Lymphokine-Activated Killer Cells in Mouse Bone Marrow Chimaeras The Relationship to Natural Killer Cells and to Alloreactive Cytotoxic T Cells

Spleen cells from mouse bone marrow chimaeras were cultured in vitro in mixed lymphocyte cultures (MLC) or in the presence of interleukin 2 (IL-2) without the added alloantigen. Precursors for the nonspecific cytotoxic cells (in this study: lymphokine-activated killer (LAK) cells) lysing natural killer (NK) cell-sensitive YAC-1 lymphoma could be found 10-12 days after the bone marrow reconstitution, simultaneously with the appearance of the NK activity. The ability of LAK cells to lyse NK-resistant tumour targets as well was demonstrated using the P 815 mastocytoma cell line; reactivity against this target was demonstrable 1 week later than the appearance on YAC-1 lysing cells. Phenotypically LAK cells derived from spleen cell cultures of bone marrow chimaeras did not differ from LAK cells derived from normal spleen cell cultures: precursors resided within the Thy 1-, asialo-GM1+ cell population, and effectors expressed both of these antigens. Splenic NK cells of early bone marrow chimaeras (up to 14-18 days after the bone marrow reconstitution) were Thy 1+ cells, and thus LAK cells of bone marrow chimaeras were not derived from these Thy 1+ NK cells. The treatment of effector cells with anti-Thy 1 antibody plus complement (C) abolished the lytic activity totally. However, these cells were not cytotoxic T cells, since alloreactivity, as an indication of the T-cell cytotoxicity, could not be demonstrated until 4-5 weeks after the bone marrow reconstitution.     

Highly Increased Natural Killer Cell Number and Lytic Activity in the Murine Peripheral Blood and Lungs after Interferon Induction in Vivo

We have studied the effect of interferon in vivo (induced by polyinosinic-polycytidylic acid (pIC] on the natural killer (NK) cell lytic activity and on the numbers of large granular lymphocytes (LGL) and target-binding cells (TBC) in different lymphoid compartments. One day after the pIC induction the spleen contained two- to four-fold increased lytic activity without a significant change in percentages of TBC and LGL. In the peripheral blood the lytic activity was 12- to 40-fold higher, and a concomitant clear increase in the number of LGL and TBC was seen. In the lungs the increase in lytic activity was 8- to 16-fold and in the number of LGLs ca. 3-fold. These results demonstrate that not only the lytic activity of the pre-existing NK cells but also the total amount of NK cells is clearly elevated in the body, but this could be seen more significantly in other highly NK-active organs than the spleen.     

Effect of Biostim (RU 41.740) on natural killer cell generation from bone marrow precursors

We have evaluated the possible effect of RU 41.740 (Biostim), a mixture of two glycoproteins extracted from K. pneumoniae, on the in vitro interleukin-2 (IL-2)-induced generation of NK cells from bone marrow (BM) precursors and on the in vivo reconstitution of splenic NK activity in lethally irradiated (9 Gy) and BM reconstituted mice. Our results show that RU 41.740 is able to augment the generation of NK cells when added (1-0.01 micrograms/ml) to normal or 5-fluorouracil-resistant BM, cultured in the presence of recombinant IL-2. Also, in vivo treatment of lethally irradiated mice, transplanted with syngeneic BM cells, with RU 41.740 (1-0.1 mg/kg i.v.) from day 0 through day 4 after BM graft, resulted in a significant augmentation of NK activity reconstitution.     

Susceptibility of natural killer cell activity of old rats to stress.

