The phenotype of the neoplastic cells of hairy cell leukemia studied with monoclonal antibodies

Eighteen cases of hairy cell leukemia were studied with a battery of polyclonal anti-Ig and nine monoclonal antibodies to determine the lineage of the hairy cells (HC) and the stage of their maturation arrest. Hairy cells tend to nonspecifically bind many antisera and precautions had to be taken to avoid nonspecific fluorescence of the cells. All but one case was reactive with one light chain type and one or more heavy chain isotype antisera as reported before. All cases studied were positive for monomorphic HLA-DR determinants, using monoclonal antibody 7.2. Most cases tested (6/7) were positive with the B-lineage related antibody PI 153/3. While most cases (15/18) were nonreactive with the B-lineage related antibody BA-1, they became positive (5/5) following in vitro culture. Seven out of nine cases were reactive with OKM1. Common acute lymphoblastic leukemia antigen (CALLA) was absent in all (15) cases tested and the ALL associated structure p24/BA-2 was absent from 16 of 18 cases. HC from none of the cases were clearly positive with the T-cell antibodies 9.6., or TA-1, whereas in only 1/18 the cells reacted with T101. The results of this study support the B cell lineage of most HC, and show the presence of multiple phenotypes. In combination with the surface Ib present on the cells, a hierarchy of phenotypes is postulated, with SIg-ormu delta, BA- 1+, PI 153/3+, HLA-DR+ being the most immature, and SIg(alpha) gamma, BA-1-, PI 153/3-, HLA-DR+ the most mature.     

Acid alpha-naphthyl acetate esterase in hairy cell leukemia cells and other cells of the hematopoietic system

Summary The activity of-naphthyl acetate esterase at an acid pH (ANAE) was investigated in 10 cases of hairy cell leukemia. All 10 cases, including two cases with only a few tartrate-resistant acid phosphatase-reactive cells, revealed a moderate or strong ANAE reaction. There was a characteristic pattern of activity consisting of small, medium-sized, or large distinct granules often distributed in a semicircle in the cytoplasm, but sparing the nucleus of hairy cells. This reaction pattern was not found in T cells, T cell-derived leukemias, normal B cells, a number of B-cell lymphomas, myeloid cells, myeloid leukemias (including monocyte-derived leukemias), reticulum cell sarcoma, or malignant reticulosis. The cells from some B-cell lymphomas and plasmacytoma showed a relatively homogeneous pattern of fine or moderately coarse ANAE-positive granules that was similar to that of hairy cells only in some cases of plasmacytoma. Thus, fine to coarse granular ANAE reactivity is characteristic of hairy cells and is of potential diagnostic value for hairy cell leukemia.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 111, Project CL1.     

Simultaneous demonstration of the Philadelphia chromosome in T,B, and myeloid cells

A patient presented with lymphoblastic lymphoma in lymph-nodes and chronic myeloge-nous leukemia (CML) in marrow and peripheral blood. All marrow and unstimulated peripheral blood cells contained the Philadelphia chromosome{t(9:22)}. Lymphoma cells were analyzed by flow cytometry and were identified as T cells (CD2+CD5+CD7+CD34+). All fresh lymphoma cells contained the t(9:22) translocation. Cultures of purified peripheral blood T and B cells and specifically stimulated NK cells revealed that 59% of the B cells, 10% of the NK cells, and none of the normal T cells contained the translocation. The lack of translocation in normal peripheral T cells is attributed to their long lifespan. No rearrangement of immunoglobulin or T cell receptor beta or gamma genes was found in either the leukemia or lymphoma cells. Analysis of the DNA from cryopreserved lymphoma biopsy showed clonal rearrangement within the common breakpoint cluster region of the bcr gene identical to the bcr rearrangement in DNA from leukemia blood cells. The data support the concept that T and B cells originate in the patient's totipotent stem cell from which the CML is also derived. © 1993 Wiley-Liss, Inc.     

