Lymphokine-activated Killer Cytotoxicity Research Articles

Specific antitumor activity of anti-CA125 CAR-T lymphocytes against CA125-positive and CA125-negative cells

Aim. To evaluate the antitumor efficacy of our developed drug based on cytotoxic T lymphocytes genetically modified with a chimeric antigen receptor (CAR) specific to the CA125 antigen in relation to both CA125-positive and CA125negative cell cultures.Materials and methods. We performed an in vitro study on CA125-positive human ovarian cancer cells (OVCAR-3, OVKATE) and CA125 negative cells (breast cancer MCF 7, embryonic kidney HEK293). Cytotoxic effects on tumor cells were evaluated after 0, 4, 8 and 24 hours using the 3’-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Lactate Dehydrogenase (LDH) tests. We also studied the changes in the number of cells “in real time” when exposed to transfected lymphocytes using the RTCA iCELLIgence device (ACEA Biosciences, USA). Lymphokineactivated killer (LAK) cells were used as a specificity control.Results. The study demonstrated that anti-CA125 CAR-T lymphocytes exhibited a pronounced cytotoxic effect on OVCAR-3 and OVKATE cell cultures, exceeding the effect of LAK by 1.3 times. The cell population in the experimental samples decreased by 70 ± 4%, which exceeded the LAK effect by 9 ± 8.2%. With regard to the MCF-7 cell line, the cytotoxic effect of anti-CA125 CAR-T lymphocytes was minimal as evidenced by a 25.8% decrease in the relative number of live cells in comparison to the LAK cytotoxicity of 68%. Real-time monitoring of cell proliferation and viability proved a high specific cytotoxic effect of anti-CA125 CAR-T lymphocytes against tumor cultures expressing CA-125, while inferior to LAK in cultures not expressing CA125 (MCF-7, HEK293).Conclusions. The use of anti-CA125 CAR-T lymphocytes against CA125-positive tumor cell lines OVCAR-3 and OVKATE demonstrated a pronounced specific cytotoxic effect exceeding the cytotoxic effect of LAK, which was not achieved against CA125-negative MCF-7 and HEK293 cells.

Immunologic Studies in Humans.

Immunologic Studies in Humans.

Activity of Continuous Infusion + Pulse Interleukin-2 with Famotidine in Metastatic Melanoma

High-dose interleukin-2 (IL-2), given via continuous intravenous (i.v.) infusion, induces lymphokine-activated killer (LAK) cell cytotoxicity against tumor cells. These LAKs exhibit enhanced cytotoxicity against tumor cells in vitro when they are subsequently pulsed with additional IL-2. Famotidine may increase LAK cytotoxicity against neoplastic cells by allowing for greater IL-2 uptake at the IL-2 receptor on lymphocytes. Twenty-three (23) patients received famotidine 20 mg i.v. twice per day and continuous-infusion IL-2 (18 MIU/m(2)/24 hours) for 72 hours, followed by a 24-hour rest, then 1-3 daily-pulse IL-2 doses of 18 MIU/m(2) over 15-30 minutes preceded by famotidine 20 mg i.v. Cycles were repeated every 3 weeks. The most common metastatic sites were lung, lymph node, and subcutaneous/soft tissue. The most common toxicities were fever, rigor, nausea/emesis, hypophosphatemia, hypotension, elevated creatinine, and pulmonary edema. There were no treatment-related deaths. One (1) complete (4%) and 9 partial responses (39%) were seen (43% total response rate; 95% confidence interval: 22%-65%). Median survival for all patients is 13 months. The combination of famotidine and high-dose continuous infusion + pulse IL-2 is active in metastatic melanoma.

Lymphokine-activated killer cytotoxicity against leukaemic blast cells

While the sensitivity of cell lines and solid tumours to lymphokine-activated killer (LAK) cell cytotoxicity is extensively reported, the ability of these generated cytotoxic cells to lyse leukaemic blast cells is controversial. This study reports the successful generation of LAK cells with specificity for leukaemic blast cells. Control donors, patients in remission and patients with active leukaemia were capable of lysing allogeneic leukaemic blast cells. One patient was also capable of lysing autologous leukaemic blast cells. Successful LAK generation was achieved by the use of high dose recombinant interleukin-2 (rIL-2) while combinations of lymphokines did not improve LAK specificity or efficacy. These findings suggest that LAK immunotherapy may be considered for the treatment of patients with acute leukaemia.

