Human bone marrow cells positive for terminal deoxynucleotidyl transferase (TdT), HLA-DR, and a T cell marker may represent prothymocytes

Recent evidence suggests that prothymocytes, which occur in a low frequency in murine bone marrow (BM), are already committed to thymocyte differentiation and discrete from precursor B cells as well as pluripotent hematopoietic stem cells. Furthermore, it was suggested that, in rodents, prothymocytes are positive for the nuclear enzyme terminal deoxynucleotidyl transferase (TdT) and a T cell surface antigen. The human prothymocyte has not been identified as yet. We analyzed human BM cells by double immunofluorescence staining for TdT and the T cell surface markers Tp41 (recognized by the monoclonal antibodies WT1 and 3A1), T11, T1, and T6. In the BM samples tested, neither T1+/TdT+ nor T6+/TdT+ cells were detected, but Tp41+/TdT+ and T11+/TdT+ cells were present in low frequencies. In childhood BM, the frequency was about two to five in 10,000, whereas in adult BM and regenerating BM, these cells were not always detectable, but if detected, their frequency was five- to 10-fold lower. In a triple staining, using fluorescein, rhodamine, and colloidal gold particles as labels, it appeared that all Tp41+/TdT+ cells were also positive for HLA-DR. These Tp41+/HLA-DR+/TdT+ cells were also detectable in low frequencies in the thymus, and occasionally Tp41+/TdT+ and T11+/TdT+ cells were detected in the peripheral blood (PB), suggesting a migration from the BM to the thymus via the PB. The malignant counterpart of the Tp41+/HLA-DR+/TdT+ cell was detected in a patient with acute lymphoblastic leukemia with the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- phenotype and germ-line immunoglobulin heavy chain genes. We postulate that the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- cell represents a human prothymocyte.     

Antigenic phenotype of TdT-positive cells in human peripheral blood

Double immunofluorescence studies for terminal deoxynucleotidyl transferase (TdT) and leucocyte surface membrane antigens have been used to characterize the small subpopulation of TdT-positive cells in human peripheral blood. The predominant antigens demonstrated were those coded for by the major histocompatibility complex, namely HLA-A,B and Ia-like antigens. A small proportion of TdT+ cells expressed antigens restricted to B lymphocytes and their precursors (BA-1+ CALLA+). In contrast, antigens associated with T-lymphocyte differentiation were not detected using a panel of T-cell-specific monoclonal antibodies. These results preclude the possibility that circulating TdT+ cells are immature cortical thymocytes that have "leaked" into the bloodstream. Although bone marrow-derived prothymocytes, which have not yet acquired T-cell lineage markers, may be included amongst this subset, the expression of B-cell related antigens by some TdT+ cells indicates the likely existence of lineage heterogeneity amongst this population of lymphoid cells. The relevance of these findings to the monitoring of human acute lymphoblastic leukaemia is discussed.     

Phenotypic heterogeneity of Gross virus-induced thymic lymphomas in the rat: cellular origins and migratory properties

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross leukemia virus (RAGV) were analyzed for a variety of differentiation markers. The neoplasms from individual rats all expressed the antigenic phenotype MP+, W3/13+, Thy-1+, RT-1+, RT-7+, W3/25-. However, approximately two-thirds of the neoplasms were positive for the OX 8 antigen, and one-third were negative. The OX 8- neoplasms only involved the thymus, whereas approximately 40% of the OX 8+ neoplasms involved the spleen as well as the thymus. Virtually all OX 8+ and OX 8- neoplastic cells contained terminal deoxynucleotidyl transferase (TdT), and both OX 8+ and OX 8- lymphomas expressed the lactate dehydrogenase (LDH)-5' isozyme and the primary, but not the secondary, ADA isozyme. This enzymatic phenotype is characteristic of thymocyte precursors, but not thymocytes. Our results therefore indicate that RAGV-induced lymphomas arise from transformed prethymic TdT+ cells which contain the LDH-5' and the primary ADA isozymes. These preleukemic cells presumably migrate to the thymus where they express the RT-7 pan-T-cell antigen and, in some instances, the OX 8 antigen during the development of overt leukemia. The OX 8+ neoplasms, being more differentiated than their OX 8- counterparts, then migrate to peripheral lymphoid tissues.     