Integrated human T-cell leukemia virus II genome in CD8 + T cells from a patient with "atypical" hairy cell leukemia: evidence for distinct T and B cell lymphoproliferative disorders

We previously reported isolation of human T-cell leukemia virus II (HTLV-II) from a second patient (N.R.A.) with atypical hairy cell leukemia. Follow-up analysis of the characteristics of the patient's HTLV-II infection over a 2-year period has revealed that the patient had two coexistant lymphoproliferative disorders. Oligoclonally integrated HTLV-II was detected in DNA extracted from the patient's peripheral blood mononuclear cells on separate occasions greater than 1 year apart, similar to integration of HTLV-I seen in adult T cell leukemia/lymphoma. Although integrated provirus was readily detected, no HTLV-II viral RNA expression was seen in fresh peripheral blood lymphoid cells. Although the patient's peripheral blood consistently contained a majority of atypical lymphoid cells with a T cell antigenic phenotype, he ultimately developed extensive pleural, hepatic and soft tissue infiltration with malignant Tac+, tartrate-resistant, acid phosphatase-positive (TRAP+) B cells of clonal origin. To further characterize the role of HTLV-II, the patient's peripheral blood mononuclear cells were fractionated into four enriched subpopulations at autopsy. Oligoclonally integrated HTLV-II was detected in DNA from a T cell-enriched fraction and a CD8+ T cell-enriched fraction, but not in a CD4+ T cell-enriched fraction, a non-T cell fraction, or in B cells obtained from the malignant pleural effusion. We conclude that the patient harbored two distinct lymphoproliferative disorders, a TRAP+, Tac+ B cell malignancy consistent with hairy cell leukemia that did not contain HTLV-II and a Tac-, CD8+ lymphoproliferative syndrome with oligoclonally integrated HTLV-II.     

Production of T lymphocyte colony-forming units from precursors in human long-term bone marrow cultures

T cell differentiation in human marrow was studied in Dexter type long- term bone marrow cultures. In these cultures, T lymphocyte colony- forming units (TL-CFU), E rosette-forming cells (E+), and T3+, T4+, and T8+ cells (assayed by indirect immunofluorescence) were found to be present for at least 7 weeks. It was investigated whether the existence of T cells in long-term culture resulted from the persistence of inoculated T lymphocytes or from the production by immature progenitors. No significant numbers of E+, T3+, T4+, or T8+ cells were detected in cultures that were established from E+ lymphocyte-depleted bone marrow, indicating little or no production of T lymphocytes from E- negative precursors. On the other hand, bone marrow cells purged of E+ lymphocytes did not contain TL-CFU, but appeared to regain high numbers of TL-CFU during Dexter culture; this suggested that an earlier step in T cell differentiation may take place in this culture system. The generation of TL-CFU in the E-negative long-term marrow cultures only occurred when an adherent stroma layer had been established in the culture flask; it did not require added mitogens or detectable interleukin 2 in the culture medium. TL-CFU in fresh marrow (TL-CFU II) are mature (E+, T3+) T cells and are capable of producing helper (T4+) and suppressor/cytotoxic (T8+) phenotype cells in colonies. The TL-CFU newly formed in E-depleted Dexter cultures (TL-CFU I) are distinct from this population, as they are E-negative and give rise to colonies of the helper type only. T3 cell depletion of the marrow inoculum prior to culture did not prevent the appearance of TL-CFU I in long-term culture; this suggests that TL-CFU I are derived from an E- and T3- precursor (pre-TL-CFU).     

Interferons regulate the in vitro differentiation of multilineage lympho-myeloid stem cells in hairy cell leukemia.

In vitro 14-day cultures of peripheral blood mononuclear cells from hairy cell leukemia patients consistently showed the presence of hematopoietic stem cells giving rise to multilineage colonies containing a high proportion of lymphoid cells associated with the myeloid and erythroid progenitors. These stem cells are not the hairy cells but appear to be pluripotent lymphomyeloid primitive stem cells persisting in this leukemia. Interferon alpha c or beta 1 did not inhibit the growth of these colonies, as they did the growth of colonies of normal hematopoietic progenitors, but markedly decreased the ratio of lymphoid to myelomonocytic cells, by increasing the formation of monocytes and other nonlymphoid cell types in these multilineage colonies. Interferon gamma did not have the same effects on differentiation.     

