Subpopulation analysis of human granular lymphocytes: associations with age, gender and cytotoxic activity

Levels of human blood granular lymphocyte subpopulations were enumerated in 105 healthy donors ranging in age from 0 to 79 years. Using double-label immunofluorescence, subpopulations of granular lymphocytes were enumerated as follows: Leu7+Leu2+, Leu7+Leu3+, Leu7+11-, Leu7+77+ and Leu7-Leu11+. The proportion of cells with granular lymphocyte morphology was determined by Giemsa staining. Natural killer (NK) cell activity against K-562 cells and lymphokine-activated killer activity against non-cultured melanoma cells were examined in parallel. Levels of total Leu7+ and Leu11+ cells increased with age (p = 0.0001) and were higher in males than females (p = 0.001). The total number of cells with granular lymphocyte morphology had an age-related increase (p = 0.001), but were not significantly higher in males than in females (p = 0.07). There was no selective increase in one granular lymphocyte subpopulation versus another since the Leu7+Leu11- (p = 0.0001), the Leu7+Leu11+ (p = 0.0001), the Leu7+Leu2+ (p = 0.0001) and the Leu7+Leu3+ (p = 0.0004) all had similar age-related increases. The one exception was the Leu7-Leu11+ (p = 0.1) granular lymphocyte subset which was low in the first decade of life but had reached maximum levels in the second decade. NK cell activity against K-562 cells was moderately increased with age (p = 0.06) with males and females exhibiting comparable activity. In contrast. lymphokine-activated killer cytotoxicity of non-cultured melanoma cells was similar in all age groups. NK cell activity was highly correlated with levels of morphologically defined granular lymphocytes (p = 0.005) and moderately with total Leu11+ cells (p = 0.06) but not with other subpopulations of granular lymphocytes.     

Characterization of human large granular lymphocyte subpopulations: comparison of the phenotype of NK cells and of interleukin 2-dependent progenitors of cytolytic effector cells

Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.     

Natural killer cells in the blood and bone marrow of the rhesus monkey

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.     

Morphologic and phenotypic analysis of canine natural killer cells: evidence for T-cell lineage

Canine natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity were studied utilizing a canine thyroid adenocarcinoma cell line and a lymphoblastoid cell line (CT-45S), respectively, as cell targets. Fractionation of peripheral blood mononuclear cells by Percoll discontinuous-gradient centrifugation resulted in a six- to sevenfold enrichment in large granular lymphocytes (LGL) in parallel with a twofold increase in NK activity (%specific lysis) in low-density fractions. Further enrichment in LGL (78 +/- 6%) and NK activity (threefold increase) was obtained by lytic treatment of low-density fractions 2 and 3 with monoclonal antibody WIG4. By means of cytolytic treatment with additional monoclonal antibodies the phenotype of canine NK cells was determined as Dly-1+, Dly-6+, 1A1+, E-11+, DT-2-, WIG4-. Some NK cells were also Ia+. NK activity was relatively radioresistant with 40% specific lysis even after irradiation with 40 Gy. Among the populations examined, the highest NK activity was found in peripheral blood mononuclear cells, followed by splenic mononuclear cells and bone marrow mononuclear cells. These results indicate that canine NK cells have the morphology of LGL, are relatively radioresistant, and express cell surface antigens suggesting a T-cell lineage.     

Production of B cell growth factor by a Leu-7+, OKM1+ non-T cell with the features of large granular lymphocytes (LGL)