Evidence for the cellular origin of Gross virus-induced leukemia in the rat: description of a unique LDH isozyme sub-band in leukemic lymphoid cells and lymphohemopoietic precursor cells

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross-leukemia virus (RAGV) were analyzed for a variety of differentiation markers to define their differentiation state and possible cellular origin. A majority of thymocytes from leukemic rats had the phenotypic characteristics of subcapsular cortical thymocytes that are the most ancestral of the thymocytes. These cells exhibited readily detectable levels of Thy-1 and histocompatibility antigens on their surfaces, they contained terminal deoxynucleotidyl transferase (TdT) and they contained low adenosine deaminase (ADA) and high purine nucleoside phosphorylase (PNP) specific activity. The leukemic thymocytes also contained a sub-band of the LDH-5 isozyme (LDH-5') that was not detected in normal thymocytes but that was present in lymphocyte-rich fractions of postnatal bone marrow, fetal and prepubertal spleen, and fetal and neonatal liver. The tissue distribution and ontogeny of LDH-5'-containing cells is similar to prethymic TdT+ cells in the rat and both TdT and LDH-5' are enriched in a subset of bone marrow "null" cells. These results suggest that TdT+ thymocyte progenitors or their precursors are the targets of leukemic transformation of RAGV.     

The production of terminal deoxynucleotidyl transferase (TdT) positive cells in cultures of human pluripotent hemopoietic progenitor cells

A transient increase in terminal deoxynucleotidyl transferase positive (TdT+) cells was observed during the early phase of (less than or equal to day 5) cultures supporting the growth of pluripotent myeloid progenitor cells (CFU-mix). T-cell growth-promoting medium and erythropoietin were not required. The rapidity with which TdT+ cells appeared in cultures and the results of cultures where TdT+ cells were high initially (greater than 800 cells/culture) were not consistent with their having been produced by proliferation of pre-existing TdT+ cells from the bone marrow inoculum. The results suggest production of TdT+ cells from a TdT-negative precursor either by altered enzyme expression or by production of TdT+ progeny.     

An in situ study of B-lymphocytopoiesis in rat bone marrow. Topographical arrangement of terminal deoxynucleotidyl transferase-positive cells and pre-B cells

To understand bone marrow (BM) as a site of B-lymphocytopoiesis, insight into the topographical arrangement of developing B cells and their relationships to the microenvironment in vivo is required. To study the spatial distribution of B lymphocyte progenitors defined by intracellular markers (cytoplasmic mu H chain and nuclear terminal deoxynucleotidyl transferase (TdT], we developed a technique to cut frozen femurs of rat, yielding cross-sections with intact subendosteal and central marrow. By using (double) immunofluorescence staining techniques we located pre-B and TdT+ cells, and IgM+ B cells in those sections. Of the B cells present in BM, one-third was accumulated in the lumen of blood sinuses. The rest were in the BM parenchyma, as were virtually all pre-B and TdT+ cells. The subendosteal area was twice as rich in pre-B and TdT+ cells as the central area, and within the subendosteal area a profound positive gradient toward the bone was evident. B cells showed an equal distribution over the center and the periphery of the BM. The distribution patterns of B lineage cells in the BM parenchyma were analyzed and shown in part to deviate from random distribution. Additional study of clonal development and microenvironmental factors in hematopoiesis will have to clarify the underlying mechanisms for the observed distribution patterns of B cell precursors in BM.     

