Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

Regulation of APO-2 ligand/trail expression in NK cells-involvement in NK cell-mediated cytotoxicity.

Apo-2L is a new member of the tumour necrosis factor (TNF) family shown to induce apoptosis in a number of tumour cell lines. Apo-2L mRNA is expressed by numerous human tissues. Here we report that Apo-2L is expressed and utilized by human Natural Killer (NK) cells. NK cells were shown to express surface Apo-2L in response to interleukin 2 (IL-2) activation, and this response was restricted to the CD3(-)population of the NK cells. Apo-2L mRNA and intracellular Apo-2L were present in both CD3(-)and CD3(+)NK cells; however, increased expression in response to IL-2 was only observed in CD3(-)CD56(+)cells. Also, IL-2-activated NK cells were shown to utilize membrane-bound Apo-2L in mediating lysis of Jurkat cells. Furthermore, Apo-2L-induced apoptosis of Jurkat cells was more rapid than FasL-induced apoptosis, indicating an important and distinct role for Apo-2L in apoptotic cell destruction. In conclusion, we report that NK cells express Apo-2L and that IL-2 activated CD3(-)NK cells utilize the Apo-2L pathway in mediating target cell lysis.     

Nucleosome-dependent escape of tumor cells from natural-killer-mediated lysis: nucleosomes are taken up by target cells and act at a postconjugation level

 Our previous data suggested that chromatin fragments released from dead cells into the extracellular medium could be involved in the impairment of natural-killer (NK)-mediated cytotoxicity reported in cancer patients. In the present study, an inhibition of the NK-mediated lysis was obtained in vitro by nucleosome addition to different tumor target cells, independently of their sensitivity to NK-mediated lysis. We observed a rapid endocytosis and degradation of nucleosomes by K562 tumor target cells and (although to a much lesser extent) a binding to a subpopulation of lymphocytes. Nucleosomes impaired neither the conjugation step nor the expression of adhesion molecules at the effector (CD11a, CD18, CD2) or target (CD54, CD58) cell surface. On the contrary, flow-cytometry analysis of the conjugation suggested that nucleosomes might stabilize the conjugates. Investigations of the killing process showed that nucleosomes decreased the NK cytotoxic potential without modifying Ca²⁺-dependent lethal-hit-delivery kinetics. The cytotoxic potential was not restored by increasing the available magnesium and calcium concentrations in the extracellular medium. Taken together, the results suggest that the inhibition of NK-mediated lysis by nucleosomes may result from alterations of the NK mechanism at the postconjugation level and after lethal-hit delivery. Hence, the inhibition could involve a delay in the recycling of effector cells, or a resistance of tumor target cells to NK cells. Received: 7 October 1996 / Accepted: 12 November 1996     

Sensitization of resting T cells to autologous natural-killer-cell-mediated lysis by phytohemagglutinin

Natural killer (NK) cells are non-T, non-B cell lymphocytes that lyse a variety of tumor and virus-infected cells. In this study, we demonstrated that phytohemagglutinin (PHA) rendered resistant autologous T cells extremely sensitive to natural-killer(NK)-cell-mediated lysis. The sensitization was very rapid and concentration-dependent (0.01–1 μg/ml); 62% and 95% of autologous T cells were lysed by interleukin-2-activated NK cells 5 min and 18 h respectively after treatment with PHA (1 μg/ml). The maximal decrease in the level of MHC class I molecules observed on T cells was 22%. Induction of susceptibility to NK-mediated lysis was correlated with the expression of activation markers on T cells treated for relatively long intervals (more than 18 h) with high concentrations of PHA (more than 0.1 μg/ml). Sensitization of T cells required RNA and protein synthesis, although DNA synthesis was not essential. We propose that this unique system is suitable for studying the mechanisms involved in recognition and killing of target cells by NK cells. Received: 11 February 1999 / Accepted: 28 July 1999     

The role of monocytes and natural killer cells in mediating antibody-dependent lysis of colorectal tumour cells