We determined an in vivo response of NK cells in young and old rats towards the suppressive effect of stress. Stress was developed by isolating rats in separate cages, but control littermates were kept together. Animals were subjected to stress for 7 days, and alterations of NK cell activities were examined in the spleen, peripheral blood (PB) and bone marrow (BM). The results showed that old rats subjected to stress had a remarkable decrease in splenic and PB-NK activity compared to old control rats, concomitant with a highly increased level of NK cell activity in BM. Suppression of the lytic activity in the spleen of stressed old rats was correlated with a decrease in the percentage of conjugate formation between splenic NK cells and target tumour cells. In contrast, stressed young rats demonstrated relatively unchanged activity of NK cells examined in different tissues compared to age-matched controls. We concluded that old animals are more sensitive to the suppressive effect of stress compared to young ones, and the mechanism of this suppression is probably due to the migration of large granular lymphocytes (LGL) from spleen and PB to other sites such as BM.     

Effect of pharmacological purging on natural killer cell number and activity in human bone marrow.

We assayed natural killer (NK) cell activity and phenotype from human bone marrow (BM) following "purge" with 4-hydroperoxycyclophosphamide (4HC) at 60 micrograms/ml for 30 min in vitro. In all cases studied, lytic activity against the K562 cell line was either significantly decreased or abolished following 4HC purge. Although NK activity was significantly affected by 4HC treatment, no major differences in the phenotype between the purged and unpurged population were seen. Further, while in vitro culture of BM with IL2 resulted in a significant increment of NK activity, no IL-2 responsive cells were found in the 4HC purged BM after 14 days of culture. This study demonstrates that pharmacological purging of bone marrow results in a persistent functional decline of NK cell activity and may serve as a useful model for the study of the ontogeny of NK cells.     

Large granular lymphocyte leukemia. A heterogeneous lymphocytic leukemia in F344 rats.

The morphology, histochemistry, cell surface antigens, and natural killer cell (NK) activity of 10 primary and 10 transplantable large granular lymphocyte (LGL) leukemias of aging F344 rats were studied. The LGL leukemia is the major cause of death of aging F344 rats. Morphologically, the LGL leukemias were composed of cells with either pleomorphic nuclei with many intracytoplasmic granules or round nuclei with few intracytoplasmic granules. The granules appeared to be lysosomes containing beta-glucuronidase and acid phosphatase and ultrastructurally developed in association with vesicles in the Golgi apparatus. Splenic natural killer cell activity against YAC-1 cells varied from case to case, and it appeared to be associated with LGL leukemia cells. Some transplantable leukemias had stable NK activity. Fluorescence-activated cell sorter (FACS) analysis of surface antigens revealed the LGL leukemias to be heterogeneous, and there was no correlation between cytotoxic activity and cell surface antigens. Although the morphologic features of cells in LGL leukemias resemble those of normal rat LGLs, differences in cytotoxic activity and surface antigens suggest that LGL tumors represent a heterogeneous group of leukemias which may serve as a model for the study of origin and lineage of normal LGL and NK cells.     

Cell Proliferation during the Maturation of Natural Killer Cells

The requirement for cell division during the maturation of natural killer (NK) cells was studied by following the appearance of donor-type NK cells in irradiated mice injecied with bone marrow cells and by blocking the cell division at different times during this development. Irradiation (700 rad) or treatment with hydroxyurea (1 mg/g body weight, twice daily) of the recipieni mice 7 days after the bone marrow cell inoculation inhibited the appearance of normal NK cell levels, suggesting that the NK cell progenitors are dividing cells. Blocking of the cell division in chimeras that had already developed high NK levels decreased the splenic NK activity, indicating the presence of a dividing NK cell population at this stage of maturation. These results are in accordance with the concept that mature NK cells are nondividing cells but are derived from actively proliferating progenitors in the bone marrow, and some of the first NK cells appearing in the spleen from the bone marrow can still be dividing.     