A non-natural nucleoside with combined therapeutic and diagnostic activities against leukemia

Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, presenting with approximately 5,000 new cases each year in the United States. An interesting enzyme implicated in this disease is terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase involved in V(D)J recombination. TdT is an excellent biomarker for ALL as it is overexpressed in ~90% of ALL patients, and these higher levels correlate with a poor prognosis. These collective features make TdT an attractive target to design new selective anti-cancer agents against ALL. In this report, we evaluate the anti-leukemia activities of two non-natural nucleotides designated 5-nitroindolyl-2'-deoxynucleoside triphosphate (5-NITP) and 3-ethynyl-5-nitroindolyl-2'-deoxynucleoside triphosphate (3-Eth-5-NITP). Using purified TdT, we demonstrate that both non-natural nucleotides are efficiently utilized as TdT substrates. However, 3-Eth-5-NITP is poorly elongated, and this observation validates its activity as a chain-terminator for blunt-end DNA synthesis. Cell-based experiments validate that the corresponding non-natural nucleoside produces robust cytostatic and cytotoxic effects against leukemia cells that overexpress TdT. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore via "click" chemistry. This reaction allows the extent of nucleotide incorporation to be quantified such that the anti-cancer effects of the corresponding non-natural nucleoside can be self-assessed. The applications of this novel nucleoside are discussed, focusing on its use as a "theranostic" agent that can improve the accuracy of dosing regimens and accelerate clinical decisions regarding therapeutic intervention.     

The kinetics of terminal deoxynucleotidyl transferase-positive cells in the mixed colony assay

Human bone marrow was treated with cytolytic monoclonal antibodies BA-1, BA-2 and BA-3 and examined for terminal deoxynucleotidyl transferase-positive (TdT+) cells during culture in the mixed colony assay. The kinetics of TdT+ cells was compared with untreated bone marrow controls over a 14-day culture period. In control cultures a progressive decline in the number of TdT+ cells occurred during the first three days and by day 4 no TdT+ cells were observed. In antibody-treated cell cultures in which all or the majority of TdT+ cells were removed, no TdT+ cells were detectable after 24 h. Both control and antibody-treated cultures showed comparable colony formation from myeloid, erythroid and multipotential progenitor cells by day 14. The results therefore suggest that the mixed colony assay does not support the growth of TdT+ cells or the production of TdT+ cells from TdT- precursors.     

Analysis of human terminal deoxynucleotidyl transferase cDNA expressible in mammalian cells

Human Molt3 cDNA library was constructed using pcD vector system which permits the expression of cDNA inserts in mammalian cells. Nearly full-length human terminal deoxynucleotidyltransferase (TdT) cDNA was cloned using a fragment of bovine TdT cDNA as a probe. The human TdT cDNA contains an open reading frame of 1,557 bp coding for 519 amino acids, including 31 bp and 341 bp from 5' and 3' untranslated regions, respectively. The TdT cDNA was transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 59,495. The cloned TdT cDNA hybridized with poly A+ RNAs of 2,100 b and 3,300 b from stable T-cell leukemia Molt3 and Molt4 cells.     

Terminal deoxynucleotidyl transferase in diagnosis of lymphomas

TdT activities were determined on 29 specimens of mononuclear blood, bone marrow or lymph node cells from 18 patients with non Hodgkin's Lymphomas, 2 Hodgkin's patients and 3 patients with non neoplastic lymph nodes. The neoplastic cells were typed using tests detecting membrane markers (E, Em, SIg), and monoclonal antibodies (MoAb). In a group of 15 patients with Low Grade Malignant Lymphoma (L. lymphocytic, centrocytic and lymphoplasmocytic) 14 cases belonged to B cell phenotype lymphoma, with 3 cases among them with a moderate TdT activity. In one case of lymphocytic lymphoma the cells had the non T, non B, TdT+ characteristics. High TdT activity was observed in both examined patients with lymphoblastic lymphoma and in cells obtained from the lymph node of one Hodgkin's lymphoma case. Although our group was of heterogenic character, our investigations confirm the value of TdT as biochemical marker of immature lymphocytes and its usefulness for differential diagnosis of malignant lymphomas.     