Monocytes and natural killer (NK) cells are known to be important effector cell populations in mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Purified monocyte and NK effector cell populations, from normal and colorectal cancer (CRC) patients, together with a number of murine (17-1A and 323/A3) and their chimaeric (c17-1A) or humanised (3622W94) equivalents, and chimaeric (c) SF25 were compared for their ability to mediate ADCC of colorectal tumour cells. The chimaeric and humanised antibodies were significantly better at mediating tumour lysis than their murine equivalents with all-effector populations. When effector cells from CRC patients were used the cSF25 antibody was significantly better than 3622W94 (P < 0.02) which, in turn, was significantly better than c17-1A (P < 0.03). Depletion of NK cells produced a decrease in specific tumour lysis with all antibodies. In addition a higher rate of NK cell death was observed in CRC patients during the assay than in normal controls. The chimaeric and humanised antibodies stained a similar percentage of the HT-29 target cells (>80%), but 3622W94 bound to significantly more cells from primary tumour biopsies than cSF-25 (P = 0.001). Together, the results suggest that NK cells are the most important effector cell type mediating ADCC in vitro, that there is some impairment of NK function in CRC patients, and that cSF25 is the most potent antibody. For use in vivo the anti-Ep-CAM antibody 3622W94 would appear to be the most suitable reagent for further study. Received: 3 June 1999 / Accepted: 22 July 1999     

CD1d1 displayed on cell size beads identifies and enriches an NK cell population negatively regulated by CD1d1

NK cells destroy microbe-infected cells while sparing healthy cells, and are controlled, in part, by inhibitory receptors specific for class I Ag-presenting molecules. CD1d1, a beta(2)-microglobulin-associated class I-like molecule, binds glycolipids and stimulates NKT cells. We previously demonstrated that target cell lysis by IL-2-activated mouse NK cells is inhibited by target cell expression of CD1d1, suggesting that IL-2-activated NK cells may express a CD1d1-specific inhibitory receptor. We now report that a significant subset of mouse IL-2-activated NK cells specifically binds cell size beads displaying either naturally expressed or recombinant CD1d1. In contrast, although tetramers of soluble recombinant CD1d1 loaded with alpha-galactosylceramide identify NKT cells, binding of this reagent to resting or IL-2-activated NK cells was undetectable, even with activated NK cells sorted with CD1d1 beads. Cytotoxicity by the CD1d1 bead-separated NK subset was strongly inhibited by CD1d1, compared with the NK cell subset not bound to CD1d1 beads. An Ab that blocks NKT cell recognition of CD1d1 also reverses CD1d1 inhibition of NK lysis, suggesting that TCRs of NKT cells and NK inhibitory receptor(s) may interact with a similar site on CD1d1. These results provide direct evidence for a physical interaction of NK cells with CD1d1, mediated by a functional, CD1d1-specific low-affinity inhibitory NK receptor. Display of ligands on cell size beads to maximize multivalent interaction may offer an alternative approach to examine NK cell receptor-ligand interactions, particularly those of lower expression and/or lower affinity/avidity that may go undetected using tetrameric reagents.     

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8⁺ T or CD4⁺ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18. Received: 7 May 1998 / Accepted: 27 May 1999     

Adoptive immunotherapy of cancer with pharmacologically activated lymph node lymphocytes: a pilot clinical trial

 Adoptive immunotherapy (AIT) of cancer with T lymphocytes may be limited by the need to activate tumor antigen-sensitized cells in vitro. In murine models, we have shown that AIT with tumor-sensitized T cells that have been pharmacologically activated with bryostatin 1 and ionomycin plus interleukin-2 can induce tumor regression. A Phase I clinical trial was carried out to assess the feasibility and toxicity associated with using tumor- or vaccine-draining lymph node cells, activated pharmacologically and expanded in culture with low-dose interleukin-2 and infused intravenously, followed by IL-2 infusion. Nine patients were entered into the trial, and six were treated as planned. Average expansion of cell numbers over 13 to 27 days in culture was 118-fold. No patient's cells reached the target cell number (2.5 × 10¹⁰). Infusion of these cells did not result in any unexpected toxicities. The toxicities observed were related to IL-2 infusion, and conformed to the expected range of side-effects. Based on these Phase I results, additional trials, with tumor antigen vaccine-sensitized DLN and technical modifications of the culture technique, are planned. Received: 18 January 2001 / Accepted: 26 April 2001     