T cell receptor (alpha, beta, gamma) gene rearrangements and expression in normal and leukemic large granular lymphocytes/natural killer cells

The large granular lymphocyte (LGL) population, which effects a natural killer (NK) function, consists of cells whose lineage derivation has not been clearly established on the basis of phenotypic and functional properties. To clarify the relationship of LGL/NK cells to T cells we studied patterns of rearrangement and expression of the T cell receptor (Ti) genes alpha, beta, and gamma in normal human LGLs; in CD8+, CD8-, Mol+, and Mol- LGL subsets; and in 17 cases of leukemic LGL proliferations (T gamma LPD). T alpha, T beta, and T gamma genes were not expressed, nor were T beta and T gamma genes rearranged in normal LGLs or LGL subsets. The T gamma LPD were divided into two groups. One group (15/17 cases) was characterized as CD3+ and displayed Ti gene rearrangements. Seven of these cases were reactive with monoclonal antibody WT31, which suggested expression of an alpha/beta heterodimer on the cell surface. The other group (2/17 cases) was CD3- with unrearranged Ti genes. These results indicate that the normal LGL/NK population is homogeneous and distinct from the normal T cell population because it does not express, and as a result, cannot effect its immune function through the T cell receptor molecules. Conversely, T gamma LPDs represent a heterogeneous group of lymphoproliferative diseases within which the CD3-, Ti- cases most likely represent the neoplastic counterpart of normal LGL cells. The more frequent CD3+ cases may be related to recently described NK-like T cells. The observations that normal LGLs maintain germline T gamma genes and that many CD3+ T gamma LPD display an alpha/beta heterodimer suggest that a T gamma-containing receptor may not be necessary for NK or NK-like cytotoxicity.     

Cytochemically demonstrable B-glucuronidase activity in normal and neoplastic human lymphoid cells

Mononuclear cell suspensions were prepared from 40 normal peripheral blood and lymphoid tissue specimens and 42 neoplastic specimens obtained from patients with malignant lymphoma and lymphocytic leukemia. These suspensions were analyzed for la antigens, surface immunoglobulin (Slg), sheep erythrocyte (E) rosette formation and, in some instances, acid alpha-naphthyl acetate esterase (ANAE) activity. The results of these studies were correlated with the expression of cytochemically demonstrable BG activity. The percentage of BG+ lymphocytes was found to be comparable, within 10%, to the percentage of E+ (T) cells in the majority of normal, non-neoplastic peripheral blood, tonsil, spleen, and lymph node specimens examined. Occasionally, the percentage of E+ cells exceeded the percentage of BG+ cells by 20% or more, suggesting the presence of an E+BG- T cell subpopulation. BG+ B lymphocytes were only demonstrated in 1 of 40 non-neoplastic lymphoid specimens. The neoplastic B cells in each of 14 B cell (la+Slg+E-) lymphomas were BG-. However, a variable proportion of the neoplastic cells isolated from 6 cases of B cell chronic lymphocytic leukemia and neoplastic plasma cells isolated from 7 cases of multiple myeloma expressed BG activity. Thus, it appears that both normal and neoplastic BG- and BG+ B lymphocyte populations exist; the latter may be related to a state of activation or a stage of B cell differentiation. The neoplastic cells isolated from 4 T cell (la-Slg-E+) malignancies were BG+ while those isolated from 3 T cell malignancies were BG-. The variable expression of BG activity by T cell malignancies may be related to T cell differentiation. Investigation of BG expression by T cell derived malignancies may prove useful in sorting out T cell phenotypes.     

