Changes in hemopoietic and regulator levels in mice during fatal or nonfatal malarial infections. I. Erythropoietic populations

Erythroid precursors BFU-E and CFU-E and erythroblasts (ERB) were monitored in the marrow and spleen of mice during fatal or nonfatal malaria. Transient depletions of marrow CFU-E and ERB without modification of BFU-E or erythropoietin (Epo) levels were found as early events in fatal infections. Before anemia development, erythropoiesis was reduced in the bone marrow but increased in the spleen. During the anemic phase, for comparable levels of anemia, plasma Epo levels were elevated to a similar degree in fatal and nonfatal malaria. In the bone marrow, CFU-E increased twofold and BFU-E were usually reduced as expected in severe anemia. ERB populations increased but remained below or within normal values, suggesting an impairment of marrow erythropoiesis related to early events following infection. In contrast, in the spleen, ERB production was strongly simulated but amplification of ERB, CFU-E, and BFU-E populations was 2.5-fold lower in fatal than in nonfatal malaria. The results suggest that a defect in amplification of splenic erythropoiesis is a crucial determinant of the fatal outcome of malarial infection. This may have been mediated by a defective stem cell migration or multiplication. Some evidence obtained during recovery stages suggested that a factor(s) other than Epo may control splenic erythropoiesis during the anemia associated with malaria.     

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Regulation of B-lymphocyte production in the bone marrow: mediation of the effects of exogenous stimulants by adoptively transferred spleen cells

The cellular mechanism by which an injection of sheep red blood cells (SRBC) results in an increased production of B lymphocytes in mouse bone marrow has been studied by adoptive cell transfer and the use of two in vivo assays of bone marrow B-cell genesis. Proliferation of B progenitor cells was examined by immunofluorescent labeling combined with mitotic arrest, while small lymphocyte renewal was measured by [3H]thymidine labeling and radioautography. In C3H/HeJ mice the administration of SRBC resulted in increased proliferation among bone marrow pre-B cells which contained cytoplasmic mu heavy chains but lacked kappa light chains and surface mu chains. The turnover of small lymphocytes also increased. These stimulatory effects were transferred to naive recipient mice by organ fragments and by cell suspensions harvested from the spleens of donor mice injected with SRBC. In contrast, spleen cells and thymus cells from saline-injected donors and thymus cells from SRBC-injected donors had no such stimulatory effects. The results demonstrate that spleen cells mediate the stimulatory effect of SRBC on bone marrow B-lymphocyte production. Spleen cell transfer provides a system to study further the cells and factors involved in the regulation by external environmental agents of the rate of primary B-cell genesis in vivo.     

The immunoregulatory role of bone marrow. II. Characterization of a suppressor cell inhibiting the in vitro antibody response.

The addition of bone marrow cells (BMC) to spleen cell cultures suppressed the antibody response in a dose-dependent manner. This suppression required viable cells. Treatment of BMC with anti-thymocyte serum did not affect the suppressive activity and BMC, but not spleen cells, from nude mice inhibited the antibody response to the same degree as marrow from normal littermates. BMC which had been depleted of macrophages with antimacrophage serum or carbonyl iron showed increased suppressor activity. Furthermore, fractionation of BMC by velocity sedimentation and resetting revealed the suppressor cell to be a medium-to-large Fc receptor-positive lymphocyte. Absence of detectable B or T cell markers on the suppressor cell indicates this cell to be an Fc-positive null lymphocyte, possibly a precursor cell, which inhibits the response of mature lymphocytes     

The influence of T lymphocyte subsets and humoral factors on colony formation by human bone marrow and blood megakaryocyte progenitor cells in vitro