B lymphocyte-associated antigens on terminal deoxynucleotidyl transferase-positive cells and pre-B cells in bone marrow of the rat

To investigate early stages of B lymphocytopoiesis in rat bone marrow (BM) before the expression of surface IgM (s mu), the populations of cytoplasmic mu-chain-positive (c mu+) pre-B cells and terminal deoxynucleotidyl transferase-positive (TdT+) cells were studied by double immunofluorescence microscopy. B lymphocytes that were s mu+ constituted 5%, c mu+s mu- pre-B cells 23%, and TdT+ cells 4% of nucleated cells in the BM of juvenile rats. TdT+ and pre-B cells ranged between 7 and 17 microns in diameter. TdT+ cells were slightly larger, with a modal diameter of 10.5 microns against 9 microns for pre-B cells. mu-Chains were absent from nearly all TdT+ cells. Their surface antigenic phenotype was studied by using a panel of mouse monoclonal antibodies (MAb) to rat B lymphocyte-associated antigens (Ig, Ia, and others) and T lymphocyte-associated antigens. Both pre-B cells and TdT+ lacked surface Ig and Ia but carried most of the other B lymphocyte-associated antigens analyzed. TdT+ and pre-B cells lacked those antigens found only on the T lineage. By using MAb HIS24 (detecting a non-Ig/Ia B lymphocyte-associated antigen) and fluorescence-activated cell sorting, TdT+ and pre-B cells were highly enriched. The results show that most TdT+ cells in rat BM are mu- but demonstrate strong similarity with pre-B cells in surface antigenic phenotype. Therefore, as suggested for man, a major proportion of rat BM TdT+ cells may be B lineage-cells before mu heavy chain gene expression.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line kappa light chain genes and germ-line T-cell receptor beta- and gamma-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Developmental abnormalities of terminal deoxynucleotidyl transferase positive bone marrow cells and thymocytes in New Zealand mice: effects of prostaglandin E1

The enzyme TdT was used as a marker with which to study the ontogeny of primitive lymphopoietic cells in NZ strain mice. A marked accumulation of abnormally large, rapidly proliferating TdT+ cells was seen in the subcapsular region of the thymus cortex in the NZB and NZB/W mice. This abnormal accumulation of TdT+ thymocytes was most pronounced in the NZB/W hybrid and persisted for at least the first 16 wk of life. In addition, significantly elevated percentages of TdT+ bone marrow cells (presumptive prothymocytes) were present in NZB, NZW, and NZB/W mice between 1 and 4 wk of age, with the highest mean peak levels occurring in the NZB strain. Treatment of both normal and adrenalectomized BALB/c and NZB/W mice with pharmacologic doses (7 to 10 mg/kg) of PGE1 caused a marked, dose-dependent decrease in thymus weight and thymus cell number within 12 to 18 hr. Histologic and cell separation studies showed that this was due to the selective depletion of PNA+ TdT+ cortical thymocytes. Similarly, PGE1 caused a reversible, dose-dependent decrease in the percentage of TdT+ bone marrow cells. In contrast, PGF2 alpha, which is not therapeutically active against autoimmunity in NZB/W mice, had no detectable effect on TdT+ bone marrow cells or thymocytes in BALB/c or NZB/W mice. These results directly document the existence of abnormalities in the development of lymphopoietic precursor cells in the bone marrow and thymus cortex of NZ strain mice prior to the onset of autoimmune phenomena. The results also raise the possibility that the therapeutic efficacy of exogenous PGE1 in autoimmune NZ strain mice may be related, at least in part, to its ability to rectify the abnormal development of these early lymphoid cells.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

Inhibition of human bone marrow lymphoid progenitor colonies by antibodies to VLA integrins.

Studies in animal models suggest that the integrin adhesion protein VLA-4 may play an important role in lymphopoiesis. The relationship between cell adhesion and lymphopoiesis in humans has been difficult to study because of the relative rarity and stringent in vitro growth requirements of lymphoid progenitors from normal adult human bone marrow. To determine the functional significance of VLA-4-mediated adhesion in human lymphopoiesis, we developed a culture system in which a bone marrow-derived adherent layer supports the formation of colonies of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursor cells from normal adult human bone marrow. Limiting dilution studies were consistent with clonal origin of these colonies. CFU-TdT were enriched in the CD34+ bone marrow fraction, consistent with CD34 expression by other hematopoietic progenitors. CD34 expression and lack of lineage-specific markers in a significant proportion of the TdT+ colony cells suggest that the TdT+ CFU may represent an uncommitted lymphoid progenitor cell. Development of TdT+ colonies required direct contact with the adherent layer and was significantly inhibited by specific anti-VLA-4 alpha chain antibody, suggesting a functional role for the previously reported VLA-4-dependent adhesion of human B cell precursors to bone marrow-derived fibroblasts.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Detection of terminal deoxynucleotidyl transferase (TdT) by flow cytometry in leukemic disorders

