Positively selected Leu-11a (CD16+) cells require the presence of accessory cells or factors for the lysis of herpes simplex virus-infected fibroblasts but not herpes simplex virus-infected Raji

Previous studies from our laboratory indicated that human NK activity against HSV-infected fibroblasts (HSV-Fs) but not K562 targets was sensitive to treatment with anti-HLA-DR plus C. In the current study, we have selected Leu-11a+ (CD-16) cells by fluorescence activated cell sorting and found that although Leu-11a enriched populations lysed K562 targets in 14-h 51Cr-release assays, they were unable to kill HSV-Fs targets unless a Leu-11a-depleted population was added back to the effectors or unless known activators of NK cells (IFN-alpha or IL-2) were added to the assays. In contrast, Leu-11a-enriched populations were able to mediate ADCC against HSV-Fs in the presence of sera from HSV-seropositive individuals without the requirement for accessory cells. We have begun preliminary characterization of the accessory cells which allow lysis of HSV-Fs by NK cells: they are HLA-DR+ cells which enrich in the light density fractions of Metrizamide density gradients. They need be present in very small numbers for lysis to take place and are not MHC restricted in that heterologous add-backs between anti-HLA-DR plus C and anti-Leu-11b plus C-treated populations are capable of target cell lysis at levels similar to those achieved with the autologous add-backs. Further, the levels of lysis in heterologous add-back experiments reflected the lytic potential of the effector rather than the accessory cell donor. Finally, although the requirement for accessory cells for NK lysis has been demonstrated for fibroblasts infected with HSV-1, CMV, and VZV, lysis of HSV-infected Raji lymphoblastoid cells is relatively accessory-cell independent, indicating that the requirement for accessory cells for lysis by NK cells is not a property of all herpesvirus-infected targets.     

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.     

Inability of interferon to protect virus-infected cells against lysis by natural killer (NK) cells correlates with NK cell-mediated antiviral effects in vivo

Murine cytomegalovirus (MCMV) is a natural killer (NK) cell-sensitive virus, whereas lymphocytic choriomeningitis virus (LCMV) is an NK cell-resistant virus. Selective depletion of NK cell activity by injection of mice with anti-asialo GM1 antibody enhanced synthesis of MCMV but not that of LCMV when mice were simultaneously infected with the two viruses. This suggests that the NK cell-mediated antiviral effects may depend on target cell susceptibility to NK cell-mediated lysis rather than the ability of a virus to induce a specialized antiviral NK cell. In support of this concept, activated NK cells isolated from either MCMV- or LCMV-infected mice had similar patterns of killing against all targets tested. Mouse embryonic fibroblasts (MEF) infected with MCMV were less sensitive to lysis by activated NK cells than either uninfected or LCMV-infected MEF. However, when MEF were pretreated with IFN, activated NK cell-mediated lysis against MCMV-infected MEF was undiminished and was much higher (up to fourfold) than that against uninfected MEF, whose sensitivity to lysis was almost totally abolished by IFN pretreatment. LCMV-infected MEF were also protected by IFN against activated NK cell-mediated lysis. During infection, the virus-induced IFN may protect uninfected and LCMV-infected cells from IFN-activated, NK cell-mediated lysis, but MCMV-infected cells may remain sensitive to lysis. This could explain how NK cells play a role in resistance to MCMV but not LCMV.     

Decay-accelerating factor expression on either effector or target cells inhibits cytotoxicity by human natural killer cells.

Previous studies have shown that freshly isolated CD16+ NK cells are deficient in the expression of decay-accelerating factor (DAF), or CD55, a membrane regulator of C3 activation. In this study we investigated the significance, for NK cell-mediated lysis, of DAF expression on the target and effector cells. The effect of DAF expression on the susceptibility of NK cell targets was investigated by several means: first, DAF- cell lines were cloned from K562; second, the cloned DAF- cells were reconstituted with exogenous purified DAF; and third, anti-DAF F(ab')2 was used to block DAF function on the DAF+ K562 cells. Consistently, the presence of DAF in the target cell membrane, either naturally occurring or experimentally incorporated, afforded the target cell protection against lysis, and this protection could be blocked with anti-DAF. Similarly, targets for antibody-dependent cell-mediated cytotoxicity with exogenous DAF incorporated in their plasma membrane became less sensitive to antibody-dependent cell-mediated cytotoxicity by NK cells compared with the same target cells without incorporated DAF in their membranes. DAF incorporated in the plasma membranes of the effector NK cells made the NK cells less effective at killing K562 targets. The known function of DAF is to regulate C3 activation, and we were able to demonstrate that the isolated NK cell is capable of releasing C3. It is also possible that the participation of DAF in NK cell function represents a new, noncomplement-dependent function for DAF.     