Antigenic phenotype of splenic hairy cells

Hairy cell leukemia, a distinct clinical and morphologic lymphoproliferative disorder, is characterized by the proliferation of mononuclear cells of uncertain derivation. Attempts to identify the cell of origin have used studies either of functional capabilities or of membrane/cytoplasmic antigens. Only a few cases have been studied via monoclonal antibodies. Frozen sections of splenic tissue involved with hairy cell leukemia were studied with a variety of monoclonal antibodies having specificity for differentiation antigens using the avidin-biotinylated peroxidase complex technique. Conventional direct and indirect immunohistochemical study was used for immunoglobulin heavy and light chains. In all but one case, the neoplastic cells expressed monoclonal immunoglobulin. Although T cells were identified in persisting periarteriolar sheaths and occasionally admixed with red blood cells in pseudosinuses, phenotypic expression of intrathymic or peripheral T cell antigens by the proliferating neoplastic cells was not observed. Conversely, expression of B1 and HLA-Dr antigens by splenic hairy cells was documented in all 10 cases. Hairy cell leukemia cells did not express either monocyte antigens (M1 and MO2) or the antigens expressed by early (J5) and intermediate (B2) B cells or plasmacytoid lymphocytes and plasma cells (T10). These immunohistochemical results with monoclonal antibodies provide further evidence that hairy cell leukemia is characterized by a combination of antigens peculiar to mature B lymphocytes.     

Lipopolysaccharide responsiveness of malignant lymphoid cells in a patient with hairy cell leukemia.

The malignant cells in a patient with hairy cell leukemia responded most evidently to lipopolysaccharide (LPS) in in vitro culture for 3 1/2 days when the conventional tritiated thymidine uptake method was used. Since the malignant cells from patients with several other forms of leukemia and the peripheral blood mononuclear cells from healthy individuals did not show a comparable degree of responsiveness to LPS, we could exclude the possibility that this response was due to effects on contaminating normal mononuclear cells or to the nonspecific conditioning effect through LPS-affected contaminating normal monocytes. Morphological changes were observed with photo- and electronmicroscopy. It is likely that the hairy cells from the patient did respond to LPS, and whether or not this phenomenon may be confined to this type of lymphoid leukemia is not being investigated.     

Participation of gamma/delta-T cells in the protection against mycobacterial infection]

To search for a potential role of T cell receptor (TcR) gamma/delta T cells in host-defense against mycobacterial infection, we analyzed the kinetics, repertoire, specificity and function of gamma/delta T cells in the peritoneal cavity, lymph node (LN) and spleen during an intraperitoneal infection with a sublethal dose (5 x 10(5)) of viable BCG in mice. The number of bacteria in the organs increased to a maximal level by 28 days after infection, and thereafter decreased gradually. Of the CD3+ cells in PEC on day 7 after infection, approximately 25% were CD4-CD8-, most of which express TcR gamma/delta on their surface. On the other hand, the PEC on day 28 contained an increased number of alpha/beta T cells which were CD4+CD8- or CD4-CD8+ and the proportion of gamma/delta T cells was reciprocally decreased. The kinetics of gamma/delta and alpha/beta T cells in the LN ad spleen during BCG infection are much the same as that seen in the PEC. The early appearing gamma/delta T cells preferentially used V gamma 1/V delta 6 although the repertoire of these T cells are diversified. The gamma/delta T cells in PEC on day 7 remarkably proliferated and produced gamma IFN and IL-2 in response to sonicated BCG or PPD derived from Mycobacterium tuberculosis but not to 65 kd heat shock protein (HSP) derived from M. bovis. These results suggest that gamma/delta T cells precede alpha/beta T cells in appearance during mycobacterial infection and the early appearing gamma/delta T cells may participate in the protection at the early stage against the mycobacterial infection.     

Hairy cell leukemia: B-lymphocyte and phagocytic properties.

The diagnosis of hairy cell leukemia was made in three patients by phase-contrast microscopy and histochemistry of the abnormal peripheral blood cells. Both IgM and IgD surface immunoglobulins were resynthesized after these cells were trypsinized and cultured. Aggregate or Fc receptors were demonstrated on hairy cells. The ability to phagocytose latex was also a property of hairy cells; however, these cells did not demonstrate nonspecific esterase activity. Stimulation by phytohemagglutinin resulted in very low incorporation of tritiated thymidine. Cytofluorographic analysis of the phytohemagglutinin-stimulated cell population revealed less than 9% of the cells in an interploid or tetraploid state. The abnormal mitogen response was largely restored when purified T lymphocytes obtained from the peripheral blood of the patients were cultured with phytohemagglutinin. Hairy cells cultured with normal allogeneic mononuclear cells did not undergo blast transformation. These data strongly suggest that the cells of at least some patients with hairy cell leukemia are B lymphocytes with phagocytic capabilities.     