Cellular and humoral influences of T lymphocytes on human megakaryocyte colony formation in vitro were assessed by using a microagar system. Megakaryocyte colony formation from nonadherent low density T lymphocyte-depleted (NALDT-) bone marrow cells was increased significantly after the addition of aplastic anemia serum (AAS) or purified megakaryocyte colony-stimulating factor (Meg-CSF). The addition of conditioned medium obtained from phytohemagglutinin-stimulated T lymphocytes replaced, at least partially, the requirement for AAS or purified Meg-CSF for the growth of megakaryocyte colonies. The cellular influence of T lymphocytes and T lymphocyte subsets on megakaryocyte colony formation was assessed by removing either T cells from nonadherent peripheral blood mononuclear cells with monoclonal OKT4, OKT8, or OKT3 antibodies plus complement, or by adding back populations of bone marrow or blood T4+ or T8+ lymphocytes, isolated by means of fluorescence-activated cell sorting, respectively, to NALDT--bone marrow or -blood cells. When sorted T cell subpopulations were added to a fixed number of NALDT--bone marrow or -peripheral blood cells in the presence of AAS or Meg-CSF, T4+ cells enhanced megakaryocyte colony formation and T8+ cells decreased it. These studies demonstrate that although the stimulation of megakaryocytic progenitor cells by Meg-CSF may not require the presence of monocytes or T lymphocytes, T4+ lymphocytes enhance and T8+ lymphocytes down-regulate megakaryocyte colony formation induced by Meg-CSF. These observations suggest that the immune system is capable of modulating the proliferative response of human megakaryocytic progenitor cells to Meg-CSF.     

Retinoic acid induces granulocytic differentiation of myeloid progenitors in congenital agranulocytosis bone marrow cells

Congenital agranulocytosis is a rare fatal infantile disease characterised by recurrent bacterial infections, persistent absence of neutrophils and maturation arrest at the promyelocyte/myelocyte stage. The effectiveness of retinoic acid in inducing differentiation of congenital agranulocytosis marrow myeloid progenitor cells was studied. Non-adherent mononuclear marrow cells were treated in an in vitro culture with retinoic acid at various concentrations from 1nM to 1 microM for seven days. Morphological and functional differentiation into mature granulocytes was induced by retinoic acid in a dose-response stimulation with a maximum response at a concentration of 1 microM. These results suggest a potential therapeutic role for retinoic acid in the treatment of congenital agranulocytosis.     

The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. II. Temporal and dose response relationships of tolerance induction in B lymphocytes of various differentiation states.

The induction of TNP-specific B lymphocyte tolerance by TNBS in sources representing various differentiation states was studied in an adoptive cell transfer system. An adoptive assay was utilized in which the delay of immunization with the T-independent antigen TNP-LPS resulted in an enhanced PFC response. TNBS induced tolerance in spleen cells which was independent of T cell activity, was dose dependent, and could be adoptively transferred. While bone marrow and spleen cells were susceptible to tolerogenesis after cell transfer, TNBS treatment of the donor induced unresponsiveness in splenocytes but not marrow cells. The tolerance dose response relationship and the effect of the temporal relationship between cell transfer and tolerogenesis were studied in B lymphocytes from various sources. Adult spleen cells were resistant to tolerance induction late in the adoptive response, and the tolerance induced by TNBS administration 1 hr after cell transfer was dose dependent. Athymic nude spleen cells and adult bone marrow cells displayed similar characteristics while fetal liver cells were somewhat more susceptible to the induction of unresponsiveness. Neonatal spleen cells were rendered tolerant at much lower doses and at any stage of the adoptive response. The hierarchy obtained in these studies in the order of decreasing resistance to tolerance induction is: adult normal and athymic nude spleen and adult bone marrow, fetal liver, and neonatal spleen. This variation in tolerogenesis appears to be due to the maturity of the cell types which may reflect differences in B lymphocyte sub-populations.     

Antibody-dependent cellular cytotoxicity effector cells lack Ia antigens.