The present study reports the characterization of a non-T cell from human peripheral blood which is capable of releasing BCGF. This BCGF-producing non-T cell had a T3-, T8-, Leu-7+, OKM1+, HLA-DR-, Leu-11- surface phenotype and was likely to belong to the so-called large granular lymphocyte (LGL) subset because: after fractionation of non-T cells according to the expression of Leu-7 or HLA-DR markers, it was found in the Leu-7+, HLA-DR- fractions that were particularly enriched in LGL; it co-purified with LGL on Percoll density gradients; and it expressed Leu-7 and OKM1 markers that are shared by a large fraction of LGL. Although co-purified with cells with potent NK capacities, the BCGF-producing cell was not cytotoxic, because treatment of Leu-7+ cells with Leu-11 monoclonal antibody and complement abolished the NK activity but left the BCGF activity unaltered. The factor released by this LGL subset was not IL 1 or IL 2 mistakenly interpreted as BCGF, because: a) cell supernatants particularly rich in BCGF activity contained very little or no IL 1 or IL 2; b) BCGF-induced B cell proliferation was not inhibitable by anti-Tac antibodies (this in spite of the expression of IL 2 receptor by a proportion of activated B cells); and c) BCGF activity was absorbed by B but not T blasts.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Cytolytic activity of human peripheral blood leukocytes against Legionella pneumophila-infected monocytes: characterization of the effector cell and augmentation by interleukin 2

The present study was an in vitro attempt to define the effector mechanisms against the intracellular bacterium Legionella pneumophila. Monocytes from human peripheral blood leukocytes (PBL) were infected in vitro with L. pneumophila and cultured for 2 days to allow intracellular replication of the bacterium. Cells were then labeled with 51Cr and used as targets in a 4-h 51Cr-release assay. We report here that autologous nonadherent PBL effectively lysed infected monocytes, and this activity was enhanced when the effector cells were precultured with IL 2 for 2 days. The IL 2-activated killer cells were also cytolytic against uninfected cultured monocytes, but cytotoxicity was higher against Legionella-infected target cells in a dose-dependent manner. The effector cells were located in Percoll density fractions that were enriched for large granular lymphocytes. The phenotype of the effector cell activated by IL 2 was determined to be OKM1+, OKT11+, partially Leu-11+, and negative for Leu-M1, OKT4, OKT8, and Leu-7, indicating that it is neither a T cell nor a monocyte, and is possibly and NK subset that is Leu-11+ and Leu-7-. Cold target inhibition studies indicated that a similar recognition structure is shared by both infected and uninfected monocytes, but differs from that on K562 tumor target cells. Thus, in addition to tumor surveillance and controlling viral infections, killer cells can be activated to provide protection against intracellular bacterial infections.     

Separation of lymphokine-activated killer cell precursors and T-cells: differential growth characteristics and apparent lack of a T-cell suppressor of LAK-cell induction

Lymphokine activated killer (LAK) cell clinical effectiveness may be limited by the total cell dose and cytotoxic activity. We have, therefore, examined methods to expand the number of LAK-cells by serial passage of unfractionated and fractionated peripheral blood lymphocytes. Human purified lymphocytes were obtained by Ficoll Hypaque gradients followed by exposure of resultant mononuclear cells to phenylalanine methyl ester to remove monocytes. Lymphocytes were then fractionated on a six-step Percoll gradient (50%, 47.5%, 45%, 42.5%, 40%, and 37.5% Percoll). Unfractionated cells and fractions were cultured in standard media (RPMI-1640, 10% human sera, antibiotics and 10 mM HEPES) containing 10 nM of recombinant Interleukin-2 (rIL-2). Lymphocytes were cultured at 1 X 10(6)/ml and recultured every 3 to 4 days in fresh standard media and rIL-2. Utilizing unfractionated and fractionated lymphocytes from seven donors we made the following observations: (1) Continued passage of unfractionated lymphocytes resulted in a loss of LAK-cell activity by greater than or equal to 14 days (e.g., percent lysis of Raji at 10:1 effector:target ratio on days 0, 4, 7, and 21 was 0.38 +/- 11, 41 +/- 17, and 8 +/- 1, n = 4, respectively). (2) LAK-cell functional precursors were predominantly confined to the lymphocytes in the upper (0-4) Percoll fractions (e.g., on day 4, the percent lysis by pooled fractions 1-4 was 63 +/- 5 vs. pooled fraction 5 plus pellet, 18 +/- 7%). (3) As expected, the upper fractions (0-4) were enriched for Leu 19 positive cells (approximately 40%) and large granular lymphocytes (LGL) by morphology (approximately 30%).(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