We applied a new technique to the detection of intracellular TdT in 26 leukemic patients, including 16 non-T acute lymphoblastic leukemia (ALL), four T-ALL, one T-lymphoblastic lymphoma in leukemia phase, one undifferentiated leukemia, one de novo lymphoblastic phase of chronic myeloid leukemia, and three acute monocytic leukemias (AMOL). Mononuclear cell suspensions were incubated in saponin to permeabilize the cell membrane. The cells were then stained by indirect immunofluorescence (IF) using anti-human TdT monoclonal antibodies and were analyzed by flow cytometry (FCM). The TdT results were compared with those obtained by biochemical TdT assay (26 cases), immunoperoxidase determination (PAP) (12 cases), and fluorescence microscopy (seven cases). The results obtained by PAP and fluorescence microscopy were 100% concordant with those obtained by FCM and biochemical assay. TdT determination by FCM allows the analysis of large numbers of cells in a fast, objective, and reliable manner, as compared with biochemical assay, PAP, and fluorescence microscopy determinations.     

Induction of phenotypic differentiation, interleukin 2 production, and PHA responsiveness of "immature" human thymocytes by interleukin 1 and phorbol ester

Small human thymocytes (ST) representing 70% of the thymocytes were isolated according to size by centrifugal elutriation. Although these ST contained approximately 30% PNA-cells, they failed to respond to lectins, indicating the existence of a PNA-ST subset that can be considered to belong to the "immature" thymocyte population. The ST were induced to proliferate if, in addition to PHA, IL 1-containing supernatants of highly purified monocyte cultures or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were present. The incubation of the ST for 90 hr with TPA or IL 1 in the absence of PHA resulted in a strong reduction in the percentage of cells reacting with the immature thymocyte markers TdT and PNA. In addition, the OKT6+ cells were partially reduced after incubation with IL 1. Concomitantly, an increase in the percentage of cells reacting with the mature T cell markers OKT1 and OKT3 was observed, whereas HLA antigens became strongly expressed on all ST. Although IL 1 or TPA were unable to induce proliferation of the ST, these substances induced IL 2 production by these cells. These shifts to cells with more "mature" phenotypes that are able to produce IL 2 were not observed if the ST were incubated with PHA or culture medium only. The responder capacity of the ST to PHA plus TPA was not significantly affected by the depletion of the more "mature" OKT3+ and OKT1+ cells. In addition, in this situation OKT1+, OKT3+, OKT6- cells were found to be generated from OKT1-, OKT3-, OKT6+ cells. Therefore, it could be excluded that the proliferative responses were due to a selective expansion of a preexisting mature T cell population. Our results indicate that TPA mimics IL 1 in the induction of differentiation of the ST to a stage in which subpopulations of these cells are able to produce IL 2 and to respond to PHA. Because only the proliferating ST were found to react with a monoclonal antibody, which is thought to be directed at the IL 2 receptor (anti-Tac), our data suggest that PHA is required for the induction of expression of receptors for IL 2 in those ST subpopulations that are able to proliferate in the presence of IL 2 generated in situ.     

