. Phenotypically, cytotoxic clones derived from JM were Leu-1+, Leu-2+, and Leu-3-, whereas those derived from EG exhibited either Leu-24 or Leu-3+ markers, the latter phenotype lacking cytotoxicity. RM exhibited mainly Leu-3+ clones with strong cytotoxicity. All were HNK-1- and HLA class II+, with less than 1% of cells of each clone stained by anti-TAC monoclonal antibody. The clones from each patient did not lyse autologous or allogeneic PBL, mitogen-induced T lymphoblasts, normal fibroblasts, cells isolated from benign neoplasms, carcinoma cells, Daudi B lymphoid cells, or K562 cells. With the exception of EG, all clones produced immune interferon in a range from 12 to 50 U/ml. The generation of long-term specific T cell clones can be used to further dissect the cellular immune response to sarcomas. Cytotoxic T cell clones have potential application for tumor immunotherapy.     

Establishment of a leukemic cell model for studying human pre-B to B cell differentiation

Reproducible models for examining early stages of human B cell differentiation are poorly developed. We now describe the establishment and characterization of a novel human leukemic cell line that recapitulates the pre-B to B cell stage of differentiation. This cell line, designated BLIN-1, was initially established in tissue culture medium containing low m.w. B cell growth factor, and consistently shows a dependency on this cytokine for optimal growth at low density. BLIN-1 cells have a 9p chromosomal abnormality, identical to the abnormality present in the leukemic blasts from the patient's original bone marrow aspirate. The immunologic phenotype of BLIN-1 is consistent with a cell arrested at the pre-B cell stage of development. Analysis of Ig gene rearrangement and Ig expression in a series of BLIN-1 subclones show that the cells spontaneously rearrange kappa light chain genes, leading to the differentiation of surface kappa-negative pre-B cells into surface kappa-positive B cells. The BLIN-1 cell line is, to our knowledge, the first defined human model for examining this critical developmental stage in human B cell ontogeny. As such, it offers a unique resource for examining variables influencing onset of kappa L chain gene rearrangement and expression.     

Activation through CD3 molecule leads a number of human T cell clones to induce IgE synthesis in vitro by B cells from allergic and nonallergic individuals

Seventy-eight clones established from tonsillar T lymphocytes of two nonallergic children were tested under different experimental conditions for their ability to induce in vitro IgE synthesis by B cells from allergic or nonallergic donors. After 24 hr preactivation with phytohemagglutinin (PHA), 11 out of 32 CD4+ clones from the first and 17 out of 36 CD4+ clones from the second tonsil donor showed the ability to induce IgE synthesis in vitro by B cells from both allergic and nonallergic individuals, whereas none of 10 CD8+ clones nor T blasts of PHA-induced cell lines obtained from unfractionated T cell suspensions of the same tonsils had such an effect. Seven of the 11 T cell clones from the first tonsil donor active on IgE production after pre-activation with PHA also induced IgE synthesis in vitro by nonallergic and allergic B cells upon stimulation with anti-CD3 monoclonal antibody. Under the same experimental conditions, virtually all of the T cell clones able to induce IgE synthesis in vitro by target B cells showed the ability to stimulate IgG and IgM production as well. T cell clones were also established from the peripheral blood of a nonallergic donor and were tested for their ability to induce IgE synthesis in autologous B cells. After preactivation with PHA, seven out of 35 CD4+ clones induced the production of detectable amounts of both IgE and IgG in autologous B cells. The addition to the cultures of PHA-stimulated unfractionated T cells inhibited in a dose-dependent manner the IgE but not the IgG synthesis induced by an autologous helper T cell clone in autologous B cells. Taken together, these data indicate that a remarkable proportion of human T cell clones upon triggering of the CD3 molecular complex were able to provide help for the synthesis of IgE in B cells from both allergic and nonallergic individuals. The successful induction of IgE synthesis by single T cell clones was apparently related to the lack of concomitant suppressor activity to which IgE-producing cells appeared to be exquisitely sensitive.     