In vitro infection of natural killer cells with different human immunodeficiency virus type 1 isolates.

Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-Fc gamma RIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considered.     

Natural killer cells in rhesus monkeys: properties of effector cells which lyse Raji targets

Spontaneously occurring natural killer cell activity of rhesus monkey peripheral blood mononuclear cells was assayed against five human cell lines, three of which were Epstein-Barr virus (EBV) positive, including the human B cell line Raji. The lytic activity to Raji cells was high, significantly higher than to any other cell line tested. Raji cells are normally insensitive to spontaneous lysis by human NK cells, and the contrasting vigor of the rhesus monkey cytolytic activity to Raji prompted us to investigate the properties of this effector cell. We found the effector cell-mediating lysis of Raji to be nonadherent and phagocytic with lytic activity slightly enhanced in the E-rosette-forming cell (ERFC+) fraction and decreased in the ERFC- fraction. Further isolation of FcIgG receptor-positive and FcIgG receptor-negative subsets by rosetting resulted in significant enrichment of NK activity to Raji in the positive fraction and a loss of activity in the negative fraction. Depletion studies with various monoclonal antibodies (mAb's) confirmed that nearly all lytic activity was contained in the CD16+ (Leu 11b+) population, while subsets of effector cells expressed CD2 (9.6) and CD8 (OKT8). Depletion of CD4 (OKT4)-, HLADR (OKIa)-, or LFA1 (MAC-1)-positive populations failed to reduce NK activity. We compared the phenotypic properties of alloimmune effector cells exhibiting specificity for allogeneic donor targets with those exhibiting lysis of Raji targets. Results indicated that allospecific cytotoxic T lymphocytes expressed a CD16-, CD2+ phenotype, a pattern distinct from that of the effector cell population recognizing Raji targets. The presence of CD2 mAb's in the culture had no effect on NK lytic activity. In contrast, mAbs CD8 and Leu 11b were inhibitory. This would suggest a functional role for CD8 and FcIgG molecules in the lysis of Raji cells by rhesus effectors. In summary, these studies describe a distinct population of effector cells in the blood of rhesus monkeys which exhibit spontaneous lytic activity to Raji cells and exhibit the properties of NK cells.     

Differential replication of human immunodeficiency virus type 1 in CD8- and CD8+ subsets of natural killer cells: relationship to cytokine production pattern.

CD8+ and CD8- subsets of peripheral blood natural killer (NK) cells were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1) and for the ability to produce various types of interferon (IFN) and tumor necrosis factor (TNF). HIV-1 was preferentially grown in CD8+ NK cells. The ability of CD8- NK cells to suppress HIV-1 replication was related to their ability to produce alpha IFN (IFN-alpha) upon viral induction. Induction with interleukin-2 resulted in IFN-gamma production in both subsets of NK cells. In the CD8+ subset, IFN-gamma and HIV-1 mutually enhanced the production of TNF alpha, leading to hyperactivation of viral replication, whereas in CD8- NK cells IFN-gamma primed HIV-induced IFN-alpha production. The dichotomous effects of IFN-gamma on HIV-1 replication were dependent on the IFN-alpha-producing ability of the cellular targets. These findings can explain the selective depletion of the CD16+ CD8+ subset that begins early in the in vivo HIV-1 infection.     

Natural killer cells discriminate between high mannose- and complex-type asparagine-linked oligosaccharides

Chinese hamster ovary cell lines with different types of N-linked oligosaccharides were tested as targets for control and lymphokine treated natural killer (NK) cells. The targets tested were parent cells, Lec1 mutants and Lec4 mutants. Due to an apparent defect in GlcNAc transferase V, Lec4 cells produce complex-type N-linked oligosaccharides devoid of GlcNAc beta(1-6) linked branches. Lec1 cells form only high mannose-type N-linked oligosaccharides because they lack GlcNAc transferase I activity. Lec1 cells are very sensitive to lysis by beta-interferon treated human NK cells, but both parent and Lec4 cells are resistant to NK lysis. The ability to discriminate between parent and Lec1 targets was demonstrated with untreated control effectors as well as those which were pretreated with either beta-interferon, gamma-interferon or interleukin-2. Both control and lymphokine-boosted NK cells exhibit much greater lytic activity against targets having only high mannose-type N-linked oligosaccharides. Five oligosaccharide structures resembling those found on N-linked glycoproteins were tested for their ability to block NK lysis of Lec1 targets. Only the high mannose-type glycopeptide from 7S soybean glycoprotein was inhibitory in the mu molar range. At the same concentration, none of the complex-type oligosaccharides had any effect on lytic activity. The results suggest that a high mannose-type N-linked oligosaccharides is recognized at some step in NK cell-mediated lysis.     