Constitutive activation of Lck and Fyn tyrosine kinases in large granular lymphocytes infected with the gamma-herpesvirus agents of malignant catarrhal fever

Large granular lymphocytes (LGL) with a T or natural killer (NK) lymphoblast morphology and indiscriminate (non-major histocompatibility complex-linked) cytotoxicity for a variety of target cells can be derived in culture from the tissues of animals infected with either alcelaphine herpesvirus-1 (AlHV-1) or ovine herpesvirus-2 (OvHV-2). In this study, LGL survival in the absence of exogenous interleukin-2 was inhibited by the protein kinase inhibitor genestein, but not the p70 s6 kinase inhibitor rapamycin. Constitutive activation of the src kinases Lck and Fyn was demonstrated in a bovine LGL line infected with OvHV-2 and in two rabbit LGL lines infected with AlHV-1. The p44 erk1 and p42 erk2 mitogen-activated protein kinases (MAPK) were also constitutively activated in the LGLs but not control T cells. Lck and Fyn kinase activity in the LGLs did not increase after mitogen (concanavalin A or concanavalin A plus phorbol ester) stimulation of the cells, in contrast to control T cells. Control T cells, but not the LGLs, proliferated after mitogen stimulation. An analysis of tyrosine phosphorylated proteins in the cells indicated that the LGLs exhibited some similarities and differences to activated control T cells. The results demonstrate that the activated phenotype of the LGLs, associated with malignant catarrhal fever virus infection and in the absence of exogenous interleukin-2, involves constitutively activated Lck and Fyn kinases. These are normally crucial for the initial activation of T cells via several cell-surface receptors (e.g. the T-cell receptor and CD2). The inability of the LGLs to proliferate in response to mitogen may be due to an inability of Lck and Fyn to become further activated after mitogen stimulation.     

Effect of a synthetic thymic factor (facteur thymique serique) on natural killer cell activity in humans

The effect of the serum thymic factor, FTS, on human NK cells was studied. NK cell activity was measured in a 51Chromium-release assay in which effector cells were peripheral blood lymphocytes (PBL) or bone marrow (BM) lymphocytes from healthy individuals and patients, and targets were K562 cells. When added in vitro in this assay, FTS modulated NK cell activity of normal PBL. Low concentrations of FTS (10(-2) ng/ml) increased NK activity (P less than 0.001) whereas higher concentrations decreased it (P less than 0.01) for 10 and 10(2) ng/ml. FTS exhibited no effect on NK cell activity of BM lymphocytes. When administered in vivo to 4 cancer patients (10 micrograms/kg i.v. every 3 days), FTS progressively increased peripheral NK activity in two patients with low pre-treatment NK values, whereas it decreased NK activity in two patients with previously normal or high NK values. The mechanism by which FTS modulates NK cell activity is still unknown but such modulation suggests that NK cells belong in part to the T-lineage.     

Development of Natural Killer Cells: Comparison of the Maturation Rates in Different Lymphoid Compartments

By means of semisyngeneic bone marrow transplantation, the appearance of donor-type natural killer (NK) cells in different organs (spleen, blood, lungs) of the recipients was studied. Seven days after the transplantation the first donor-type NK cells appeared in all these organs, and adult NK levels were reached simultaneously within a couple of days. The first NK cells to appear in every organ divided rapidly (sensitive to hydroxyurea) and contained a high proportion of Thy-1+ cells. These data suggest that NK cell precursors mature locally and simultaneously in different organs.     

Population dynamics of natural killer cells in the spleen and bone marrow of normal and leukemic mice during in vivo exposure to interleukin-2.