Recombinant interleukin 2 enhanced natural killer cell-mediated cytotoxicity in human lymphocyte subpopulations expressing the Leu 7 and Leu 11 antigens

Highly purified recombinant human interleukin 2 (rIL 2) markedly augments the natural killer (NK) cell-mediated cytotoxicity of peripheral blood lymphocytes. In this study, we examined the cellular and metabolic basis of rIL 2-mediated activation of human lymphocyte subpopulations expressing the NK cell-associated surface antigens Leu 7 and Leu 11. All rIL 2-responsive cytotoxic NK cells were found within the subset of lymphocytes expressing the Leu 11 marker, an antigen associated with the Fc-IgG receptor on human NK cells. Cells lacking the Leu 11 antigen, including cells expressing another NK cell-associated marker, Leu 7, did not express NK cell-mediated cytotoxicity either before or after rIL 2 treatment. By contrast, rIL 2 augmented the NK activity of both Leu7-,11+ and Leu 7+,11+ subpopulations. Activation of Leu 11+ NK cells resulted from a direct effect of rIL 2 on these cells and neither required nor was amplified by the presence of T lymphocytes. Enhanced NK cell-mediated cytotoxicity occurred within 4 hr after exposure to rIL 2, and was blocked by the protein synthesis inhibitor cyclohexamide, but not by the DNA synthesis inhibitor mitomycin C or 1500 rad of x-irradiation. Neither Tac antigen, a high-affinity receptor for IL 2, nor other activation markers, such as transferrin receptor or HLA-DR antigen, were detectable on a significant proportion of Leu 11+ cells, either before or after incubation with rIL 2 for 48 hr. In addition, saturating concentrations of antibodies to each of these markers had no effect on the enhancement of NK activity by rIL 2. Finally, preliminary experiments with neutralizing antibodies to gamma- and alpha-interferons also failed to prevent rIL 2 enhancement of NK cell-mediated cytotoxicity, suggesting that rIL 2 does not mediate its effect via release of these cytokines.     

Induction of interferon-gamma production by human natural killer cells stimulated by hydrogen peroxide

Interferon (IFN)-inducing activity of hydrogen peroxide in human peripheral mononuclear cells was investigated. Among the mononuclear cells, purified nonadherent cells produced IFN, but not B cells and monocytes. The maximal titer of IFN by purified nonadherent cells was observed after a 72-hr cultivation in the presence of 10(-2) mM H2O2 without affecting their viability. Furthermore, the purified nonadherent cells, but not the unpurified mononuclear cells, showed an augmented cytotoxicity to K562 when stimulated with hydrogen peroxide. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into the low and high density fractions for which natural killer (NK) cells and T cells were enriched, respectively. The NK-enriched low density fractions, but not the T cell-enriched high density fractions, showed IFN production by the stimulation of hydrogen peroxide. IFN production as well as large granular lymphocytes and HNK-1+, Leu-11+ cells of the NK-enriched fractions were abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1+) but not against T cells (OKT3) in the presence of complement. Moreover, hydrogen peroxide-inducing IFN production seems to be regulated by monocytes. The antiserum neutralizing IFN-alpha and IFN-beta failed to neutralize substantially IFN-produced NK cells. The treatment with either pH 2 or antiserum-neutralizing human IFN-gamma resulted in marked reduction, indicating that a major part of IFN was IFN-gamma. The purified nonadherent cells showed IFN production and augmented cytotoxicity when cultured separately from activated macrophages by opsonized zymosan; furthermore, both IFN production and enhancement of cytotoxicity were abrogated by catalase. These results suggest that both exogenous and endogenous hydrogen peroxide might be responsible for a part of immunoregulation.     