Increased LAK activity against HIV-infected cell lines in HIV-1+ individuals.

The role of natural killer (NK) cells and their inducible counterparts, lymphokine-activated killer (LAK) cells in AIDS with regard to HIV-1 viral immunosurveillance and the control of secondary opportunistic disease has yet to be established. In this study, we have demonstrated that LAK cells derived from all HIV-1+ groups showed striking increases in their capacity to lyse HIV-1-infected U-937 cells relative to their uninfected U-937 counterparts. Surprisingly, similarly derived LAK cells from healthy seronegative controls showed no differences in their lysis of HIV-1-infected versus uninfected U-937 cells. The differential ability of LAK effectors from seropositive donors to lyse HIV-1-infected targets was demonstrable using a number of U-937 subclones and their HIV-1-infected counterparts. Again, no differences in LAK cell-mediated lysis of HIV-1-infected and uninfected U-937 subclones were observed in seronegative individuals. Our findings that HIV-1+ individuals show selective expansion of non-MHC restricted, HIV-1-directed cytotoxic LAK cells indicate that natural immunity may indeed play a role in HIV-1 viral immunosurveillance.

Effects of interleukin-2 and interleukin-2-activated cells on in vitro myelopoiesis

Lymphokine-activated killer (LAK) cells from human peripheral blood mononuclear cells cultured with recombinant interleukin-2 (IL-2) have been used clinically in adoptive immunotherapy for cancer patients. To study the influence of LAK cells and IL-2 on haematopoiesis, an in vitro assay system for colony formation of granulocyte-macrophage progenitor cells (GM-CFC) was used. LAK cells from cultures of either human peripheral blood (PB) or human bone marrow (BM) mononuclear cells were both inhibitory to allogeneic BM-derived GM-CFC. Inhibitory activity could be transferred with supernatants from co-cultures of LAK cells and BM targets, but also from the IL-2 activated PB- or BM-derived cells alone. The inhibitory activity from the initially non-cytotoxic/non-inhibitory BM population was rapidly induced by IL-2 activation, and preceded the generation of cytotoxic LAK cells in the culture. These experiments show that inhibition of haematopoietic progenitor cells by IL-2 is not dependent on generation of cytotoxic LAK cells, but rather the result of IL-2-induced cytokine production. We conclude that the synergistic action of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) may contribute to inhibition, but that also other cytokines are responsible for the observed inhibition of BM-derived GM-CFC.

Characterization of Cord Blood Natural Killer and Lymphokine Activated Killer Lymphocytes Following Ex Vivo Cellular Engineering

Cord blood (CB) natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic cells are poorly characterized but might be used to treat minimal residual and/or recurrent malignant disease. Currently, there is no mechanism to use CB for adoptive cancer cellular immunotherapy after CB transplantation (CBT). Recognizing this as a deficiency, we hypothesized that CB aliquots could be engineered ex vivo for potential donor lymphocyte infusion after CBT. Cryopreserved CB aliquots were thawed, depleted of monocytes, and cultured in serum-free medium alone or serum-free medium with anti-CD3 and interleukins 2, 7, and 12 combined with antibody/cytokines for 48 hours. Immunophenotyping, cytotoxicity, and proliferation were evaluated. A significant expansion of CD3 + was seen, in addition to increases in lymphocyte subsets of CD8 +, CD8 +/CD25 +, and CD3 +/45RO + versus medium alone. A significant enhancement of CD3 proliferation ( P < .001), NK cytotoxicity, NK subset expansion, LAK cytotoxicity, and T-helper 1 subset expansion was also demonstrated. Significant enrichment was seen in NK CD16 +/CD56 +bright, CD16 +/CD56 +dim, CD56 +bright and CD56 +dim/KIR3DL1 +, CD56 +bright and CD56 +dim/KIR2DL1 +, CD56 +bright and CD56 +dim/KIR2DL2 + and CD94 +/NKG2a + subsets. These increases in CB NK subsets were in part secondary to augmentation of cell survival. Further, survival of NOD-SCID mice xenografted with human K562 cells and treated with CB cells expanded with antibody/cytokines was significantly higher than that in animals that received no treatment (phosphate buffered saline) and those that were treated with CB ex vivo expanded in medium alone ( P < .005, respectively). These data suggest that cryopreserved CB cells could be ex vivo engineered for potential use as adoptive cancer cellular immunotherapy for donor lymphocyte infusion after CBT.