By quantitative and functional methods, changes were assessed in NK(ASGM-1+) cell numbers and NK cell-mediated lytic function of the spleen and bone marrow of mice bearing a tumor of hemopoietic origin (FLV-induced erythroleukemia) for 9 days +/- simultaneous administration of indomethacin (10 micrograms/ml drinking water) +/- rIL-2 (3x/day, 12 x 10(3) Units/injection) during the last 4 days of tumor-bearing. Recombinant IL-2 alone during the last 4 days of tumor-bearing increased both the NK(ASGM-1+) cell numbers (p less than 0.001) and the functional activity (24-fold) of the spleen. In the bone marrow, however, no change in the numbers of NK(ASGM-1+) cells was observed relative to untreated tumor-bearing mice, but the NK cell-mediated lytic activity of that organ was augmented 30-fold. The continuous presence of indomethacin from the onset of tumor-bearing prior to rIL-2 treatment during the last 4 days of tumor-bearing, further boosted both the already high, rIL-2 driven numbers of NK(ASGM-1+) cells in the spleen (p less than 0.01), as well as splenic NK cell lytic function (2-fold). In the bone marrow, continuous presence of indomethacin prior to and during the terminal 4 days of co-administration with rIL-2 increased 3-fold the numbers of NK(ASGM-1+) cells relative to that of the bone marrow of tumor-bearing mice given rIL-2 alone, and resulted in lytic activity of that organ which was 140% of that of the rIL-2 treated, tumor-bearing mice. The results indicate that under the combined influence of indomethacin and rIL-2, the production of NK(ASGM-1+) cells was augmented in the bone marrow of tumor-bearing mice, export of immature NK(ASGM-1+) cells from the bone marrow was increased, and import of immature NK(ASGM-1+) cells by the spleen was increased. The increased NK(ASGM-1+) cell numbers in each organ was reflected in increased lytic function.     

Inhibition of NK Cell Generation by Corynebacterium Parvum

Treatment of mice with Corynebacterium parvum (Cp) resulted in a substantial decrease of splenic NK activity associated with a reduced number of LGL. Cp also inhibited in vitro augmentation of NK cytotoxicity by IFN or IL-2 as well as generation of LAK activity. Localization experiments by using radiolabelled LGL indicated that the lower number of LGL in the spleen was not attributable to a Cp-induced alteration of LGL homing. Finally Cp was found to affect the ability of bone marrow cells to reconstitute NK activity in lethally irradiated mice, indicating that it can interfere with development of NK cells from bone marrow progenitors.     

Suppression of human immunoglobulin synthesis by interleukin-4 in tandem with interleukin-2 through large granular lymphocytes

Recent studies have shown that interleukin (IL)-4 can affect secretion of immunoglobulins (Igs) or activation of cytotoxic cells by IL-2, while other studies have shown that natural killer (NK) cells/large granular lymphocytes (LGLs) can also affect Ig synthesis. Therefore, we examined the effect of IL-4 with and without IL-2 or human NK/LGLs on pokeweed mitogen (PWM)-stimulated production of IgM and IgG. We found that when IL-4 and/or IL-2 were incubated with peripheral blood lymphocytes and PWM for 7 days and an enzyme-linked immunosorbent assay was run to measure Ig synthesis, IL-4 with IL-2 caused a greater suppression of Ig synthesis than either cytokine alone. A further experiment was done to determine the effect IL-4 and IL-2 would have on LGL suppression of Ig synthesis. IL-4 and IL-2 alone and in combination, when added to LGL, caused the LGL to suppress Ig synthesis to a greater extent than alone. We conclude that IL-4 acts on NK/LGLs separately and jointly with IL-2, to suppress Ig synthesis (IgM and IgG).     

Immune Reconstitution Kinetics as an Early Predictor for Mortality using Various Hematopoietic Stem Cell Sources in Children

The severity of complications of allogeneic hematopoietic stem cell transplantation (HSCT) is governed mainly by the status of immune reconstitution. In this study, we investigated differences in immune reconstitution with different cell sources and the association between the kinetics of immune reconstitution and mortality. Immunophenotyping was performed every 2 weeks in children who had undergone HSCT between 2004 and 2008 at University Medical Center Utrecht. Lymphocyte reconstitution in the first 90 days after HSCT was studied in relation to mortality in 3 HSCT groups: matched sibling bone marrow (BM) recipients (35 patients), unrelated BM recipients (32 patients), and unrelated cord blood recipients (36 patients). The median age of recipients was 5.9 years (range, 0.1-21 years). The nature and speed of T cell, B cell, and natural killer (NK) cell reconstitution were highly dependent on the cell source. In the first 90 days after HSCT, faster B cell and NK cell reconstitution and delayed T cell reconstitution were shown in unrelated cord blood recipients compared with matched sibling BM and unrelated BM recipients. Of the lymphocyte subsets investigated, a large number of NK cells and a more rapid CD4⁺ immune reconstitution over time, resulting in sustained higher CD4⁺ counts, were the only predictors of a lower mortality risk in all cell sources. The final model showed that during the first 90 days, patients with an area under the CD4⁺ cell receiver- operating curve of >4,300 cells/day and no peak in CD4⁺ cell counts had the highest likelihood of survival (hazard ratio for mortality, 0.2; 95% confidence interval, 0.06-0.5). Our data indicate that CD4⁺ kinetics may be used to identify patients at greatest risk for mortality early after HSCT.     