T cells expressing the gamma delta T cell receptor induce apoptosis in cardiac myocytes

OBJECTIVE: Enterovirus infections are major etiological factors in myocarditis and dilated cardiomyopathy. Using an experimental murine model of this disease, previous studies have shown that myocarditis susceptibility depends upon activation of T lymphocytes expressing the gamma delta T cell receptor (TcR), and that only mouse strains which accumulate gamma delta T cells in the myocardium show apoptosis of myocytes or evidence of dilated cardiomyopathy-like disease. The objective of the present studies is to demonstrate that gamma delta T cells directly induce greater Fas-dependent apoptosis of cultured myocytes than T cells expressing the alpha beta TcR. METHODS: Bl.Tg.E alpha mice were infected for 7 days with coxsackievirus B3 (CVB3). Hearts were removed and were either formalin-fixed, sectioned and stained with hematoxylin and eosin for inflammation, and using TdT-TUNEL for apoptosis, or were minced and collagenase digested for isolation of gamma delta+ and alpha beta+ T cells using immunomagnetic bead separation. Neonatal cultures of cardiac myocytes were isolated from mice less than 2 days old by collagenase and pancreatin digestion, and were either untreated or infected with virus. Levels of Fas (CD95) were measured using FITC-conjugated hamster anti-mouse Fas monoclonal antibody and flow cytometry. Susceptibility of myocytes to Fas-dependent killing was measured by 51Cr-release by labeled myocytes incubated for 4 h on either 3T3-mock or 3T3-FasL transfected cell monolayers. Killing by T cells was also measured in a 4 h 51Cr-release assay. Fas-dependent and perforin-dependent cytotoxicity was determined by specific blocking using either Fas-Fc or concanamycin A. RESULTS: Virally infected myocyte cultures showed significantly enhanced Fas expression compared to uninfected cells, with maximal upregulation of Fas occurring 18-24 h after virus infection. Both infected and uninfected myocytes were selectively killed by FasL-transfected 3T3 cells but not by mock control cells. Approximately 38% of CD3+ lymphocytes isolated from the heart express the gamma delta TcR with the remainder expressing the alpha beta TcR. Both gamma delta+ and alpha beta+ T cells lysed myocyte targets. Blocking studies indicate that gamma delta+ T cells induced predominantly Fas-mediated killing, while alpha beta+ cell produced more perforin-mediated death, although these effectors were capable of Fas-dependent killing as well. CONCLUSIONS: These studies demonstrate that T cells expressing the gamma delta TcR are more effective mediators of myocyte apoptosis than alpha beta+ T cells in vitro and suggests that these effectors may be primarily responsible for myocardial injury associated with dilated cardiomyopathy-like signs during coxsackievirus B3-induced myocarditis.     

Constant activation of the RAF-MEK-ERK pathway as a diagnostic and therapeutic target in hairy cell leukemia

The BRAF-V600E mutation defines genetically hairy cell leukemia among B-cell leukemias and lymphomas. In solid tumors, BRAF-V600E is known to aberrantly activate the oncogenic MEK-ERK pathway, and targeted BRAF and/or MEK inhibitors have shown remarkable efficacy in clinical trials in melanoma patients. However, the MEK-ERK pathway status in hairy cell leukemia has not been thoroughly investigated. We assessed phospho-ERK expression in 37 patients with hairy cell leukemia and 44 patients with neoplasms mimicking hairy cell leukemia (40 splenic marginal zone lymphoma, 2 hairy cell leukemia-variant and 2 splenic lymphoma/leukemia unclassifiable) using immunohistochemistry on routine biopsies and/or Western blotting on purified leukemic cells, and correlated the phospho-ERK status with the BRAF-V600E mutation status. Besides confirming the constant presence of BRAF-V600E in all patients with hairy cell leukemia, we observed ubiquitous phospho-ERK expression in this malignancy. Conversely, all 44 cases with neoplasms mimicking hairy cell leukemia were devoid of BRAF-V600E and none expressed phospho-ERK. Furthermore, the two exceptionally rare cases of non-hairy cell leukemia unclassifiable chronic B-cell neoplasms previously reported to be BRAF-V600E⁺ on allele-specific polymerase chain reaction lacked phospho-ERK expression as well, suggesting the presence of the mutation in only a small part of the leukemic clone in these cases. In conclusion, our findings support the use of phospho-ERK immunohistochemistry in the differential diagnosis between hairy cell leukemia and its mimics, and establish the MEK-ERK pathway as a rational therapeutic target in this malignancy.     

