Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs

Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hi CD16(-), CD56lo CD16(-), CD56+CD16+ and CD56(-)CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-gamma was preferentially produced by CD56+CD16(-) NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16(-) and CD56+/-CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

Effects of resveratrol on human immune cell function.

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol found in grapes and grape products such as red wine, has been reported to exhibit a wide range of biological and pharmacological activities both in vitro and in vivo. Because many of the biological activities of resveratrol, like the inhibition of cyclooxygenase, induction of CD95 signaling-dependent apoptosis, effects on cell division cycle and modulation of NF-kB activation, suggest a possible effect on the immune system, we evaluated the in vitro effects of resveratrol in three immune response models: i) development of cytokine-producing CD4+ and CD8+ T cells induced by stimulation of peripheral blood mononuclear cells (PBMC) with anti-CD3/anti-CD28; ii) specific antigen-induced generation of cytotoxic T lymphocytes; iii) natural killer (NK) activity of PBMC. The results showed that in vitro exposure to resveratrol produces a biphasic effect on the anti-CD3/anti-CD28-induced development of both IFN-gamma- IL2- and IL4-producing CD8+ and CD4+ T cells, with stimulation at low resveratrol concentrations and suppression at high concentrations. Similarly, the compound was found to induce a significant enhancement at low concentrations and suppression at high concentrations of both CTL and NK cell cytotoxic activity. On the whole, the results of the study indicate that resveratrol modulates several human immune cell functions and suggest that this activity may be related to its effects on cytokine production by both CD4+ and CD8+ T cells.     

Characterization of porcine T lymphocytes and their immune response against viral antigens.

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4+CD8- T helper cells and CD4-CD8+ cytolytic T lymphocytes, extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-CD4-CD8- T cell receptor (TcR)-gamma delta+ T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4+CD8- and CD4+CD8+. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4+CD8+ T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4-CD8+ T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6- cells which showed spontaneous cytolytic activity and CD6+ MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.     

Long-term proliferation of functional human NK cells,with conversion of CD56<Superscript>dim</Superscript> NK cells to a CD56<Superscript>bright</Superscript> phenotype,induced by carcinoma cells co-expressing 4-1BBL and IL-12

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL + IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21 day) NK cell proliferation. The expanded NK cells are predominantly CD56^bright, and we show that isolated CD56^dimCD16⁺ NK cells can switch to a CD56^brightCD16⁻ phenotype and proliferate in response to 4-1BBL + IL-12. Whereas 4-1BB upregulation on NK cells in response to 4-1BBL required ‘help’ from other PBMC, it could be induced on isolated NK cells by IL-12, but only in the presence of target (OVCAR-3) cells. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL + IL-12 and subsequent resting until day 21, NK cells remained predominantly CD56^bright and retained both high cytotoxic capability against K562 targets and enhanced ability to produce IFNγ relative to NK cells in PBMC. These data support the concept that NK cells could contribute to anti-tumour activity of 4-1BB agonists in humans and suggest that combining 4-1BB-stimulation with IL-12 could be beneficial for ex vivo or in vivo expansion and activation of NK cells for cancer immunotherapy.     

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.     

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.     

Glycosylation profile and biological activity of Remicade® compared with Flixabi® and Remsima®

As biosimilars enter the market, comparisons of product quality are needed. Manufacturing differences may lead to differences in critical quality attributes, which affect efficacy. Therefore, critical quality attributes (structure and biological activity) of Remicade® and of 2 biosimilar products (Flixabi®/Renflexis® and Remsima®/Inflectra®) were determined. We assessed binding to tumor necrosis factor in a fluorescence competitive binding assay; potency in a luciferase reporter gene assay; percentages of galactosylated glycan, afucose plus high mannosylated glycans, and charged glycan; FcγRIIIa (CD16) binding (assessed by 3 methods); and antibody-dependent cell-mediated cytotoxicity (ADCC) in the NK92-CD16a cell line and in peripheral blood mononuclear cells (PBMC). The results of Fab-related activity were similar for all products. Compared with Remicade®, Flixabi® had a lower percentage of charged glycan, and Remsima® had a higher percentage of galactosylated glycan and a lower percentage of afucose plus high mannosylated glycans. Whereas Remsima® and Remicade® are expressed in a Sp2/0 cell line, Flixabi® is expressed in a CHO cell line. Despite this difference, galactosylated glycans from the 3 products were not correlated with the expression system. The results of all 3 methods used in this study indicated that FcγRIIIa binding was lower with Remsima® than with Remicade®. The percentage of ADCC in NK92-CD16a cells was lower with Remsima® and higher with Flixabi® compared with Remicade®, but was similar for all 3 products in PBMC. Surface expression of CD16 was 5.7-fold greater on NK92-CD16a cells than on PBMC. Combined percentages of afucosylated and high mannosylated glycans were positively correlated with FcγRIIIa binding and ADCC in NK92-CD16 cells, while no correlation was observed in PBMC.     