NK-leukocyte chemotactic factor (NK-LCF): a large granular lymphocyte (LGL) granule-associated chemotactic factor

A chemotactic factor was identified in the supernatants of human large granular lymphocytes (LGL) activated by a glutaraldehyde-fixed NK-sensitive tumor, K562. The factor stimulated migration of human LGL, rat alveolar macrophage (RAM), and human monocytes and neutrophils (PMN). The locomotor response was chemotactic and chemokinetic on the basis of unidirectional migration in concentration gradients. The cell producing the factor was detected exclusively in LGL-rich Percoll fraction coincident with the peak of NK lytic activity and HNK-1+ cells. The monoclonal phenotype of the cell was HNK-1+, partially OKT-11+, OKM-1-, OKT-3-, OKT-4-, and OKT-8-. The factor was released by LGL within 20 min of incubation with Sr++, a cation that is able to induce LGL degranulation. A powerful chemoattractant was also detected in the granules of the rat LGL leukemia, RNK. Chemotactic activity coincided with granule enzyme beta-glucuronidase and cytolysin after RNK nitrogen cavitation and Percoll fractionation of subcellular constituents. The RNK granule chemoattractant induced unidirectional migration of human LGL and was also active against rat alveolar macrophages and human PMN. Anti-RNK granule antibody conjugated to Sepharose 4B was able to deplete the chemotactic activity from both K562-induced LGL supernatants and solubilized RNK granules. These observations indicate that a leukocyte chemotactic factor (NK-LCF) is present in NK cell granules and is probably released after tumor-induced granule exocytosis.     

Morphologic, phenotypic, and cytotoxic analyses of C57BL/6 murine non-parenchymal liver cells: new evidence associating murine liver large granular lymphocytes with monocyte precursors and implications for tumor immunotherapy.

Non-parenchymal liver cells (NPCs) have been implicated in murine host resistance to hepatic metastases. We have examined the relative cell number, morphology, phenotype, and cytotoxic potential of Percoll fractionated C57BL/6 murine liver NPCs. Low density (Percoll fractions 2 and 3) cells showed a large granular lymphocyte morphology and made up 76% of all NPCs recoverable, while high density (fractions 5 and 6) showed a small lymphocyte morphology and made up 10% of all NPCs. Low density cells demonstrated the following phenotype: 14% of the cells demonstrated the Thy 1.2 marker; 12%, the Lyt-2 marker; 67%, the L3T4 marker; 74%, the asialo GM1 marker; 30%, the 49H.8 marker; and 65%, the F4/80 marker. The high density cells expressed the same markers on 71%, 21%, 33%, 68%, 37%, and 19% of their cell surface, respectively. There were no differences phenotypically between high density NPCs and splenocytes except for the F4/80 expression (fractions 5 and 6 NPCs, F4/80 expression 19%, fresh splenocytes 60%). Dual color analysis of L3T4+ NPCs documented that fractions 2 and 3 cells also expressed the F4/80 marker on 85% of their cell surface and the Thy 1.2 marker on 11% of their cell surface. The high density fractions 5 and 6 L3T4+ cells expressed the F4/80 marker on 16% of their cell surface, and the Thy 1.2 marker on 89% of their cell surface. Cytotoxicity against YAC-1 [a natural killer (NK) sensitive target], MCA-102 (a NK resistant target), and WEHI-164 (a natural cytotoxicity target) were similar for fractions 2 and 3, and 5 and 6 cells. Based upon the expression of the F4/80 marker on L3T4+ cells that are Thy 1.2 negative and appear to be similar to LGLs morphologically (fractions 2 and 3 NPCs), we propose that these cells are monocyte precursors while fractions 5 and 6 cells are small lymphocytes. These findings with liver LGLs support the need for the evaluation of monocyte directed biological response modifiers in therapeutic models of murine hepatic metastases.     