Phenotypic features and proliferative activity of B cell progenitors in X-linked agammaglobulinemia

In this study, we applied mAb and heterologous antisera in double marker combinations to investigate the phenotype and the proliferative activity of immature B lineage cells in XLA. Bone marrow (BM) samples from eight male adult patients with no circulating B lymphocytes were studied. The proportions and the phenotype of the earliest identifiable B cell progenitors, expressing nuclear terminal deoxynucleotidyl transferase (TdT), cytoplasmic CD22, and membrane CD19 and CD10 were identical to those observed in normal BM. In XLA these cells represented 1.2% to 22% of BM mononuclear cells; 5% to 42% and 1% to 45% of such cells weakly expressed CD20 and CD37, respectively, and invariably lacked CD13 and CD33. Cytoplasmic mu+ sIg- pre-B cells were seen in low numbers (0.1% to 0.3%) in four samples and were undetectable in the remaining four. Consequently, the ratio TdT+/c mu+ was greater than 100 in five out of eight samples studied in contrast to the less than 10 values seen in normal individuals. The proliferative activity of B lineage progenitor cells was studied by using Ki67 and anti-bromodeoxyuridine mAb. Although the proliferation of TdT+ cells in XLA was comparable with that seen in normal BM samples (24% to 59% of TdT+ were Ki67+ and 11% to 27% incorporated bromodeoxyuridine), this was dramatically reduced in the c mu+ cells (no c mu+, Ki67+ seen in three samples where pre-B cells were observed). Thus, the abnormalities of B cell differentiation in XLA are first seen at the c mu+ pre-B stage and suggest a maturation block in the transition between TdT+, c mu- pre-pre-B cells and c mu+ pre-B cells. The severity of this block may be variable, allowing the generation of a near normal number of pre-B cells in some patients, which nevertheless have a defective proliferative activity. Finally, our study further supports the concept that the effects of the "XLA gene" are confined within the B lineage by demonstrating that the proportions of T cells bearing TCR-alpha beta and TCR-gamma delta in XLA are similar to those seen in normal individuals.     

Defective lymphopoiesis in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mutant mice. II. Description of a microenvironmental defect for the generation of terminal deoxynucleotidyltransferase-positive bone marrow cells in vitro

We have presented evidence in a previous paper that the development of prothymocytes, pre-B cells, and TdT+ lymphoid precursor cells in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mice is defective. In the present study, we have used a selective culture system that supports the generation of rat- and mouse-origin TdT+ bone marrow lymphoid cells in vitro to further investigate the early stages of lymphopoiesis in me/me and mev/mev mice. The results demonstrate that bone marrow stromal cell feeder layers derived from me/me and mev/mev mice do not support the growth of rat TdT+ cells in vitro, whereas stromal cell feeder layers from heterozygous (+/-) littermates and wild type (+/+) control mice do. Moreover, composite feeder layers formed by mixing as few as one part me/me and mev/mev bone marrow cells with 7 to 9 parts +/- littermate bone marrow cells also fail to effectively support the generation of TdT+ cells in vitro. In contrast to me/me and mev/mev mice, other mutant mouse models of autoimmune (NZB, NZB/W), immunodeficient (nu/nu), and hemopoietic (W/Wv, Sl/Sld) disorders form feeder layers that support normal to elevated levels of TdT+ cell growth in vitro. Thus, to date, only the me/me and mev/mev mutant mice have been found to lack the appropriate microenvironment for the generation of TdT+ bone marrow cells. Histologic analysis of the stromal cell feeder layers that are formed in our culture system shows that multilayered cellular patches, which normally are the most active sites of TdT+ cell development in vitro, are absent in feeder layers of me/me and mev/mev cells. Moreover, feeder layers from mev/mev mice contain a population of MAC 1+, basophilic, nonvacuolated, macrophage-like cells; whereas feeder layers from control mice contain MAC 1+, eosinophilic, vacuolated macrophage-like cells. Stromal cell feeder layers formed by mixtures of me/me or mev/mev and control mouse bone marrow cells contain numerous multilayered cellular patches and vacuolated mononuclear cells, but also contain large numbers of basophilic mononuclear cells. These composite feeder layers have a disproportionately reduced capacity to support the generation of TdT+ cells in vitro. Although the stromal microenvironment of me/me and mev/mev bone marrow does not support the growth of TdT+ cells in vivo or in vitro, the bone marrow from these mutant mice contains detectable numbers of pre-TdT+ cells. Thus, when cultured on normal mouse feeder layers, mutant mouse bone marrow rapidly generates TdT+ cells in vitro, albeit at significantly reduced levels as compared to +/- littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)