Targeting of human dendritic cells by autologous NK cells

NK cells have the capacity to spontaneously kill tumor cell lines, in particular cell lines of hemopoietic origin. In contrast, they do not generally kill nontransformed autologous cells. However, here we demonstrate that short-term activated polyclonal human NK cells, as well as human NK cell lines, efficiently lyse autologous dendritic cells (DC) derived from peripheral blood monocytes as well as Langerhans-like cells derived from CD34+ stem cells isolated from umbilical cord blood. Lysis of autologous DC by short-term activated NK cells and NK cell lines was dependent on granule exocytosis, since total abrogation of lysis was observed in the presence of EGTA. Induction of DC maturation by LPS, monocyte conditioned media (MCM), or stimulation through CD40 ligand (CD40L) rendered the DC less susceptible to lysis by NK cells. Infection of DC with influenza virus was likewise associated with a reduced susceptibility to lysis by NK cells. Thus, susceptibility to lysis by autologous NK cells is a particular property of immature DC. The present results are discussed in relation to the ability of DC to interact with NK cells and to the ability of NK cells to regulate development of specific immunity.     

Bone marrow cell modulation and inhibition of myelopoiesis by large granular lymphocytes and natural killer cells

Non-adherent Percoll-separated large granular lymphocytes (LGLs) fractionated by fluorescence-activated cell sorter into CD16+ CD4- natural killer (NK) cells and CD16- CD4+ T cells, were co-cultured with bone marrow (BM) cells previously depleted of adherent T and/or NK cells by immunoadsorption (panning) and plated in a clonogenic assay to assess myeloid colony formation (CFU-gm growth). LGLs, NK cells and LGL T cells [low buoyant density (LBD) T cells] each significantly reduced colony-stimulating factor (CSF)-dependent CFU-gm growth to 70% of control values (p less than 0.05). Non-LGL T cells [high buoyant density (HBD) T cells] did not affect this growth. Incubation of the effector cells with human recombinant interleukin 2 prior to co-culturing did not alter these findings. The supernatants obtained from LGLs, NK cells and LBD T cells co-cultured with BM cells also inhibited CFU-gm growth to 70% of the control, whereas supernatants from effector cells which were not co-cultured with BM had no such effect. These supernatants from the LGL:BM co-cultured cells possessed NK cytotoxic factor (NKCF), but lacked alpha and gamma interferons, tissue necrosis factor-alpha, and prostaglandin E2. These results suggest that BM cells stimulate LGLs to produce NKCF, and that LGLs, CD16+ NK cells, and CD4+ CD16- LBD T cells activated by contact with BM cells inhibit CFU-gm growth.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.     

IFN increases class I MHC antigen expression on adenovirus-infected human cells without inducing resistance to natural killer cell killing.

It has been previously shown that unstimulated NK cells cannot preferentially lyse adenovirus serotypes 2 and 5-infected human cells. In this study, the ability of IFN to promote the selective NK cell-mediated lysis of adenovirus-infected human cells was determined. The relationship between target cell susceptibility to NK cell-mediated killing and class I Ag expression was also analyzed through the use of adenovirus serotype 2 and 5 mutants that do not make the adenovirus early region 3 19-kDa class I binding protein. IFN induced the selective lysis of adenovirus serotype 2 and 5-infected human cells by activating NK cells (IFN-alpha) and protecting uninfected, but not adenovirus-infected cells, from NK cell-mediated lysis (IFN-gamma). IFN-gamma increased the expression of class I Ag on the surface of cells infected with the adenovirus early region 3 deletion mutants, dl327 or dl801, to a level equal to or greater than that expressed on uninfected cells. Despite the increased expression of class I Ag, IFN-gamma could not protect these adenovirus-infected cells from NK cell-mediated lysis. Thus, dl327 or dl801 infection prevented IFN-gamma's induction of cytolytic resistance to NK cell-mediated killing but left IFN-gamma's induction of class I Ag intact. Surface class I Ag levels were substantially higher on IFN-gamma-treated, dl327-, and dl801-infected cells in comparison to cells infected with wild type adenovirus serotype 5. Again, higher target cell levels of class I Ag did not correlate with increased resistance to NK cell-mediated lysis because there was equivalent NK cell-mediated killing of IFN-gamma-treated adenovirus serotype 5-, dl327-, or dl801-infected cells. Thus, IFN-gamma only protects uninfected cells from NK cell-mediated killing, irrespective of target class I Ag levels, and thereby concentrates NK lytic activity on just adenovirus-infected cells. These data demonstrate that IFN-gamma's ability to protect target cells from NK cell-mediated cytolysis is unrelated to IFN-gamma's induction of surface class I MHC Ag.     