GP120 specific cellular cytotoxicity in HIV-1 seropositive individuals. Evidence for circulating CD16+ effector cells armed in vivo with cytophilic antibody

Fresh circulating PBMC from HIV-1 seropositive individuals have been found to mediate specific, non-MHC restricted lysis of targets expressing the major envelope glycoprotein of HIV-1, gp120, in 6-h 51Cr release assays. This gp120 specific cell-mediated cytotoxicity (CMC) is broadly reactive against target cells infected with a wide range of viral isolates, is IL-2 augmentable, and is mediated by a CD16+, Leu-7+, CD15-, CD3- population of NK/K cells. The presence of FcR (CD16) on these cells suggested that the lytic specificity for gp120 might be directed by cytophilic antibody bound to the cell surface. Affinity purified F(ab')2 antibody fragments specific for the Fc and F(ab')2 portions of human IgG were used in attempts to block gp120 specific lysis. A 1/50 dilution of these antibodies inhibited gp120 specific cytolytic activity by more than 90% while exhibiting a minimal effect on NK/K cell lysis of K562 targets. The blocking activity of these fragments demonstrates the direct involvement of cytophilic antibody in CMC. In attempts to isolate this cytophilic anti-HIV-1 antibody, short 56 degrees C incubations were used to dissociate antibodies from the surface of PBMC of seropositive individuals. The supernatants generated in this manner exhibited specific gp120 activity in antibody-dependent cellular cytotoxicity assays. The ability of Staphylococcal protein A to remove this activity confirms the presence of cytophilic antibody on freshly isolated PBMC. Selective enrichment of specific cell subpopulations revealed the origin of the cytophilic antibody to be CD16+ NK/K cells and not B cells, T cells, or monocytes/macrophages. These studies show that the gp120-specific CMC seen in HIV-1 seropositive individuals is directed by cytophilic antibody bound to circulating CD16+ NK/K cells and represents a form of direct antibody-dependent cellular cytotoxicity which may provide a primary cytotoxic host defense.     

The human MHC-restricted cellular response to herpes simplex virus type 1 is mediated by CD4+, CD8- T cells and is restricted to the DR region of the MHC complex

The nature of the in vitro human cytotoxic T-cell responder population to HSV type 1 (HSV-1) was studied. In 5-day HSV-1-stimulated cultures that contained MHC-restricted activity, two phenotypically distinct populations of cells were present that were capable of lysing HSV-1-infected B cell lines in a 5-h 51Cr-release assay. The first was CD4+, CD8-, CD16- cell typical of class II-restricted T cells, whereas the other population bore a CD4-, CD8-, CD16+ NK-cell phenotype. Elimination of the NK cell fraction from bulk cultures by using anti-CD16 plus C frequently resulted in cell populations that killed in an Ag-specific, HLA-DR-restricted fashion. In some cases the anti-CD16-pretreated cultures retained a killing population that was unrestricted to MHC products. In no instance were any cytotoxic T cells that were restricted to class I Ag in evidence. Limiting dilution analysis of precursor frequency indicated that about 1 in 4000 to 1 in 8000 cells from peripheral blood are specific for HSV-1 in seropositive individuals. Comparisons of HLA class I-matched and HLA class II-matched targets with the autologous target by using limiting dilution analysis yielded results entirely consistent with those obtained in the bulk culture assay system.     

Activation of cloned human natural killer cells via Fc gamma RIII

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.     