Lymphocyte therapy of renal cell carcinoma

During the past 20 years, there has been considerable interest in lymphocyte therapy as a treatment for renal cell carcinoma. There is no therapeutic role for B-lymphocyte therapy, but their products, monoclonal antibodies, now have widespread clinical applications. The major types of autologous lymphocyte therapy that have been explored in clinical trials are cytotoxic lymphokine-activated killer cells, which are natural killer cells and T-cells that have been stimulated in vitro by interleukin-2 or other similar cytokines; cytotoxic and noncytotoxic tumor infiltrating lymphocytes, which are T-cells derived from tumor tissue; other tumor antigen-stimulated T-lymphocytes derived from regional lymph nodes or peripheral blood; and noncytotoxic lymphocytes of the memory/helper phenotype. More recently, allogeneic immune therapy using nonmyeloablative hematopoietic stem cell transplant and/or donor lymphocyte therapy has also shown promise.

Gs Protein-Coupled Adenosine Receptor Signaling and Lytic Function of Activated NK Cells

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.

Differential Regulation of MHC Class‐I‐Restricted and Unrestricted Cytotoxicity by the Us3 Protein Kinase of Herpes Simplex Virus‐1

Modulation of cytotoxic responses by viral immunoevasins plays an important role in the establishment of latent and persistent viral infections. Together with MHC class-I-restricted CD8T-lymphocytes, non-MHC-restricted natural killer (NK) and lymphokine-activated killer (LAK) cells participate in this anti-viral control. The Us3 protein kinase of herpes simplex virus-1 (HSV-1) inhibits CD8T-cell cytotoxicity, which correlates with the inhibition of granzyme-B (GrB)-induced activation of pro-apoptotic Bid. We have investigated the effect of Us3 on NK and LAK cytotoxicity, because these effectors are believed to share common mechanisms for inducing cell death. We show that, in contrast to their lower sensitivity to CD8T-cell lysis, HSV-1-infected cells are lysed by NK cells or LAK cells as efficiently as the uninfected controls. Both CD8T and NK/LAK effectors were dependent on the activity of GrB and were efficiently blocked by means of treatment with a GrB inhibitor. However, unlike CD8T cells, LAK cells and NK cells failed to induce Bid cleavage, suggesting that various GrB downstream targets be involved in the induction of cell lysis. This finding explains their various sensitivities to viral modulation, which is likely to be important for the respective role of MHC-restricted and non-restricted effectors in the control of HSV-1 infection.

Mycophenolate mofetil does not suppress the graft-versus-leukemia effect or the activity of lymphokine-activated killer (LAK) cells in a murine model.

Graft-versus-leukemia (GVL) effect is an essential component in the course of allogeneic stem cell transplantation (SCT). However, both prevention and treatment of established graft-versus-host disease (GVHD), including with drugs such as cyclosporine, can suppress GVL effects. Mycophenolate mofetil (MMF) is becoming a standard of care in SCT recipients for better prevention of GVHD as well as for promoting stem cell engraftment. To evaluate the effect of MMF, an immunosuppressive drug increasingly used for prevention of GVHD, on disease recurrence following SCT in a preclinical animal model. Since GVL effects may be also induced by alloreactive natural killer (NK) cells, the goal was to investigate the effects of MMF on the activity of lymphokine-activated killer (LAK) cells. MMF was administered by daily intraperitoneal injection starting at day 1 post-SCT. Cytotoxic LAK activity was measured by 5-h 35S-release assay, and GVL was tested by the appearance of BCL1 leukemia in a semi-mismatched (C57BL/6 donors to [BALB/c x C57BL/6] F1 recipients) murine model. A dosage regimen of 28-200 mg/kg per day MMF had no negative effect on either cytotoxic LAK activity or GVL (as measured by finding of leukemic cells in recipient spleen by PCR or the appearance of clinical leukemia with adoptive transfer). These results suggest that MMF does not impair GVL effects or reduce LAK cell activity in mice.