Long-term treatment with low doses of interleukin-2 and interferon-α: immunological effects in advanced renal cell cancer

We aimed to determine the immunological effects of low doses of recombinant interleukin-2 (rIL-2) and recombinant interferon-α (rIFN-α) in patients bearing advanced renal cell carcinoma. Methods: Twenty-seven patients received therapeutic cycles consisting of subcutaneous rIL-2 for 5 days per week and intramuscular rIFN-α twice weekly, for 4 consecutive weeks. The cycle was repeated indefinitely at regular 4-month intervals, for all patients. rIL-2 (1 × 10⁶ IU/m²) was administered every 12 h on days 1 and 2 and once a day on days 3–5 of each week; rIFN-α (1.8 × 10⁶ IU/m²) was given on days 3 and 5. In the enrolled patients, total and differential white blood cell counts, phenotypic analysis of some lymphocyte subsets, and soluble IL-2 receptor (sIL-2R), were investigated before and after each of the first six cycles of therapy (about 24 months of follow-up). Results: The cycles of immunotherapy induced a significant increase of total lymphocytes (37%, P < 0.001), eosinophils (222%, P < 0.001), CD25+ cells (27%, P=0.004), sIL-2R (174%, P < 0.001) and natural killer (NK) cells (CD3-CD56+) (61%, P < 0.001); the subset that expresses CD56 with high density (CD56+ bright) expanded more (233%, P < 0.001) than the subset expressing the same marker with low density (CD56+ dimmer) (15%, P=0.043). Unlike the previous subsets, the treatment decreased significantly T-lymphocytes with NK cell marker (CD3+ CD56+) (28%, P=0.011). No significant differences of effectiveness were found among the subsequent treatment cycles, except for CD25+ cells and sIL-2R (P=0.036 and P=0.005, respectively): the increase induced by immunotherapy was maximum after the first cycle and decreased progressively thereafter. Conclusions: Long-term repeated cycles of low-dose immunotherapy induced repeated and significant expansion of one of the most important lymphocyte subsets for the non-MHC-restricted immune response to the tumour mass: CD3–CD56+ cells. Received: 8 November 2000 / Accepted: 11 January 2001     

NK cells lyse T regulatory cells that expand in response to an intracellular pathogen

We evaluated the capacity of NK cells to influence expansion of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) in response to microbial Ags, using Mycobacterium tuberculosis as a model. We previously found that Tregs expand when CD4(+) cells and monocytes are exposed to M. tuberculosis. Addition of NK cells that were activated by monokines (IL-12, IL-15, and IL-18) or by exposure to M. tuberculosis-stimulated monocytes reduced Treg expansion in response to M. tuberculosis. NK cell inhibition of Treg expansion was not mediated through IFN-gamma. Activated NK cells lysed expanded, but not freshly isolated Tregs. Although monokines increased NK cell expression of the activating receptors NKp46, NKG2D, 2B4, CD16, and DNAM-1, only anti-NKG2D and anti-NKp46 inhibited NK cell lysis of expanded Tregs. Of five NKG2D ligands, only UL16-binding protein 1 (ULBP1) was up-regulated on M. tuberculosis-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs. M. tuberculosis-stimulated monocytes activated NK cells to lyse expanded Tregs, and this was also inhibited by anti-NKG2D and anti-ULBP1, confirming the physiological relevance of this effect. Our study identifies a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response.     

Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways

NK cells lyse certain tumor cell targets but the effector cell surface molecules responsible for this reactivity remain uncertain. The allotypic NK1.1 Ag is the most specific serologic marker on murine cells that display non-MHC-restricted cytolysis of tumor cell targets, but no function has been previously ascribed to this Ag. In this report, we demonstrate that, in the presence of a mAb specific for the NK1.1 Ag (mAb PK136), freshly isolated and IL-2-activated NK cells from C57BL/6 mice can be induced to lyse an otherwise resistant target cell, Daudi. This phenomenon is effector and mAb specific because NK cells derived from BALB/c mice do not express the NK1.1 Ag and cannot be triggered by mAb PK136. We demonstrate that IL-2 activated but not freshly isolated NK cells express the Ly-6 and VEA Ag, originally described as T cell activation Ag. Moreover, mAb specific for Ly-6 and VEA induce target cell lysis by IL-2 activated but not freshly isolated NK cells. These mAb effects are specific, concentration dependent, and display kinetics that are similar to spontaneous cytolysis of NK-sensitive targets. The Fc portion of the activating antibodies and only FcR bearing target cells participate in mAb-induced activation, consistent with the mechanism of redirected lysis. Finally, analysis of Daudi cells transfected with beta 2-microglobulin gene demonstrate that the expression of MHC class I Ag by the target cell does not affect its sensitivity to mAb-induced lysis by NK cells. These data demonstrate that the NK1.1 Ag is functionally active on both freshly isolated and IL-2-activated NK cells and that IL-2-activated NK cells possess additional pathways of specific stimulation.     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

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.     

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.     

Interleukin-12 increases bispecific-antibody-mediated natural killer cell cytotoxicity against human tumors

 The combination of CD16/CD30 bispecific monoclonal antibodies (bi-mAb) and unstimulated human resting natural killer (NK) cells can cure about 50% of mice with severe combined immunodeficiency (SCID) bearing subcutaneously growing established Hodgkin’s lymphoma. As interleukin-2 (IL-2) and IL-12 have been shown to increase NK cell activity, we tested the capacity of these cytokines to increase bi-mAb-mediated NK cell cytotoxicity against two types of human tumors (Hodgkin’s disease and colorectal carcinoma). Unstimulated NK cells needed a three- to five-times higher antibody concentration than cytokine-stimulated NK cells to exert similar levels of bi-mAb-mediated cytotoxicity. The augmented tumor cell lysis was achieved with IL-12 at considerably lower concentrations than with IL-2 and was associated with a significantly increased bi-mAb-mediated intracellular Ca²⁺ mobilization. The efficiency of IL-12 in this setting together with its low toxicity make it the ideal candidate for a combination therapy with NK-cell-activating bi-mAb in human tumors that are resistant to standard treatment. Received: 26 July 1995 / Accepted: 16 November 1995     

Immunization of mice with fucosyl-GM1 conjugated with keyhole limpet hemocyanin results in antibodies against human small-cell lung cancer cells

Fucosyl-GM1 (Fuc-GM1) [Fucα1 → 2Galβ1 → 3GalNAcβ1 → 4(NeuAcα2-3)Galβ1 → 4Glcβ1 → O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein keyhole limpet hemocyanin (KLH) or covalently linked with KLH, plus immunological adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC. Received: 25 March 1999 / Accepted: 5 August 1999     

A bispecific single-chain antibody directed against EpCAM/CD3 in combination with the cytokines interferon α and interleukin-2 efficiently retargets T and CD3+CD56+ natural-killer-like T lymphocytes to EpCAM-expressing tumor cells