A dual-targeting triplebody mediates preferential redirected lysis of antigen double-positive over single-positive leukemic cells

The single-chain triplebody HLA-ds16-hu19 consists of three single-chain Fv (scFv) antibody fragments connected in a single polypeptide chain. This protein with dual-targeting capacity mediated preferential lysis of antigen double-positive (dp) over single-positive (sp) leukemic cells by recruitment of natural killer (NK) cells as effectors. The two distal scFv modules were specific for the histocompatibility protein HLA-DR and the lymphoid antigen CD19, the central one for the Fc gamma receptor CD16. In antibody-dependent cellular cytotoxicity (ADCC) experiments with a mixture of leukemic target cells comprising both HLA-DR sp HuT-78 or Kasumi-1 cells and (HLA-DR plus CD19) dp SEM cells, the triplebody mediated preferential lysis of the dp cells even when the sp cells were present in ≤20-fold numerical excess. The triplebody promoted equal lysis of SEM cells at 2.5-fold and 19.5-fold lower concentrations than the parental antibodies specific for HLA-DR and CD19, respectively. Finally, the triplebody also eliminated primary leukemic cells at lower concentrations than an equimolar mixture of bispecific single-chain Fv fragments (bsscFvs) separately addressing each target antigen (hu19-ds16 and HLA-ds16). The increased selectivity of targeting and the preferential lysis of dp over sp cells achieved by dual-targeting open attractive new perspectives for the use of dual-targeting agents in cancer therapy.     

Phenotypic and cytolytic activity of Macaca nemestrina natural killer cells isolated from blood and expanded in vitro

Natural killer (NK) cells from nonhuman primates have not been completely characterized, and methods for expanding nonhuman primates NK cells in vitro have been described only in rhesus species. The purpose of this report was to characterize NK cells in pigtail macaques (Macaca nemestrina), a species that is frequently used in studies of transplantation biology/immunology, virology, vaccine development, and reproductive biology. NK cells from Macaca nemestrina peripheral blood were best defined by the expression of CD16 and CD8alpha, and the absence of CD3. Subsets of these cells express CD56, NKp30, and NKp46. An enhanced ability to kill K562 cells was not present in fluorescence activated cell sorted (FACS)-purified CD16-/CD3+ and CD16-/CD56+ cells isolated from fresh peripheral blood. However, FACS-purified CD16+/CD3- and CD16+/CD56- cells were highly efficient killers of K562 cells. Macaca nemestrina NK cells can be expanded by in vitro culturing of FACS-purified CD16+/CD2-/CD3-/CD56- cells, or from peripheral blood cells depleted of cells expressing CD3, CD4, and HLA-DR. Cells in these cultures expand 70-fold after 21 days of culturing. After culturing, these cells express high levels of natural cytotoxicity receptors (NCRs) NKp30 and NKp46. NK cell populations obtained from FACS-purified CD16+/CD3-, CD16+/CD56- cells and CD3/CD4/HLA-DR-depleted cells were highly efficient killers of K562 cells. These data suggest that a population of highly enriched cytolytic NK cells can be obtained from purified CD16+/CD3- and CD16+/CD56- cells obtained from peripheral blood, as well as from cells that have been cultured and expanded from peripheral blood that is depleted of CD3/CD4/HLA-DR-expressing cells.     

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP

NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.     

Induction of human NK cell-mediated cytotoxicity by CD40 triggering on antigen presenting cells

Engagement of CD40 on antigen presenting cells (APC) is central to the initiation of cell-mediated immune response. Here, we investigated the ability of CD40 ligation on APC to induce NK cell-mediated cytotoxicity in the human system and the mechanism(s) underlying this process. We showed that APC (consisting in adherent peripheral blood mononuclear cells) (PBMC), pre-stimulated with anti-CD40 monoclonal antibodies and co-cultured with autologous non-adherent PBMC for 5-9 days, induced CD3-/CD56+ NK cell-mediated cytotoxicity as well as CD3+/CD56+ T cell-mediated unrestricted cytotoxic activity. The generation of NK cell-mediated cytotoxicity was independent on cell-to-cell contact between CD40-triggered APC and NK cells. Moreover, we found that IL-12 did not play a role in NK cells induction by anti-CD40 priming, while IL-2 and IL-15 did play a role. Our results provide an insight into the mechanism by which NK cells are activated in peripheral blood and useful informations for therapeutic application of anti-CD40 antibodies.     