Tumor-infiltrating lymphocyte secretion of IL-6 antagonizes tumor-derived TGF-beta 1 and restores the lymphokine-activated killing activity.

IL-6 is a multifunctional cytokine that regulates cell growth, differentiation, and cell survival. Many tumor cells produce TGF-beta1, which allows them to evade CTL-mediated immune responses. IL-6 antagonizes TGF-beta1 inhibition of CD3 cell activation. However, whether IL-6 restores NK activity, which also is suppressed by TGF-beta1, is not known. We used canine transmissible venereal tumor (CTVT), which produces TGF-beta1, as a model to determine whether IL-6 restores lymphokine-activated killer (LAK) activity. During the progression phase, CTVT cells stop expressing MHC molecules. During the regression phase, the number of surface MHC molecules increases dramatically on about one-third of tumor cells. Tumor cells that stop expressing MHC should be targeted by NK cells. In this study, we found that TGF-beta1 secreted by CTVT cells suppressed LAK cytotoxicity. Interestingly, tumor-infiltrating lymphocytes (TIL) isolated from regressing CTVT secrete high concentrations of IL-6 and antagonize the anti-LAK activity of tumor cell TGF-beta1. TIL also produce IL-6 during progression phase, but the concentration is too low to block the anti-LAK activity of TGF-beta1. There is probably a threshold concentration of IL-6 needed to reverse TGF-beta1-inhibited LAK activity. In addition, in the absence of TGF-beta1, IL-6 derived from TIL does not promote the activity of LAK. This new mechanism, in which TIL manufacture high concentrations of IL-6 to block tumor TGF-beta1 anti-LAK activity, has potential applications in cancer immunotherapy and tumor prognosis.

Lipoprotein lipase protects bovine endothelial cells from human NK cytotoxic activity

Human lipoprotein lipase (LPL), in a dose dependent fashion, significantly inhibited spontaneous human natural killer (NK) cells, but not lymphokine-activated killer (LAK) cytotoxic activity against bovine pulmonary endothelial cells. The effect was dependent on endothelial heparan sulfate (HS) sites, since heparitinase reverted it. When HS is added before LPL, NK and LAK cytotoxicity are markedly reduced. Endothelial and NK cell priming, with LPL and HS + LPL, significantly induced CD40 and CD154 expression, respectively. Furthermore, CD40 expression was inversely proportional to lytic units ( R 2=0.9, P<0.001). Treating endothelial cells simultaneously with indomethacin, CD154 fusion protein, and Wortmanin prevented the CD40 effect increasing xenograft rejection. LPL and HS + LPL protect bovine endothelial cells from NK cytotoxicity by inducing CD40, CD154 expression, and secretion of soluble factors. The high, non-modulated expression of adhesion receptors and the low number of HS sites account for the minor effect of CD40 in LAK cytotoxic responses against bovine endothelial cells.

Laparotomy and laparoscopy differentially accelerate experimental flank tumour growth.

Surgery depresses host tumoricidal activity and may increase tumour growth. This study compared the effects of laparoscopy with laparotomy on extraperitoneal tumour growth and immune function in a murine model. C57BL/6 female mice aged 8-10 weeks had tumours induced in the right flank (n=45) and were randomized to undergo halothane anaesthesia only, laparoscopy or laparotomy. Flank tumour volume was assessed over 10 days. A second group of animals (n=540) were randomized to undergo the same procedures and killed at 24, 48 and 96 h. Splenocytes were harvested for natural killer (NK) cell and lymphokine activated killer (LAK) cell cytotoxicity studies. There was a significant increase in flank tumour growth in the first 48 h after laparotomy and laparoscopy compared with controls (P < 0.01). By 96 h the difference was only significant in the laparotomy group (P< 0.01). Both NK and LAK cell cytotoxicities were suppressed significantly (P < or = 0.03) from 24 h up to 96 h following laparotomy compared with control and laparoscopy groups. There was also a significant suppression in the laparoscopy group compared with controls in the first 48 h after operation (P < or = 0.02). Extraperitoneal tumour growth was significantly accelerated after laparotomy and correlated with significantly suppressed NK and LAK cytotoxicity for at least 4 days after operation. Laparoscopy had a shorter, less profound effect on tumour growth and immune function.