Lipopolysaccharide stimulates the proliferation of human CD56+CD3- NK cells: a regulatory role of monocytes and IL-10

NK cells recognize and kill tumor cells and normal cells, and these play an important role in immune defense in cancer, infectious disease, and autoimmunity. NK killing is regulated by positive or negative signals derived from the interaction of surface receptors with ligands on the target cells. However, the mechanisms controlling the proliferation and maintenance of NK cells in normal human individuals are less clearly defined. In this study, using an entirely autologous system, we demonstrate that human peripheral blood CD3-CD56+, killer cell-inhibitory receptor (KIR)-expressing cells proliferate and expand in response to LPS. These responses are enhanced in the presence of anti-IL-10 receptor-blocking Abs or on the removal of CD14+ cells from the cultures. This enhancement is also reflected in substantial increases in cytolytic activity and IFN-gamma production. The negative effect of CD14+ cells may also be IL-10 mediated, IL-10 being lost from the culture supernatants of CD14-depleted PBMC and rIL-10 reversing the effect of this depletion. On the other hand, mRNA for the p35 and p40 subunits of IL-12 is still induced in CD14-depleted cultures. The expansion of CD3-CD56+ cells was also inhibited by CTLA4-Ig, indicating a role for CD80/86. B lymphocytes were not required for the expansion of CD3-CD56+ cells, whereas removal of MHC class II+ cells from CD14-depleted cultures resulted in a complete abrogation of these responses. Expansion of CD3-CD56+ cells was reconstituted in MHC class II-depleted cell cultures by adding back monocyte-derived dendritic cells. These results indicate that the responses of CD3-CD56+ NK cells to LPS may be driven by a MHC class II+ B7+ CD14- peripheral population, most likely blood dendritic cells.     

Induction of NK cell activity against fresh human leukemia in culture with interleukin 2

Natural killer (NK) cells have been implicated in defense against malignancies, especially leukemia. Because patients with leukemia and preleukemic disorders manifest low NK activity, it is possible that NK cell impairment may contribute to leukemogenesis. In view of this possibility, it was important to characterize the NK cell defect of leukemic patients and to design new approaches for its correction. Analysis of the mechanism of NK cell defect demonstrated that NK cells of leukemic patients were impaired in their tumor-binding and lytic activity and did not display ability to recycle or to produce cytotoxic factor. However, deficient NK activity could be corrected by culture of peripheral blood effector cells with IL 2. IL 2-activated NK cells manifested restoration of all measured parameters of the cytotoxic mechanism, as exemplified by normalized tumor-binding and lytic activity, as well as the rate of lysis and ability to recycle. Importantly, such in vitro stimulated cytotoxic cells displayed reactivity against fresh leukemic cells of autologous as well as allogeneic origin. Another interesting observation from these studies was that the NK activity was also induced in the leukemic bone marrow, a tissue with a very low frequency of cytotoxic NK cells. It is important to note that cultured NK cells did not represent a stationary cell population, but proliferated in vitro quite actively (doubling time 3 to 6 days) for at least 5 wk. Characterization of the in vitro generated cytotoxic cells indicated that these cells displayed large granular lymphocyte morphology and CD16 and Leu-19 cell surface phenotype. Our data demonstrate that the NK cell defect of leukemic patients is not a permanent phenomenon, but can be reversed in culture with IL 2, and that fully cytotoxic NK cells can be maintained and expanded in vitro. Thus, it is reasonable to suggest that adoptive transfer of autologous NK cells to the patients may represent a promising new therapy for treatment of leukemia.     

Regulation of human cell engraftment and development of EBV-related lymphoproliferative disorders in Hu-PBL-scid mice

Human PBMC engraft in mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation (Hu-PBL-scid mice). Hu-PBL-NOD-scid mice generate 5- to 10-fold higher levels of human cells than do Hu-PBL-C.B-17-scid mice, and Hu-PBL-NOD-scid beta2-microglobulin-null (NOD-scid-B2mnull) mice support even higher levels of engraftment, particularly CD4+ T cells. The basis for increased engraftment of human PBMC and the functional capabilities of these cells in NOD-scid and NOD-scid-B2mnull mice are unknown. We now report that human cell proliferation in NOD-scid mice increased after in vivo depletion of NK cells. Human cell engraftment depended on CD4+ cells and required CD40-CD154 interaction, but engrafted CD4+ cells rapidly became nonresponsive to anti-CD3 Ab stimulation. Depletion of human CD8+ cells led to increased human CD4+ and CD20+ cell engraftment and increased levels of human Ig. We further document that Hu-PBL-NOD-scid mice are resistant to development of human EBV-related lymphoproliferative disorders. These disorders, however, develop rapidly following depletion of human CD8+ cells and are prevented by re-engraftment of CD8+ T cells. These data demonstrate that 1) murine NK cells regulate human cell engraftment in scid recipients; 2) human CD4+ cells are required for human CD8+ cell engraftment; and 3) once engrafted, human CD8+ cells regulate human CD4+ and CD20+ cell expansion, Ig levels, and outgrowth of EBV-related lymphoproliferative disorders. We propose that the Hu-PBL-NOD-scid model is suitable for the in vivo analysis of immunoregulatory interactions between human CD4+ and CD8+ cells.     

IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury

NK cells promptly disappear from the circulation of patients treated with high dose i.v. rIL-2. To further study this process, we evaluated the effects of IL-2 (1000 U/ml) on normal donor PBMC incubated for 1 h on cultured human saphenous vein endothelial cells (EC). Although the NK activity of non-adherent PBMC recovered from flasks coated only with fibronectin increased in the presence of supplemental IL-2, the activity of cells recovered from flasks coated with EC decreased when IL-2 was added to the medium. The percentage of NK (CD16+) cells among the EC-non-adherent PBMC was reduced relative to that of the input cells when IL-2 was added. The percentage of CD16+ cells in the EC-adherent PBMC, as well as their NK activity, increased in the presence of added IL-2. Although EC had no effect on the lysis of labeled K-562 cells by unstimulated PBMC in cold target competition experiments, they were able to compete in cytolytic assays using PBMC previously activated by exposure to IL-2 for 1 h. EC were not lysed by these briefly activated PBMC in 3-h cytotoxicity assays but were lysed by these effectors in 18-h assays and in 3-h assays using PBMC pre-activated by more prolonged culture with IL-2. The ability of IL-2 to induce NK cell adhesion to EC was not blocked by a mixture of neutralizing antisera raised against rTNF-alpha, rIL-1 alpha, and rIL-1 beta, factors known to promote leukocyte adhesion to EC. We conclude that IL-2 rapidly induces NK cell adhesion to EC and propose that this effect accounts for the disappearance of circulating NK cells after the infusion of high doses of IL-2. In addition, these results suggest that NK cells activated by IL-2 in vivo may injure the endothelium and contribute to the extravasation of plasma and the retention of fluid characteristic of IL-2 treatment.     

Phenotypic characterization of lymphokine-activated killer cells from human lymph node lymphocytes

We previously reported that lymphokine-activated killer (LAK) activity can be generated in human lymph node lymphocytes (LNL) at the same level as that in peripheral blood lymphocytes (PBL), despite the absence of active natural killer (NK) cells. In the present study, we investigated the surface phenotype of LNL-LAK cells by fractionation of lymphocytes, using a panning method. LNL isolated from lung cancer patients were cultured in the presence of recombinant interleukin 2 for 8 days and separated into T cells and non-T cells according to the expression of CD3 antigen. LAK effectors were enriched in the CD3- non-T cells. However, the CD3+ cells also mediated a low but substantial level of LAK activity, which was attributed to a CD8+ T-cell subset. Further investigation of the CD3- cells revealed that most of the CD3- effector cells expressed neither B-cell (CD20) nor NK-cell (CD16) markers. Precursors of this CD3-CD20-CD16- (null) population appeared to be also CD3-, CD20-, and CD16-. From these results, we would stress the significant contribution of CD3-CD20-CD16- null cells to the LAK phenomenon, which has not been focused on in PBL.     

Cutting edge: engagement of CD160 by its HLA-C physiological ligand triggers a unique cytokine profile secretion in the cytotoxic peripheral blood NK cell subset

CD160 is an Ig-like activating NK cell receptor expressed on the majority of circulating NK cells. This population corresponds to the nonproliferating, highly cytolytic, CD56dimCD16+ subset. CD160 engagement by HLA-C molecules mediates cytotoxic function. In this study, we report that upon specific activation by the physiological ligand HLA-C, or Ab cross-linking, CD160+ peripheral blood NK cells produce IFN-gamma, TNF-alpha, and IL-6. This unique CD160-mediated cytokine production differs from the one observed after CD16 engagement whose expression is also restricted to the CD56dim cytotoxic NK cell subset. As already reported for the CD160-mediated cytotoxic effector function, CD160-mediated cytokine production by peripheral blood-NK cells is negatively controlled by the killer Ig-like receptor CD158b. Thus, the CD160 receptor represents a unique triggering surface molecule expressed by cytotoxic NK cells that participates in the inflammatory response and determines the type of subsequent specific immunity.     