 Cytokine-induced killer cells (CIK), generated in vitro from peripheral blood mononuclear cells (PBMC) by addition of interferon γ (IFNγ), interleukin-2 (IL-2), IL-1 and a monoclonal antibody (mAb) against CD3, are highly efficient cytotoxic effector cells with the CD3⁺CD56⁺ phenotype. In this study, we evaluated whether the cytotoxicity of these natural-killer-like T lymphocytes against the colorectal tumor cell line HT29 can be enhanced by the addition of a bispecific single-chain antibody (bsAb) directed against EpCAM/CD3. For determination of bsAb-redirected cellular cytotoxicity we used a new flow-cytometric assay, which directly counts viable tumor cells and can assess long-term cytotoxicity. We found that this bsAb induced distinct cytotoxicity at a concentration above 100 ng/ml with both PBMC and CIK at an effector-to-target cell ratio as low as 1:1. CIK cells revealed higher bsAb-redirected cytotoxicity than PBMC. Cellular cytotoxicity appeared after 24 h whereas PBMC showed the highest bsAb-redirected cytotoxicity after 72 h. The addition of the cytokines IL-2 and IFNα but not granulocyte/macrophage-colony-stimulating factor enhanced bsAb-redirected cytotoxicity of both PBMC and CIK. When the bsAb was combined with the murine mAb BR55-2, which recognizes the Lewis^y antigen, bsAb-redirected cytotoxicity was partly augmented, whereas murine mAb 17-1A, which binds to EpCAM as well, slightly suppressed bsAb-redirected cytotoxicity induced by the bsAb. We conclude that CIK generated in vitro or in vivo combined with this new EpCAM/CD3 bsAb and the cytokine IL-2 should be evaluated for the treatment of EpCAM-expressing tumors. Received: 9 December 1999 / Accepted: 18 May 2000     

Leukemic priming of resting NK cells is killer Ig-like receptor independent but requires CD15-mediated CD2 ligation and natural cytotoxicity receptors

Resting human NK cells require a two-stage activation process that we have previously described as "priming" and "triggering." NK-sensitive tumor cells provide both priming and triggering signals. NK-resistant tumors evade lysis, mostly by failure to prime; however, we recently reported a tumor cell line (CTV-1) that primes resting NK cells but fails to trigger lysis. In this article, we report two additional leukemia cell lines that prime NK cells but are resistant to lysis. Tumor-mediated NK priming is via CD2 binding to a ligand within CD15 on the tumor cell. NK-resistant RAJI cells became susceptible to NK lysis following transfection and expression of CD15. Blockade of CD15 on K562 cells or on CD15(+) RAJI cells significantly inhibited lysis, as did blockade of CD2 on resting NK cells. NK priming via CD2 induced CD16 shedding, releasing CD3ζ to the CD2, leading to its phosphorylation and the subsequent phosphorylation of linker for activation of T cells and STAT-5 and synthesis of IFN-γ. Blockade of C-type lectin receptors significantly suppressed the tumor-mediated priming of NK cells, whereas blockade of Ig-superfamily-like receptors had no effect at the NK-priming stage. Tumor priming of resting NK cells was irrespective of HLA expression, and blockade of HLA-killer Ig-like receptor interactions did not influence the incidence or degree of priming. However, CD15-CD2 interactions were critical for NK priming and were required, even in the absence of HLA-mediated NK inhibition. Tumor-mediated priming led to a sustained primed state, and the activated NK cells retained the ability to lyse NK-resistant tumors, even after cryopreservation.     

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.     

HER-2/neu peptide specificity in the recognition of HLA-A2 by natural killer cells

Although natural killer (NK) cells have been described as non-MHC-restricted, new evidence suggests that NK activity can be either up- or down-regulated after interaction with the peptide–MHC-class-I complex expressed on target cells. However, the epitope(s) recognized by NK cells have remained ill-defined. We investigated NK cell recognition of synthetic peptides representing a portion of a self-protein encoded by the HER-2/neu (HER-2) proto-oncogene and presented by HLA-A2. HER-2 nonapeptides C85, E89, and E75 were found partially to protect T2 targets from lysis by freshly isolated and interleukin-2(IL-2)-activated NK cells (either HLA-A2⁺ or A2⁻). This inhibition was not solely due to changes in the level of HLA-A2 expression or conformation of serological HLA-A2 epitopes. Using single-amino-acid variants at position 1 (P1) of two HER-2 peptides, we observed that protection of targets was dependent on the sequence and the side-chain. These results suggest similarities in the mechanism of target recognition by NK and T cells. This information may be important for understanding the mechanisms of tumor escape from immunosurveillance and could help explain the aggressiveness of HER-2-overexpressing tumor cells. Received: 16 March 1999 / Accepted: 3 June 1999