HLA-restricted lysis of herpes simplex virus-infected monocytes and macrophages mediated by CD4+ and CD8+ T lymphocytes

Freshly isolated human peripheral blood monocytes and in vitro monocyte-derived macrophages were infected with HSV type 1 and used as target cells in a cell-mediated cytotoxicity assay. PBMC from both HSV-immune and non-immune donors were stimulated in vitro for 5 days with UV-inactivated HSV Ag and used as effector cells. Effectors from HSV-immune donors mediated virus-specific lysis of both monocyte and macrophage targets, whereas effectors from non-immune donors failed to mediate target cell lysis. Mean virus-specific lysis of autologous monocytes was (8.5 +/- (+/- 2.0)%) compared to a threefold greater virus-specific lysis of autologous macrophages (24.7 (+/- 4.3)%). More than 70% of this lysis was mediated by CD16- T lymphocytes. Further analysis demonstrated that the majority of the lysis against autologous and allogeneic targets was HLA-DR-restricted and mediated by CD4+ CTL. However, CD8+ CTL also contributed to the lysis of autologous targets as well as allogeneic targets having a common HLA-A and/or -B determinant. The HLA-restricted cytotoxicity was virus-specific as HSV-infected, but not CMV-infected, cells were lysed. CTL-mediated lysis of HSV-infected monocytes and macrophages may be of significance in the anti-viral and immunoregulatory host response.     

Generation of lymphokine-activated killer activity in T cells. Possible regulatory circuits.

CD4+ and CD8+ T cells do not develop significant lymphokine-activated killer (LAK) activity when PBL are cultured with IL-2 or even when they are activated with a T cell stimulus such as OKT3 mAb. The possibility that a T cell regulatory mechanism prevents the development of LAK activity by CD4+ or CD8+ cells in OKT3 mAb and IL-2 cultures was tested by depleting CD8+ or CD4+ cells from PBL before stimulation with OKT3 and IL-2. Under these conditions, the remaining CD4+ and CD8+ cells were able to generate non-MHC-restricted lysis of NK-resistant tumor targets. Our data suggested that a regulatory signal was present in the culture to prevent the development of lytic function by T cells. T cells removed from the PBL cultures were, upon culture with IL-2, able to generate high LAK activity, suggesting that inhibition of the CD4+ or CD8+ T cell-mediated LAK activity was an active ongoing process, which blocked the lysis at the level of the activated cell and not the precursor cell. Mixing experiments demonstrated that the CD4+ or the CD8+ cells isolated from the PBL cultures were able to inhibit the development of lytic function in the CD4-depleted and CD8-depleted cultures. Transforming growth factor-beta (TGF-beta) has been shown to block LAK activity of NK cells in IL-2-stimulated cultures. When TGF-beta was added to CD4(+)- or CD8(+)-depleted cultures, it also inhibited LAK activity of T cells in a dose-dependent fashion, without interfering with T cell growth. Lytic activity returned to activated levels when TGF-beta was removed from the culture medium, thereby demonstrating the reversibility of TGF-beta inhibition.     

A dual-targeting triplebody mediates preferential redirected lysis of antigen double-positive over single-positive leukemic cells

The single-chain triplebody HLA-ds16-hu19 consists of three single-chain Fv (scFv) antibody fragments connected in a single polypeptide chain. This protein with dual-targeting capacity mediated preferential lysis of antigen double-positive (dp) over single-positive (sp) leukemic cells by recruitment of natural killer (NK) cells as effectors. The two distal scFv modules were specific for the histocompatibility protein HLA-DR and the lymphoid antigen CD19, the central one for the Fc gamma receptor CD16. In antibody-dependent cellular cytotoxicity (ADCC) experiments with a mixture of leukemic target cells comprising both HLA-DR sp HuT-78 or Kasumi-1 cells and (HLA-DR plus CD19) dp SEM cells, the triplebody mediated preferential lysis of the dp cells even when the sp cells were present in ≤20-fold numerical excess. The triplebody promoted equal lysis of SEM cells at 2.5-fold and 19.5-fold lower concentrations than the parental antibodies specific for HLA-DR and CD19, respectively. Finally, the triplebody also eliminated primary leukemic cells at lower concentrations than an equimolar mixture of bispecific single-chain Fv fragments (bsscFvs) separately addressing each target antigen (hu19-ds16 and HLA-ds16). The increased selectivity of targeting and the preferential lysis of dp over sp cells achieved by dual-targeting open attractive new perspectives for the use of dual-targeting agents in cancer therapy.     

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.