Synthesis of a peroxidase activity by the cells of hairy cell leukemia: a study by ultrastructural cytochemistry

The nature of cells present in the blood, marrow, and spleen of patients with hairy cell leukemia is largely debated. These cells have been tentatively categorized on the basis of either monocytic or lymphocytic markers, and the accumulating data points to the fact that they share some characteristics of both cell types. Although hairy cells are known to lack myeloperoxidase-positive granules, present in normal human monocytes, we investigated the possible presence of other peroxidase activities differing from the granule-bound myeloperoxidase. The study was carried out with several methods based on the incubation of fixed and unfixed cells in the presence of diaminobenzidine and hydrogen peroxide. A peroxidase activity was found in hairy cells, located always in the endoplasmic reticulum but not in the Golgi apparatus or in any granule. By its cytochemical characteristics it appears to be closely related to that of tissue macrophages, activated blood monocytes, and other nonlymphocytic hematopoietic cells. This peroxidase is not found in lymphocytes with B or T phenotypes.     

Prostaglandin E counteracts the gamma interferon induction of major histocompatibility complex class-II antigens on U937 cells and induction of responsiveness of U937 colony-forming cells to suppression by lactoferrin, transferrin, acidic isoferritins, and prostaglandin E

The established human monoblast or early monocyte cell line, U937, was evaluated for modulating influences of prostaglandin E2 (PGE2) on human gamma interferon (HuIFN gamma) induction of MHC class-II (Ia) antigens on U937 cells and the HuIFN gamma induction of responsiveness of U937 colony-forming cells (CFC) to inhibition by lactoferrin (LF), transferrin (TF), acidic isoferritins (AIF), and prostaglandin E (PGE). U937 CFC were induced to a state of responsiveness to the suppressive influences of PGE by HuIFN gamma. When MHC class-II antigens were induced on U937 cells and the cells sorted on the fluorescence activated cell sorter (FACS) IV into positive and negative cells, colony formation by the MHC class-II antigen+ population of cells was suppressed by LF, TF, AIF, and PGE2. Colony formation by the sorted population of MHC class-II antigen- cells was not influenced significantly by LF, TF, AIF, or PGE2. When PGE was present in the suspension culture for 72 h with U937 cells exposed to HuIFN gamma plus indomethacin, it blocked the induction of MHC class-II antigens as well as the associated inhibition of U937 CFC by LF, TF, AIF, and PGE2.     

In vitro expansion and analysis of cloned cytotoxic T cells derived from patients with chronic T gamma lymphoproliferative disorders