Phenotypic characterization of cynomolgus monkey natural killer cells

Natural killer (NK) activity of cynomolgus monkey peripheral blood lymphocytes (PBL) was determined using B95-8 cells as target cells. Examination for the reactivity of human NK-related monoclonal antibodies (mAbs), anti-Leu-7, anti-Leu-11b, anti-NKH1A, and NC-1, with cynomolgus PBL revealed that Leu-11b (CD16) was the only antigen expressed on cynomolgus PBL. The percentage of Leu-11b-positive (Leu-11b+) cells correlated well with the level of NK activity when PBL taken from 21 monkeys were tested. After depletion of Fc receptor-positive (FcR+) cells, NK activity was lost concomitantly with the disappearance of Leu-11b+ cells. These results show that cynomolgus NK cells are mainly FcR+ which can be detected by mAb directed to Leu-11b. Cynomolgus PBL were separated by Ficoll-Hypaque centrifugation after E rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, and NK activities of both E rosette-forming (E+) and nonforming (E-) fractions were determined. The high level of killing was observed in the E- fraction, suggesting that the majority of cynomolgus NK cells was contained in the E- fraction. The separation of PBL by Percoll discontinuous density gradient showed cynomolgus NK cells were enriched in the low density fractions.     

Estrogen and antiestrogen modulation of the levels of mouse natural killer activity and large granular lymphocytes

We investigated the time course of the 17β-estradiol effect on mouse natural killer (NK) activity and the number of splenic large granular lymphocytes (LGL), a cell population recently associated with natural cytotoxicity and enriched in low density fractions of Percoll discontinuous density gradients. After 7 days of in vivo treatment with estrogen, an increased cytotoxicity against YAC-1 lymphoma cells was observed using only as effectors cells recovered from higher density fractions, usually devoid of NK activity. In contrast, after a 30-day treatment, augmented NK activity and an increase in LGL number were observed in the lower density Percoll fractions. Similar results were observed after a 30-day treatment with the antiestrogen tamoxifen. The cytotoxicity of both low density and high density splenocyte fractions was totally abrogated by treatment with antiserum to asialo GM₁ plus complement, whereas anti-Thy 1.2 antibody treatment only partially decreased the reactivity. Further estrogen administration up to 60 days decreased both NK activity and LGL number. It is concluded that estradiol can induce opposite effects on NK activity depending on the time of treatment, with stimulation of NK activity during the first 30 days after treatment followed by depressed NK activity 1 month later.     

Characterization of natural killer cells and their precursors in the murine bone marrow

We have fractionated murine bone marrow cells according to their density on bovine serum albumin (BSA) gradient and studied (a) the NK activity against YAC-1 targets, (b) the proportion of asialo GM1+ lymphocytes, (c) and the presence of large granular lymphocytes (LGL) in the different fractions (A, B, C, D). The NK activity was found mainly in the C fraction, but the proportion of asialo GM1+ cells was the same in every fraction. No LGLs were found in the bone marrow. Cells from the various fractions were also transplanted into irradiated recipients. Seven days later the highest NK activity was found in the spleens of mice injected with cells from the A + B fractions indicating that the immediate precursors for NK cells reside in the low density fractions of the BSA gradient. Mice transplanted with C or D fractions needed longer time to develop normal NK levels. The treatment of bone marrow cells before transplantation with anti-asialo GM1+ complement did not inhibit the development of NK activity, so it can be concluded that the precursor for NK is asialo GM1-.     

Isolation and identification of normal killers from Syrian hamsters

Natural killers (NK cells) possessing cytotoxic activity were isolated from the blood, spleen and bone marrow of Syrian hamsters in discontinuous Percoll density gradient. These cells were morphologically identified as granular lymphocytes (GL). The largest amount of GL was isolated from the fraction containing 52-55% of Percoll. Cytotoxic activity of cells in this fraction was the highest, as compared to nonfractionated control or cells in other Percoll fractions.     