Surface immunoglobulin (sIg)-positive and sIg-negative subpopulations of macrophage-depleted murine splenic lymphocytes were obtained by Sephadex anti-Fab immunoabsorbent fractionation. These lymphocyte subpopulations were analyzed for the presence of Thy 1 and Ia alloantigens and also for Fc receptors by fluorescence microscopy. Concurrently, these lymphocyte subpopulations were studied for effector cell activity in antibody-dependent cellular cytotoxicity (ADCC). Effector cells mediating ADCC were contained in the sIg-negative lymphocyte subpopulation and sIg-positive lymphocytes did not mediate cytotoxicity. The majority of sIg-positive lymphocytes were found to bear Ia antigens and Fc receptors, and these cell surface structures were associated in that treatment of these cells with anti-Ia sera inhibited binding of complexed immunoglobulin to Fc receptors. In contrast, most sIg-negative, Thy 1-negative lymphocytes lacked Ia Antigens, and the Fc receptors detected on such cells were not blocked by anti-Ia sera. In addition, a small subpopulation of sIg-negative, Ia antigen-positive, Fc receptor-positive lymphocytes was found. Elimination of this subpopulation of Ia antigen-positive cells from sIg-negative lymphocytes, by treatment with anti-Ia serum and complement, did not diminish ADCC effector cell activity in the resultant cell population when compared with untreated sIg-negative lymphocytes. Thus, in murine spleen, nonphagocytic mononuclear cells that lack both sIg and Ia antigens were shown to mediate ADCC.     

An evaluation of the maternal natural killer cell population during the course of murine pregnancy

Earlier work from this laboratory revealed an increase in the level of null (Thy-1-, IgM-) lymphocytes in the maternal lymphoid organs during the first pregnancy in the mouse that was more pronounced during allogeneic pregnancy than during syngeneic pregnancy. In view of the suggestive evidence for the bone marrow origin of these null cells, the functional significance of the null cell rise was explored in this study by an examination of (1) splenic NK activity as measured by the 51Cr-release assay using YAC-1 lymphoma targets, (2) the incidence of NK lineage cells in the spleen as measured by the ability of splenic null lymphocytes to bind YAC-1 lymphoma targets, and (3) the possible presence of a NK target structure on placental trophoblast cells, studied with a cold target competition assay. Results revealed that the absolute levels of null lymphocytes, NK lineage cells, and NK activity in the spleen increased moderately and nearly at the same time during syngeneic pregnancy. During allogeneic pregnancy all three parameters increased more significantly, the rise in the levels of NK activity and NK lineage cells somewhat preceding the null cell rise, suggesting preferential recruitment of active NK cells within the null lymphocyte population of the spleen. Trophoblast cells appeared to share NK target structures with YAC-1 lymphoma cells, suggesting that a rise in the NK cell level in both pregnancy types may represent a response of the mother to such target structures. Since the density of such moieties was similar for homozygous and heterozygous trophoblasts, a higher NK cell response during allogeneic pregnancy is considered to result indirectly from an alloreactive response of the mother to the paternally encoded antigens on the fetoplacental unit, possibly from a stimulation of interferon producing cells such as macrophages. Nevertheless, such a response appears to be harmless for the allogeneic conceptus.     

Induction of allospecific suppressor T cells in vitro.

Incubation of mouse thymic lymphocytes with irradiated allogeneic spleen cells gave rise to suppressor cells. The suppressor activity was assayed by adding the incubated cell mixture to a mixed lymphocyte culture (MLC) in which the responder cells were syngeneic with the sensitized thymocytes and the stimulator cells were syngeneic with the sensitizing spleen cells. Such addition suppressed significantly thymidine incorporation in the mixed lymphocyte reaction (MLR). The suppressor cells were found to carry the θ antigen and to function allospecifically, as shown by cross-testing in three allogeneic combinations. Our data suggest that these cells may originate from immature cortisone-sensitive thymic lymphocytes and also provide some preliminary information concerning their mode of action.     