Effects of different sample preparations on enumeration of large granular lymphocytes (LGLs), and demonstration of a sex difference of LGLs

Peripheral natural killer (NK) cells were identified by their morphologic appearance as large granular lymphocytes (LGLs) in a cytocentrifuged or spun blood film but not in thin wedge blood film. The authors studied the effect of different methods of sample preparation and staining on the enumeration of peripheral human LGLs as a routine test. In the blood film made from anticoagulated venous blood, LGLs were scattered in a field and sometimes deformed by compression of erythrocytes. In a blood film made from mononuclear cells, LGLs were shrunken, rendering it difficult to always identify cytoplasmic granules. In contrast, LGLs were easily and accurately identified in a blood film made from leukocyte-rich plasma, displaying none of the above artifacts. As well, the percentage of LGLs obtained was similar to that obtained from a blood film made from anticoagulated venous blood. In addition, May-Grünwald-Giemsa (MGG) stained azurophilic granules more clearly than did Giemsa above. As a result of these observations, a blood film that was made from leukocyte-rich plasma and that was stained by MGG was considered to be most suitable for the routine enumeration of LGL. Using this method, the authors found a significant correlation between the percentages of LGLs and Leu-7+ cells in the same subjects (r = 0.71; n = 22; P less than 0.001) and also a significant sex difference in the percentages of peripheral LGLs, which were significantly lower in women (17.0 +/- 3.6%; n = 35; P less than 0.05) than in men (19.5 +/- 5.3%; n = 20). Furthermore, the percentage of LGLs with abundant cytoplasmic granules, which might have greater NK activity, was also significantly lower in women (15.9 +/- 3.1%; n = 35; P less than 0.01) than in men (17.9 +/- 4.0%; n = 20).     

Natural killer (NK) cell activity in human long-term bone marrow cultures (LTBMC): effects of interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the progenitor cells.

Human bone marrow-derived progenitor cells were studied in a long-term bone marrow culture system (LTBMC) dependent on an autologous stroma cell layer. The establishment of the stromal cell layer was facilitated by using marrow obtained from small pieces of sternum, which was cultured for 4 weeks without addition of exogenous growth factors. After this period, the response of LTBMC to two different cytokines [recombinant human interleukin-2 (rhIL-2) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF)] was investigated. Our results show proliferation in response to both cytokines and induction of differentiation of cells able to bind IL-2 and/or GM-CSF again. The two cytokines also generate cells responding to rhGM-CSF by colony formation. However, a difference with respect to morphology, phenotype and cytotoxic function of cells in the LTBMC, was noted between the two cytokines. Cells with large granular lymphocyte (LGL) morphology and cytotoxic activity against K562 and Daudi were generated only in the rhIL-2-supplemented LTBMC. This was compatible with a higher frequency of cells expressing the CD56+ phenotype in the IL-2-stimulated LTBMC as compared to the GM-CSF supplemented LTBMC. Our results also demonstrate the existence of a population of myeloid progenitor cells (CD33+) with ability to bind IL-2 in fresh bone marrow (BM).     