Age-associated alterations in human natural killer cells. 2. Increased frequency of selective NK subsets

We have analyzed the peripheral blood lymphocytes from healthy volunteers (20 to 94 years) for the expression of natural killer (NK) cell surface markers, NK activity, and B-cell proliferative response. An increase (2- to 3.5-fold) in relative percentage and absolute number of lymphocytes expressing Leu-7 (HNK-1) or Leu-11a (CD 16) antigen was found in the elderly group (greater than 80 years) as compared to young adults (less than 40 years). A two-color immunofluorescence analysis revealed that the age-associated increment was both progressive and selective; the actual increase occurred in Leu-7+11a+ and Leu-7+11a- populations (subsets with variable and weak NK activity) but not in the Leu-7-11a+ (most active) subset. There is a corresponding decrease in the 7-11a- cells. The ratios of 7+11a+/7-11a+ and 7+11a-/7-11a+ cells doubled with advancing age. Linear regression analysis suggests that the 7-11a+ cells are highly preserved through human senescence and the ratio of 7+11a- cells to the most stable subset, 7-11a+, could expand nearly 100-fold from birth to old age. Further analysis of Leu-7+ cells for the coexpression of Leu-11c (an epitope of Leu-11a) confirmed a similar pattern of changes in 7+11c+ and 7+11c- NK subsets with advancing age. The frequency of Leu-11+ (epitopes 11a+ or 11c+), but not of the subsets of 7+ phenotype (7+11a- or 7+11c-), correlates well with the NK activity (spontaneous killing of K562 tumor cell line). The 7+11c+ cells may directly or indirectly be responsible for the increase in NK activity observed with a majority of aged donors. The inverse relationship observed between the mitogenic response of lymphocytes to pokeweed mitogen (PWM) and the initial frequency of 7+11a-, but not other phenotypes, raises a potential functional significance for the expansion of the 7+11a-(7+11c-) subset. These age-associated NK phenotypic changes provide a cellular basis for our observations on age-associated increase in NK activity and decrease in mitogenic response to PWM.     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

Generation of large granular T lymphocytes in vivo during viral infection

Cytolytic lymphocytes were isolated from the spleens of lymphocytic choriomeningitis virus (LCMV)-infected mice and were characterized in regards to function, cell size, antigen phenotype, and cell morphology. Only 2% of the Lyt-2+ cells from uninfected mice were large granular lymphocytes (LGL), whereas 21% of the Lyt-2+ cells isolated 7 days postinfection were LGL. The day 7 Lyt-2+ populations contained all of the LCMV-specific, class I histocompatibility antigen-restricted cytotoxic T lymphocyte (CTL) activity, but no natural killer (NK) cell activity. The NK cell activity was consistently recovered in Lyt-2- populations isolated from both control mice and mice on day 7 postinfection. The LGL isolated on day 7 postinfection were concluded to be predominantly T cells and not NK cells because 1) the proportions of LGL in fractionated cell populations 7 days postinfection correlated with levels of CTL-mediated lysis but not NK cell-mediated lysis, 2) they were recovered in the Lyt-2+ population, and 3) antibody to asialo GM1, known to eliminate NK cell-mediated lysis but not T cell-mediated lysis, dramatically reduced NK cell LGL numbers in vivo on day 3 postinfection but only marginally affected LGL numbers on day 7. Virus-induced inflammation elicited a 50-fold increase in LGL numbers in the peritoneum on day 7 postinfection. The peritoneal exudate LGL were also associated with CTL activity and were resistant to treatment with antibody to asialo GM1. These results indicate that in vivo-generated CTL have the morphology of LGL and that the appearance of cytoplasmic granules correlates with the ability of cells to mediate lysis. To focus on cells being stimulated during infections, activated blast cells were separated from small resting cells by centrifugal elutriation. Coincidental with the peak in overall spleen leukocyte cytotoxic activity, the peaks of blast NK cells and CTL were at days 3 and 7 postinfection respectively. More than 50% of the blast lymphocytes isolated on either day 3 or day 7 postinfection were LGL. The CTL activity in the blast populations on day 7 postinfection was mediated by Lyt-2+ cells, and 37 to 64% of these Lyt-2+ blast cells were LGL. Cytolytic NK cell and CTL LGL could not be distinguished by morphology or by cell densities, because they overlapped in low density Percoll gradient fractions. Since this technique has been used to enrich for LGL, these data indicate that heterogeneity in LGL populations may result from the presence of both CTL and NK cell LGL.