Plasmodium berghei: influence on granulopoiesis and macrophage production in BALB/c mice

Granulocyte and macrophage progenitor cells forming colonies in vitro (GM-CFC) from bone marrow, spleen, and peripheral blood of BALB/c mice infected with Plasmodium berghei were cultured at various times postinfection in a viscous, 0.8% methylcellulose system. The numbers of GM-CFCs from bone marrow increased gradually during the first week of infection, reaching a maximum around the tenth day of the disease. Subsequently, a rise of GM-CFCs in cultures of nucleated cells from the peripheral blood was observed and, with some delay, in spleen cell cultures also, with a maximum around the end of the second week. After the tenth day of malaria infection a fall of colony frequency in bone marrow-derived cells took place, leading to subnormal values of GM-CFCs during the third week of infection. Subsequently, a decrease in the spleen cell cultures followed, but colony numbers did not fall to normal values. The general increase in GM-CFCs in the different organs was preceded by a rise in serum levels of colony-stimulating activity (CSA), attaining a maximum 1 week after P. berghei inoculation. During the following period the CSA levels fell and reached normal values around the seventeenth day of the disease. Chemotherapy with chloroquine started on the fifteenth day of infection, when GM-CFCs in the bone marrow have dropped to normal values, stopped their further decrease. In the spleen a gradual normalization took more than 2 weeks. A challenge infection evoked an elevation of GM-CFC numbers in the bone marrow and in the spleen during the first 10 days in only about 50% of immune mice. The reaction was immediate in some animals, but generally lower and of shorter duration than during primary infection. The results have indicated that a lethal P. berghei infection in mice caused a transient increase in production of CSA followed by a general recruitment of GM-CFCs in all hemopoietic organs.     

Impact of placental Plasmodium falciparum malaria on pregnancy and perinatal outcome in sub-Saharan Africa: part III: placental malaria, maternal health, and public health

Plasmodium falciparum infections of the placenta remain a major medical challenge among pregnant women in sub-Saharan Africa. A number of factors influence the prevalence of placental malaria in pregnant women, including maternal age, gravidity, use of prophylaxis, nutrition, host genetics, and level of anti-parasite immunity, as well as parasite genetics and transmission rates [1]. Maternal anemia has been shown to be one of the major complications of placental malaria in sub-Saharan Africa. The mechanisms by which malaria causes anemia are fairly well understood. The pathophysiology of malaria-associated anemia is multifactorial. The most likely mechanisms include (i) hemolysis or the direct destruction of parasitized red blood cells that occurs both intravascularly and by sequestration in the microcirculation, mainly in the spleen; (ii) specific/nonspecific immune responses, whereby red cell survival is shortened; (iii) nonspecific, defective, red cell production, which depresses erythropoiesis, inhibits reticulocyte release, and prematurely destructs red cells during maturation in the bone marrow; and (iv) hypersplenism associated with a reduction in all three blood cell series, that is, causing not only anemia but also thrombocytopenia and leucopenia [2,3]. The relationship between maternal anemia with obstetric factors, however, is not fully understood, and, thus, evaluating the link between malaria, obstetric disorders, and maternal death has been recommended [4]. There have been efforts to quantify the contribution of malaria to maternal morbidity and mortality with the expectation that this would provide the evidence necessary to improve the effectiveness of advocacy to incorporate malaria prevention strategies in Safe Motherhood Programs [5,6]. The effects of placental malaria on maternal health can better be understood when considered in relation with various maternal parameters, including maternal age, parity, peripheral malaria infection, anemia, and HIV infection.     