Immunological properties of mononuclear cells from blood stem cell harvests following mobilization with erythropoietin + G-CSF in cancer patients

Although the recombinant granulocyte colony-stimulating factor (G-CSF) is a good CD34 cell mobilizer, the effects of G-CSF mobilization on the immune effector function of the individual is not always optimal. We studied the functional and phenotypic properties of peripheral blood stem cells (PBSC) collected from 15 cancer patients mobilized with G-CSF plus recombinant erythropoietin (EPO). The patients received EPO (300 U/kg) and (G-CSF 1 microg/kg) per day as mobilizing cytokines and an autologous graft product was collected with at least daily apheresis procedures until a target number of CD34 cells and mononuclear cells were obtained. Mononuclear cells from the first four PBSC collections were tested for their natural killer (NK), activated NK and lymphokine activated killer (LAK) cytotoxicity in vitro against K562 and Raji tumor target cells. There was a significant increase in NK, activated NK and LAK cytotoxicity in EPO + G-CSF mobilized cells when compared to mononuclear cells from premobilization blood baseline values. Although there was no increase in CD3+ T cells, there was a significant increase in myeloid cells (CD14+), B-cells (CD20+) and NK cells (CD56+) following mobilization. There was no difference in T cell response to the mitogens PHA and Con-A, but there was an increase in B-cell response to PWM following mobilization. Thus, the combination of EPO + G-CSF not only mobilized hematopoietic precursor cells but also increased the number of immune effector cells in the PBSC collections.

The immunostimulating activities of anti-tumor polysaccharide from K1 capsular (polysaccharide) antigen isolated from Klebsiella pneumoniae

We have previously shown that K1 capsular polysaccharide antigen (K1CPS) of Klebsiella exhibits anti-tumor activities. In the present study, we examined the effect of K1CPS on cytotoxic effector cells. We found that K1CPS could activate many cytotoxic effector cells including alloreactive cytotoxic T cells and tumor-infiltrating lymphocytes (TILs). Moreover, K1CPS could increase the anti-tumor activity of lymphokine-activated killer (LAK) cells, both in vitro and in vivo. The i.p. injection of K1CPS in low dose could enhance the LAK cytotoxicity and the effect was further potentiated by coculture of LAK cells with K1CPS and low concentration of murine rIL-2 in vitro. The phenotypic characterization revealed that K1CPS might contribute to the increase in CD3+ LAK cell subpopulation by its in vivo priming effect. In addition, the K1CPS-treated LAK cells were able to inhibit the growth of WEHI-164 tumor cells in vivo in Winn-type inhibition assay. Subcutaneous (s.c.) and intraperitoneal (i.p.) adoptive infusion of LAK cells (splenocytes from K1CPS-treated WEHI-164-bearing mice cultured with K1CPS-plus-rIL-2) into WEHI-164 sarcoma-bearing mice could slightly cause regression in terms of tumor diameter, and more significantly in sarcoma weight.

Up-modulation of interferon-gamma mediates the enhancement of spontanous cytotoxicity in prolactin-activated natural killer cells.

Prolactin (PRL) has been shown to participate in lymphocyte activation. In particular, the constitutive natural killer (NK) and the lymphokine-activated killer (LAK) cytotoxicity of CD56+ CD16+ cells is increased by its physiological to supraphysiological concentrations. As PRL has been shown to up-regulate the production of interferon-gamma (IFN-gamma) by peripheral blood mononuclear cells, we studied its effect on IFN-gamma production by NK cells as a possible mechanism of autocrine activation of cytotoxicity. Released and intracellular IFN-gamma, as well as IFN-gamma mRNA expression, were increased by pituitary and recombinant human PRL, which stimulated optimal NK and LAK cytotoxicity. Treatment with blocking anti-IFN-gamma monoclonal antibody (mAb) selectively affected PRL-increased killing of K562 targets, demonstrating that PRL-mediated enhancement of spontaneous cytotoxicity depends, at least in part, on up-regulation of IFN-gamma.

Cytocidal activity of PBL, LAK, and IDEC-C2B8 and expression of HLA class 1, ICAM-1, and CD20 in vincristine-resistant hematologic cell lines.