Phenotypic and functional differences in NK and LAK cells in the peripheral blood of sooty mangabeys and rhesus macaques

Greater than 75% of the sooty mangabey monkeys at the Yerkes Regional Primate Research Center are naturally infected with SIV without any apparent clinical symptomology. On the other hand, experimental infection of rhesus macaques with SIV results in a clinical syndrome similar to human AIDS. These differences with regard to SIV infection prompted us to examine the natural immunosurveillance system of peripheral blood mononuclear cells (PBMC) from SIV-infected and uninfected monkeys of these two species. Phenotypic and functional studies of precursor and effector NK and LAK cells in the PBMC from these two species were carried out using monoclonal reagents, flow microfluorometry (FMF), and the standard in vitro 51Cr release assay against prototype K562 (NK sensitive) and RAJI (NK resistant, LAK susceptible) target cell lines. Data indicate that both NK and LAK cell activities in the PBMC of sooty mangabeys were significantly (P less than 0.01) greater than those in rhesus macaques. The predominant NK effector cells and LAK cell precursors were shown to be Leu 19-CD8+ in the PBMC of sooty mangabeys and Leu19+ CD8- in the PBMC of rhesus macaques as determined by panning depletion techniques and FMF analysis. On the other hand, the predominant LAK effector cells were found to be dual marked Leu 19+ CD8+ in rhesus macaques and Leu 19- CD8+ in sooty mangabeys. These qualitative and quantitative differences were not due to SIV infection of these two species since PBMC from both SIV-seropositive and virus-positive and SIV-sero-negative and virus-negative monkeys gave similar results. Moreover, of importance is the finding that the functional NK and LAK precursor cells are CD8+ and CD8- in sooty mangabeys and rhesus macaques, respectively. These data may have implications for the natural SIV/SMM virus-positive asymptomatic state of sooty mangabeys and may provide useful tools for tracing the ontogeny and lineage derivation of NK and LAK cells.     

Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenotypic characteristics and tendency to apoptosis of peripheral blood mononuclear cells from HIV+ long term non progressors

The aim of this study was to analyze (i) phenotype, (ii) in vitro spontaneous and induced apoptosis, (iii) glutathione (GSH) intracellular content and (iv) inhibitors of apoptosis of potential therapeutical use in peripheral blood mononuclear cells (PBMC) from HIV+ long term non progressors (LTNP), in comparison with progressors (HIV+P) and seronegative controls (HIV-). Three groups of subjects were studied: 15 HIV+P (patients losing >150 CD4+/year), 9 LTNP (subjects infected by HIV for at least 7 years without clinical and immunological signs of progression, with a mean of 898 CD4+/microL) and 18 HIV-. All subjects were living in a large community for former drug addicts, and were matched for age and sex. We used flow cytometry for analyzing PBMC phenotype and apoptosis; high performance liquid chromatography for measuring intracellular GSH content. PBMC phenotype of LTNP shared characteristics with those of both HIV- and HIV+P. Indeed, LTNP showed a normal number CD4+ cells (an inclusion criteria), but significantly increased numbers of CD8+ lymphocytes, activated T cells, CD19+, CD5+ B lymphocytes and CD57+ cells, as well as a decrease in CD19+, CD5- B lymphocytes and CD16+ cells. In LTNP, spontaneous apoptosis was similar to that of HIV- and significantly lower than that of HIV+P. Adding interleukin-2 (IL-2) or nicotinamide (NAM) significantly decreased spontaneous apoptosis in LTNP and HIV+P. Pokeweed mitogen-induced apoptosis was also similar in LTNP and HIV-, but significantly lower than that of HIV+P. In HIV+P, but also in LTNP, spontaneous apoptosis was inversely correlated to the absolute number and percentage of CD4+ cells and directly correlated to the number and percentage of activated T cells present in peripheral blood. GSH intracellular content was greatly decreased in PBMC from HIV+P and slightly, but significantly, reduced in LTNP. Adding 2-deoxy-D-ribose, an agent provoking apoptosis through GSH depletion, to quiescent PBMC resulted in similar levels of massive cell death in the three groups. This phenomenon was equally prevented in the three groups by N-acetyl-cysteine but not by IL-2. A complex immunological situation seems to occur in LTNP. Indeed, PBMC from LTNP are characterized by a normal in vitro tendency to undergo apoptosis despite the presence of a strong activation of their immune system, unexpectedly similar to that of HIV+P. Our data suggest that NAM and IL-2 are possible candidates for reducing spontaneous apoptosis in HIV infection.