Lymphohemopoiesis in culture is prevented by interaction with adherent bone marrow cells from mutant viable motheaten mice

Mice with the recessive "motheaten" (me) or "viable motheaten" (mev) mutations have severe immunologic disturbances and die at an early age. The function of hemopoietic progenitor cells and microenvironmental elements that regulate their growth and differentiation were studied in mev mice with two types of long term bone marrow cultures. Cells from bone marrow of homozygous defective mev/mev mice were non-productive under conditions that normally support replication of stem cells and production of neutrophil granulocytes. Similarly, in a different culture system, lymphocytes were produced from normal littermate, but not mev/mev bone marrow. Initial overgrowth of cells having macrophage-like characteristics occurred in both culture systems with marrow from defective mice. Co-cultures of normal and defective bone marrow cells were always non-productive. In contrast, supernatants of mev/mev bone marrow cultures did not have a detrimental effect on cultures of normal cells, implying that the suppression was cell-associated. Furthermore, there was no evidence for abnormal release of granulocyte or macrophage growth factors in mev bone marrow cultures. A small population of cells in mev/mev bone marrow cultures were morphologically similar to "stromal" cells that support lymphohemopoiesis. Certain culture strategies could be used to enrich for these. mev/mev stromal cells had affinity for normal lymphocytes; however, they did not support lymphocyte growth. The long term bone marrow cultures thus reveal an apparent imbalance in the regulatory mechanisms affected by these single gene mutations. This is manifested by preferential or aberrant growth of one type of adherent cell and a possible functional abnormality of stromal cells. mev mice could provide an ideal model for investigating cell-associated molecules that normally limit progenitor cell replication.     

Characterization of the normal lymphocyte population cytolytic to Burkitt's lymphoma cells of the EB2 cell line.

The normal human lymphocyte population which exhibits "spontaneous" cytolysis of EB2 Burkitt's lymphoma cells has been characterized. The effector cell has EA and EAC' receptors but lacks E receptors and probably surface Ig. "Spontaneous" anti-EB2 cytotoxicity was not reduced by preincubation of the effector cells with plastic or iron carbonyl or by passage through cotton wool or agarose columns but was reduced by passage through nylon wool columns. Thymocytes were not cytotoxic to EB2 cells, and chronic lymphocytic leukaemia cells (of B cell characteristics) had reduced cytotoxicity compared with normal lymphocytes. Cells from various lymphoid organs of rats and guinea-pigs were also cytotoxic to EB2 cells with reactivity in spleen greater than or equal to blood greater than lymph nodes. Spleen cells from neonatally thymectomized rats had increased cytotoxicity compared with normal rat spleen cells, suggesting that T lymphocytes are not essential. The effector cell in rat spleen did not adhere to cotton wool or agarose columns, indicating some resemblance to its counterpart in human peripheral blood.     

Granulocyte and macrophage proliferation after injection of antitumor active and inactive strains of Corynebacterium parvum

Summary Corparvax, a strain of Corynebacterium parvum with strong antitumor activity, had a greater and more prolonged effect of increasing the production of granulocytes and macrophages than did a weak antitumor strain, CN5888. Following the injection of Coparvax to mice, there was a prompt and sustained increase in serum granulocyte/macrophage colony-stimulating activity, an increase in the number of spleen granulocyte/macrophage progenitor cells, an increased rate of proliferation of the bone marrow granulocyte/macrophage progenitor cells and an increase in the number of blood granulocytes and monocytes. The time courses of the increased rates of proliferation of granulocyte/macrophage progenitor cells following the injection of Coparvax were different in the bone marrow and the spleen, suggesting that local microenvironmental factors were also important.If immunostimulants such as C. parvum are to be used in chemoimmunotherapy programs, the kinetics of the increased proliferative rate of the granulocyte/macrophage progenitor cells may be important, since the more rapidly proliferating cells will be more affected by cell cycle-active chemotherapeutic agents.with the technical assistance of Beverly M. Dunne and L. Atherton     

Regulation of bone marrow lymphocyte production: III. Increased production of B and non-B lymphocytes after administering systemic antigens