Measles-specific T cell clones derived from a twin with multiple sclerosis: genetic restriction studies

The association between multiple sclerosis (MS) and HLA-DR2 suggests that the disease may be associated with an aberrant immune response, likely directed against an antigen of either viral or host origin. We have used measles virus-specific T cell clones derived from a patient with MS to study genetic restriction patterns of antigen presentation by macrophage-enriched (E-) populations. Twenty-two clones proliferated in response to measles-infected Vero cells but not to mumps-infected or uninfected Veros. E- cells from both the autologous subject and her healthy, measles nonresponder identical twin were capable of presenting antigen to all clones. Studies with E- cells obtained from a panel of cell donors demonstrated clones which recognized antigen in association with D2/DR2, DR4, subgroups of DR4, and SB3. Three clones recognized antigen only in association with the autologous or twin's cells, but not with other sets of HLA-matched E-cells obtained from healthy donors or from other patients with MS. These studies indicate that the differing responses to measles virus demonstrated by these two identical twins are not explained by alterations in the interactions between antigen-presenting cells and T cells. Furthermore, at the clonal level, no preferential role is seen for HLA-DR2 as the restricting element for presentation of measles virus to these clones.     

The progenitor cell inhibition assay to measure the anti-leukemic reactivity of T cell clones against acute and chronic myeloid leukemia

Adoptive immunotherapy with donor T lymphocytes may be used as a treatment for relapsed leukemia after allogeneic hematopoietic stem cell transplantation (SCT). In vitro selected and expanded anti-leukemic T cells may be more effective in inducing a response in vivo. To identify the anti-leukemic reactivity of in vitro generated T cells, standard target cell read-out assays like the 51Cr-release assay are not always appropriate. We developed an assay in which the ability of T cells to antigen specifically inhibit the in vitro growth of leukemic progenitor cells in the presence of cytokines can be measured. This assay allows the evaluation of the cytolytic or suppressive potential of leukemia reactive T cells for prolonged periods of time. The assay is based on inhibition of [3H]thymidine incorporation by the leukemic progenitor cells induced by multiple hematopoietic growth factors. T cell clones with a known specificity were used to compare the analytic potential of the new assay with those of other cytotoxicity assays. Based on the results of the T cell clones, a modification of a limiting dilution assay was developed to identify anti-leukemic allogeneic T cells in HLA identical donor-recipient combinations selected on their ability to inhibit the in vitro growth of CML or AML progenitor cells, to be used for the generation of leukemia-reactive CTL lines for clinical use.     

Unique T cell Ia antigen expressed on a hybrid cell line producing antigen-specific augmenting T cell factor

Hybrid cell lines were established by fusion between keyhole limpet hemocyanin(KLH) binding T cells of A/J mice and an AKR T cell tumor line, BW5147. Hybrids were selected for the presence of Ia antigen and KLH-specific augmenting activity of their extracts in the secondary antibody response. The detailed phenotypic and functional analysis of 1 of these clones, FL10, is reported here. The hybrid was positive for both Thy1.1 and Thy1.2 antigens and possessed the Lyt-1+,2-,3- phenotype. Both VH and Ia determinants were detected on their cell surface. The IA locus was mapped in the I-A subregion, but the Ia specificities were serologically distinct from those of B cell Ia antigen. This was demonstrated by the fact that anti-Ia antiserum preabsorbed with B cells could react with the hybrid cells, whereas none of the monoclonal anti-Ia specific for private and public determinations of Iak could. The extract from the cell line specifically augmented the in vitro secondary antibody response against dinitrophenylated KLH, and this activity was removed by absorption with antigen and conventional anti-Ia antisera. The results indicate that the cell line, FL10, carries Ia antigen unique to the T cell, which is associated with the antigen-specific augmenting molecule.     

Regulation of cell surface receptors for different hematopoietic growth factors on myeloid leukemic cells.

There are clones of myeloid leukemic cells which are different from normal myeloid cells in that they have become independent of hematopoietic growth factor for cell viability and growth. The ability of these clones to bind three types of hematopoietic growth factors (MGI-1GM = GM-CSF, IL-3 = multi-CSF and MGI-1M = M-CSF = CSF-1) was measured using the method of quantitative absorption at 1 degree C and low pH elution of cell-bound biological activity. Results of binding to normal myeloid and lymphoid cells were similar to those obtained by radioreceptor assays. The results indicate that the number of receptors on different clones of these leukemic cells varied from 0 to 1,300 per cell. The receptors have a high binding affinity. Receptors for different growth factors can be independently expressed in different clones. There was no relationship between expression of receptors for these growth factors and the phenotype of the leukemic cells regarding their ability to be induced to differentiate. The number of receptors on the leukemic cells was lower than on normal mature macrophages. Myeloid leukemic cells induced to differentiate by normal myeloid cell differentiation factor MGI-2 (= DF), or by low doses of actinomycin D or cytosine arabinoside, showed an up-regulation of the number of MGI-1GM and IL-3 receptors. Induction of differentiation of leukemic cells by MGI-2 also induced production and secretion of the growth factor MGI-1GM, and this induced MGI-1GM saturated the up-regulated MGI-1GM receptors. It is suggested that up-regulation of these receptors during differentiation is required for the functioning of differentiated cells.     