This study was designed to determine whether the cytocidal activity of immunotherapy such as cytotoxic peripheral blood lymphocytes (PBL), lymphokine-activated killer (LAK) cells, and chimeric anti-CD20 mouse/human monoclonal antibody, IDEC-C2B8, overcome vincristine (VCR) resistance in cultured cell lines derived from human leukemia/lymphoma. In addition, the relation between the susceptibility to these immunotherapies and the expression levels of HLA class 1 and ICAM-1 as well as CD20 on the cell surface was analyzed. Three of six VCR-resistant cell lines were less susceptible to PBL cytotoxicity compared with wild-type cells, whereas the susceptibility was kept in the other three VCR-resistant cell lines. Four of six VCR-resistant cell lines were less susceptible to LAK activity and the other two cell lines were as sensitive to LAK cells as their wild-type counterparts. There was no correlation between the susceptibility for PBL cytotoxicity and the expression of HLA class 1 in both wild and VCR-resistant cells. In contrast, ICAM-1 in the two cell lines that showed decreased susceptibility for LAK cytotoxicity disappeared, although that in one cell line increased. IDEC-C2B8 was effective only against B-cell lines expressing CD20. One cell line in which the expression of CD20 increased was nearly six times more sensitive to IDEC-C2B8 than wild type. Thus, we concluded that the resistance to VCR in some tumor cell lines is associated with modified susceptibility for immunotherapies by the different expression of target molecules from those of wild-type counterparts.

The Induction of Nitric Oxide by Interleukin-12 and Tumor Necrosis Factor-α in Human Natural Killer Cells: Relationship With the Regulation of Lytic Activity

We have investigated the interleukin-12 (IL-12) and tumor necrosis factor-α (TNFα)-induced regulation of human natural killer (NK) cell function and their relationship with nitric oxide (NO) generation. We demonstrate that both cytokines were efficient to trigger the transcription of the inducible nitric oxide synthase (iNOS) mRNA, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis and intracytoplasmic fluorescence showed that iNOS protein was also induced by both cytokines. However, our data indicate that NO does not play a significant role in the effector phase of the cytotoxic activity mediated by NK-stimulated cells, inasmuch as the lytic activity was not affected in the presence of specific NO synthase inhibitors. When aminoguanidine (AMG), an inhibitor of iNOS, was added during the afferent phase of NK stimulation with IL-12 and TNFα, a subsequent increase in the lytic potential of the effector cells towards the NK-sensitive target cells (K562) and lymphokine-activated killer (LAK) target cells (Daudi) was observed. Conversely, the addition of chemical NO donors during the afferent step resulted in a dose-dependent inhibition of the NK and LAK cytotoxicity. Our data suggest that the enhancement of NK-cell cytotoxic activity resulting from iNOS inhibition may be correlated, at least in part, to an increase in interferon-γ production and granzyme B expression.© 1998 by The American Society of Hematology.

The Induction of Nitric Oxide by Interleukin-12 and Tumor Necrosis Factor- in Human Natural Killer Cells: Relationship With the Regulation of Lytic Activity

We have investigated the interleukin-12 (IL-12) and tumor necrosis factor- (TNF)-induced regulation of human natural killer (NK) cell function and their relationship with nitric oxide (NO) generation. We demonstrate that both cytokines were efficient to trigger the transcription of the inducible nitric oxide synthase (iNOS) mRNA, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis and intracytoplasmic fluorescence showed that iNOS protein was also induced by both cytokines. However, our data indicate that NO does not play a significant role in the effector phase of the cytotoxic activity mediated by NK-stimulated cells, inasmuch as the lytic activity was not affected in the presence of specific NO synthase inhibitors. When aminoguanidine (AMG), an inhibitor of iNOS, was added during the afferent phase of NK stimulation with IL-12 and TNF, a subsequent increase in the lytic potential of the effector cells towards the NK-sensitive target cells (K562) and lymphokine-activated killer (LAK) target cells (Daudi) was observed. Conversely, the addition of chemical NO donors during the afferent step resulted in a dose-dependent inhibition of the NK and LAK cytotoxicity. Our data suggest that the enhancement of NK-cell cytotoxic activity resulting from iNOS inhibition may be correlated, at least in part, to an increase in interferon-γ production and granzyme B expression.© 1998 by The American Society of Hematology.