To examine the influence of exogenous stimuli on the genesis of lymphocytes in mouse bone marrow, the production rate and subsets of marrow lymphocytes were examined after a systemic injection of sheep red blood cells (SRBC). Radioautographic analysis after either pulse labeling or infusion of [³H]thymidine revealed a pronounced increase in the number of newly formed small lymphocytes appearing in the marrow, maximal 4–5 days after SRBC injection and dose related. The resulting expansion of the marrow lymphocyte population included both immature B cells and null cells, as shown by cell surface and cytoplasmic markers. Similar stimulation of marrow lymphocyte production followed an injection of either bovine serum albumin or mineral oil. No comparable stimulation occurred in either the thymus or the spleen. The results demonstrate that antigens and nonspecific irritants can exert a central effect in the bone marrow, producing a surge in the production of both primary B and non-B lymphocytes. The possible role of external stimulants in determining the normal rate of bone marrow lymphocyte production is discussed.     

Effects of sodium periodate modification of lymphocytes on the sensitization and lytic phases of T cell-mediated lympholysis.

Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.     

Response of multipotent (CFU) and granulocyte (diffusion chamber assay) progenitor cells and differentiating cells of murine haematopoietic tissues to a perturbation of the steady state

Regulation of haematopoiesis was investigated by studying the response of haematopoietic tissues of mice to a perturbation of the steady state by vinblastine (VLB). Progenitor cells were quantified ly limiting dilution analysis of diffusion chamber cultures of haematopoietic cells and by the spleen colony technique. The diffusion chamber technique appears to assay granulocyte progenitor cells and those multipotent progenitor cells that become committed to granulopoiesis during chamber culture. The spleen colony technique probably assays multipotent progenitor cells. Decaying oscillatory responses to VLB were observed for progenitor cells as well as for differentiating cells in bone marrow. The period lengths of the diffusion chamber progenitor cell oscillations might indicate that these were induced by humoral feedback signal(s) from nonproliferative granulocytes. The oscillations of the multipotent progenitor cells of bone marrow were less pronounced and were earlier damped than those of the granulocyte progenitor cells. This may support the hypotesis that multipotent progenitor cells are regulated by more efficient mechanisms, which may depend on short range cell-cell interactions rather than long range humoral regulators.     

Normal hematopoietic progenitor cells and malignant lymphohematopoietic cells show different susceptibility to direct cell-mediated MHC-non-restricted lysis by T cell receptor-/CD3-, T cell receptor gamma delta+/CD3+ and T cell receptor-alpha beta+/CD3+ lymphocytes

To evaluate the capability of NK cells and cytotoxic T lymphocytes to interact with normal hematopoietic progenitor cells (HPC), as compared to neoplastic lymphohematopoietic cells, we investigated inhibition of colony growth of these cell populations in semi-solid culture systems, after incubation with cloned cytotoxic effector cells. Three different types of cloned effector cells were investigated: TCR-/CD3- NK cells, TCR-gamma delta+/CD3+ cells, and TCR-alpha beta+/CD3+ cytotoxic T lymphocytes. Effector cells showed differential levels of tumor cell colony inhibition, but no MHC-non-restricted lysis of normal HPC was observed. Pre-stimulation of normal HPC by culturing on established stromal layers had no effect. Cell-mediated lysis of HPC only occurred by Ag-specific MHC-restricted lysis by CTL, or by antibody-dependent cellular cytotoxicity. In cell mixing experiments, irradiated tumor cells, but not normal bone marrow cells inhibited tumor cell lysis. Furthermore, cloned effector lymphocytes were able to specifically eliminate malignant cells from tumor contaminated bone marrow without damaging normal HPC. When fresh leukemic cells were used as targets, growth of acute myeloblastic leukemia colonies was inhibited after incubation with several cytotoxic effector clones, whereas chronic myeloid leukemia precursor cells showed limited sensitivity to MHC-non-restricted cytolysis. These results indicate that MHC-non-restricted cytolysis by NK cells is selectively directed against neoplastic cells and not against normal HPC.