Tumor cells stimulate interleukin 1 (IL-1) production from enriched large granular lymphocytes

When enriched large granular lymphocytes (LGLs) were activated by K562 cells substantial amounts of interleukin 1 (IL-1) could be detected in the supernatants as measured by the mouse thymocyte assay. IL-1 could also be produced by LGLs treated with other tumor cells according to their ability to be lysed by LGLs. Therefore PLC/PRF/5 hepatoma cells which were moderately sensitive to LGL attack stimulated moderate amounts of IL-1 from the LGLs. Yac-1 cells and human fibroblasts which are resistant to LGL cytolysis did not activate LGLs to produce significant amounts of IL-1. IL-1 was shown to be produced by LGLs and not by the stimulatory tumor cells. Interferon-alpha (IFN), which reduced the susceptibility of target cells to be lysed by LGLs, also inhibited their ability to stimulate IL-1 production from LGLs. The IL-1 stimulatory effect of tumor cells on LGLs could not be attributed to Mycoplasma or endotoxin contamination. It is suggested that LGLs release IL-1 when they encounter susceptible cells and that release of this cytokine is important in the subsequent lysis of target cells.     

Phenotypical and functional heterogeneity of the large granular lymphocytes increased after various treatments in a patient with combined immunodeficiency

A boy with combined immunodeficiency having low natural killer (NK)-cell activity received thymopoietin pentapeptide (TP-5) treatment, transplanted with T cell-depleted HLA-haploidentical bone marrow (BMT) cells from his father and with thymus tissue from an infant at different times during the first year of life. He showed a marked increase in large granular lymphocytes (LGL) both during the treatment with TP-5 and after BMT. The LGL generated following TP-5 injection had a T3⁺ Leu 11^– surface phenotype and low NK activity. In contrast, the LGL appearing after BMT showed T3^–, Leu7⁺, and/or Leu11⁺ surface phenotypes, had high NK- and K-cell activities, and were lymphokine-activated killer (LAK)-cell precursors. These killer activities were assigned to the Leu7^– Leu11⁺ subset and proved to be of recipient origin. LGL proliferation following BMT was accompanied by neutropenia, which was improved in association with a reduction in the number of LGL and the appearance of T cells of BMT donor origin following thymus transplantation. This suggested the inhibition of granulopoiesis by the LGL and anin vitro study revealed that the Leu7⁺ Leu11^– subset of LGL suppressed the growth of granulocyte/macrophage colony-forming units. These results indicated that phenotypically different LGL could be generated by different treatments and that the LGL showing NK activity were distinct from those regulating granulopoiesis. It was also suggested that the generation of LGL was controlled by T cells.     

Spontaneous alloreactivity of natural killer (NK) and lymphokine-activated killer (LAK) cells from athymic rats against normal haemic cells. NK cells stimulate syngeneic but inhibit allogeneic haemopoiesis.

We wanted to re-examine the hypotheses that natural killer (NK) cells preferentially react with immature cells, and that they are not directed against major histocompatibility complex (MHC) gene products. Rat marrow cells could be separated according to maturity on a four-step discontinuous density gradient of Percoll. Almost all the immature bone marrow cells with progenitor activity, as measured in vivo in a diffusion chamber assay or in vitro in a granulocyte/macrophage colony-forming assay, resided within the lighter density cell fraction (density approximately 1.065). The higher density cells (density approximately 1.082) contained mainly the more mature, non-proliferative cells within the granulocyte series. NK and lymphokine-activated killer (LAK) cells from athymic rats, being devoid of T cells, efficiently killed low- as well as high-density bone marrow cells from a fully allogeneic and a MHC congenic rat strain, while little or no killing was observed against syngeneic bone marrow cell fractions. LAK cells also effectively inhibited granulocyte/macrophage colony formation from allogeneic bone marrow precursors in vitro, while stimulating colony formation from syngeneic bone marrow cells. The NK-mediated killing of allogeneic bone marrow cells was effectively inhibited by NK-sensitive tumour cells, while there was much less inhibition of the killing of tumour cells by allogeneic bone marrow cells. We conclude that NK cells recognize MHC incompatibilities on both immature and mature allogeneic bone marrow cells through recognition systems not related to T-cell receptors, and that allospecific killing can explain the contrasting effect of NK cells on allogeneic and syngeneic haematopoiesis.