Patients with T gamma lymphocytosis are a heterogeneous group, clearly distinguishable from other patients with chronic T lymphoproliferative disorders and usually without proven malignancy. We have attempted in vitro cloning of lymphocytes from three patients with an expansion of phenotypically and functionally different types of T gamma cells. One had T3+ B73.1+ T4 T8+ OKM1+ T gamma cells exerting antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell cytotoxicity; another had T3+B73.1-T4-T8+OKM1-ADCC+NK- and a third had T3-B73.1+T4-T8- OKM1+ADCC+NK+ cells. On morphological characterization, most of the mononuclear cells of these patients resembled large granular lymphocytes (LGLs). Although lymphocytes of these patients showed almost no proliferative response capacity after stimulation with mitogens, they shared the capacity to proliferate after stimulation with Epstein-Barr virus-transformed lymphoblastoid B (B-LCL) feeder cells. Stable clones were established by this procedure. Clones from patient 3 exerted cytolytic activity against a broad spectrum of tumor cell lines, including fresh biopsy specimens of melanoma tumor target cells. All of these clones (termed activated killer [AK] cells) had the surface phenotype T3-, T4-, T8- or +, HNK1-, OKM1-, Lyt3+, WT1+ and showed ADCC in addition to AK cell cytotoxicity. Most of them were B73.1+ and expressed IgG-Fc receptors. They most likely belong to the T cell lineage, since they express IL2 receptors as recognized by the Tac antibody and did not bind monoclonal antibodies directed against monocytes or granulocytes. Thus lymphocytes with the functional and phenotypical characteristics of T gamma cells can be cloned and expanded in vitro from the peripheral lymphocytes of these patients by using the appropriate stimulus. Our results indicate that, of the heterogeneous population of NK cells, the T3- cells are more rapidly expanded than T3+ subsets. It is discussed whether or not our culture system might selectively induce proliferation in "normal" T cells rather than aberrant ones.     

Lack of T cell antigen expression on hairy cells of B cell origin after in vitro exposure to PHA

The malignant monoclonal population in hairy cell leukemia (HCL) has been variously ascribed to be of myeloid, B, or even T cell origin. Recent data have been interpreted as suggesting that hairy cells (HC) may concomitantly or serially express both B and T surface determinants, a phenomenon which, if verified, would be unique among the lymphoproliferative malignancies. Data described here, however, demonstrate that (1) at least the majority of HCL are phenotypically of B cell derivation, and (2) the initial B cell phenotype is retained and solely expressed on cultured as well as phytohemagglutinin (PHA) activated monoclonal malignant HC.     

The generation of human natural killer cells from CD34+/DR- primitive progenitors in long-term bone marrow culture

We have adapted the stroma-dependent long-term bone marrow culture (LTBMC) system to study the development of human natural killer cells (NK) from the CD34+/HLA-DR- (CD34+/DR-) BM mononuclear cell (BMMNC) population. The CD34+/DR- population does not express any known antigens associated with myeloid or lymphoid lineage and has been shown by us and others to contain primitive hematopoietic progenitors capable of both self-renewal and differentiation to myeloid lineage. CD34+/DR- cells obtained from normal human BM by fluorescence-activated cell sorting were plated on allogeneic, irradiated BM stromal layers. After 5 weeks of culture in the presence of media containing recombinant interleukin-2 and human serum, 147- +/- 21-fold expansion of cells with the morphologic appearance of large granular lymphocytes was observed. Cultured cells (84.8% +/- 1.5%) expressed the characteristic CD56+/CD3- phenotype of NK. A proportion of CD56+/CD3- cells expressed other markers of lymphoid lineage that have been associated with mature NK, including CD2 (7.8% +/- 1.2%), CD7 (19.5% +/- 2.8), CD8 (3.1% +/- 1.0%), and CD16 (4.5% +/- 1.3%). The cultured cells did not express other antigens associated with T-lymphocyte (CD3, CD5, T-cell receptor [TCR] alpha/beta and TCR gamma/delta), B-lymphocyte (CD19), myeloid (MY8, CD33, and CD71), or monocytoid (CD14 and CD15) lineage and did not express the CD34 antigen associated with hematopoietic progenitors present on the starting population. This NK population was cytotoxic against both K562 (E:T 20:1; 79% +/- 1.9%) and Raji (E:T 20:1; 38% +/- 5.7%) target cell lines. The NK progenitor frequency in the CD34+/DR- cell population determined by limiting dilution of CD34+DR- on stromal layers followed by a functional chromium release assay against K562 targets was 1:169 +/- 50 CD34+/DR- cells. The data suggest that human LTBMC developed to study myeloid differentiation can be modified to study the origin and development of the NK and possibly other lymphoid lineages. Modified cultures show that cells with morphologic, phenotypic, and functional characteristics of NK can be derived from a population of BMMNC with the phenotype of primitive hematopoietic progenitors and without phenotypic evidence of lymphoid- or myeloid- lineage commitment. Further studies will address the cell of origin and the ontogeny of human NK and other lymphoid lineages.