Identification of a human endothelial cell antigen with monoclonal antibody 44G4 produced against a pre-B leukemic cell line

Staining of a variety of human tissue sections (lymph node, tonsil, spleen, thymus, kidney, lung, and liver) by the indirect immunoperoxidase method indicated that mAb 44G4, produced against a human pre-B leukemic cell line, was strongly reactive with vascular endothelium. All other cell types observed in these tissues were unreactive. Immunofluorescence staining of endothelial cells isolated from umbilical cord vein and grown in culture confirmed that mAb 44G4 recognized a surface membrane component of vascular endothelium. Granulocytes, monocytes, B and T lymphocytes, and T lymphocytes cultured in the presence of PHA for 72 h did not express the 44G4 Ag. mAb 44G4 reacted weakly with leukemic cells from 28 of 41 patients with non-T cell acute lymphocytic leukemia and 4 of 7 patients with acute myelocytic leukemia, whereas 8 of 10 cases of T cell acute lymphocytic leukemia were negative. Moderate reactivity with leukemic cell lines of pre-B and myelomonocytic origin was also observed. The level of 44G4 Ag on umbilical endothelial cells was three to five times that of leukemic cell lines and 25 times the average levels observed on leukemic cells isolated from patients. Immunoprecipitation of lysates prepared from surface-iodinated endothelial cells and the immunizing pre-B leukemic cell line revealed that the 44G4 Ag from both cell types was composed of two subunits of apparent m.w. 95,000 linked by disulfide bond(s). Comparison of the cellular localization and subunit structure of 44G4 to that of known Ag suggests that it represents a previously undescribed marker of endothelial cells.     

Cell-to-cell heterogeneity in the expression of carbohydrate-based epitopes of a mucin-type glycoprotein on the surface of human mammary carcinoma cells

A large, O-linked glycoprotein, termed PAS-O, is a major differentiation antigen on the surface of normal lactating breast epithelia and is also found on the surface of many mammary tumors and mammary carcinoma cell lines. A characteristic feature of populations of tumor cells that express PAS-O is the cell-to-cell heterogeneity with respect to the presence or absence of the molecule. In this study, we used the human mammary carcinoma line 734B and a set of six monoclonal antibodies reactive with PAS-O to study the basis of this heterogeneity. Extensive Western blot analysis of antibody binding to PAS-O in milk fat globule membranes and in skim milk revealed that the antibodies all recognized different epitopes of PAS-O. Moreover, the epitopes were destroyed by periodic acid oxidation, demonstrating their oligosaccharide basis. All six monoclonal antibodies stained the 734B cells heterogeneously. In addition, five clones derived from the parent 734B population also exhibited heterogeneity in the expression of each of the epitopes. An analysis of staining of the 734B clones revealed that, in some cases, certain cells within the cloned population stained with one monoclonal antibody but not with another antibody. Significantly, though, when the 734B cells were treated with neuraminidase prior to antibody staining, most of the heterogeneity was eliminated, and all but one of the monoclonal antibodies stained 90-100% of the cells. This increase in cell staining was matched by an increase in PAS-O staining on Western blots. We conclude that heterogeneity in PAS-O expression on 734B cells is due partly to masking of epitopes by sialic acid and a variation (on a cell-to-cell basis) in the extent of PAS-O sialylation.     

Detection of minimal disease in acute leukemia using flow cytometry: studies in a rat model for human acute leukemia

The study was made using a rat model for human acute myelocytic leukemia (BNML), which shows striking similarities with human acute myelocytic leukemia (AML). A monoclonal antibody (MCA-Rm124), raised against BNML cells, allowed the recognition of the leukemic cell fraction. The discriminative capacity of the monoclonal antibody is based on differences in labeling intensities between normal and leukemic cells. After i.v. cellular transfer of leukemia, the growth of the leukemic cell population in the bone marrow, the liver, and the spleen was monitored using MCA-Rm124 and flow cytometry. For the bone marrow and the liver, a clonogenic assay for leukemic cells was used to quantify the cell content in these organs. A good correlation was found between the bioassay-derived data and the flow-cytometry-derived data. The doubling times of the leukemic cell population were not equal for the two organs studied, indicating that a number of different processes contribute to the net cell production per organ. Apart from their application in the detection of residual leukemia, monoclonal antibodies might be employed in the analysis of the growth kinetics of the "invisible" leukemic cell population.