Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse uterus during the peri-implantation period

In this study the SEB-activated LAK cytotoxicity was identified and characterized in human peripheral blood lymphocytes (PBMC). After 3 days of SEB stimulation, the PBMC acquired a cytotoxicity against traditional LAK targets, K-562 and Daudi, beside that human glomerular endothelial cells (HGEC) were effectively lysed. The magnetic separation of SEB-stimulated CD5+ T cells revealed that the dominant LAK cytotoxicity remained in the CD5- lymphocyte fraction. The major part of the SEB-generated cytotoxicity of CD5- cells could be blocked with specific antibodies to IL-2 and IFN-gamma. The IFN-gamma pretreatment of HGEC reduced the target sensitivity, but because of the upregulation of MHC class II on HGEC surface, these cells were able to present SEB to CD5+ cells. These results suggested that in bacterial superantigen-mediated infection, the non-T (NK cells-derived) LAK cells might have a primary pathogenic role, and the adverse effect of IFN-gamma, that was massively secreted from superantigen-stimulated cells, requires greater consideration.     

Role of prolactin in the modulation of NK and LAK cell activity after short- or long-term morphine administration in neoplastic patients

In a previous work we demonstrated that chronic in vivo antalgic therapy of cancer patients with morphine reduced the endogenous cytotoxic activity of natural killer (NK) cells, while increasing the development of lymphokine activated killer (LAK) cell cytotoxicity. In order to investigate the mechanisms by which morphine affects NK and LAK cell function further, we evaluated the modulation exerted by short- or long-term morphine administration on either NK/LAK cell cytotoxicities or plasma levels of prolactin (PRL) and other immunomodulating neurohormones. An intravenous morphine injection (10 mg) significantly increased the plasma levels of PRL, reduced the cytotoxic activity of NK cells, and increased the development of LAK cell activity 30 min after drug injection in neoplastic patients. The administration of bromocriptine before the injection of morphine prevented both PRL augmentation and the increase in LAK cell activation, although it did not prevent the inhibition of NK cytotoxicity. The chronic oral administration of morphine (90 +/- 30 mg/day for 1 month) also resulted in higher PRL levels; the NK and LAK cell activities were, respectively, lower than or higher than those found in neoplastic patients untreated with morphine. The plasma levels of thyrotropin (TSH), adrenocorticotropic hormone (ACTH) and cortisol were not significantly modified in either short- or long-term experiments. The absolute number and the percentages of lymphocyte populations, as well as the percentage of IL-2 receptors, were not modified after short-term morphine administration whereas little changes of T lymphocyte populations and NK cell number were observed after oral treatment with morphine. In vitro morphine did not affect the development of LAK cell activity. In conclusion, our findings indicate that morphine reduces NK cytotoxicity and increases the development of LAK cell cytotoxicity after short- and long-term administration. The effect of morphine on LAK cell activation but not on NK cell reduction is related to the modulation of PRL levels determined by the opioid drug.     

Transforming growth factor-beta inhibits interferon-gamma secretion by lymphokine-activated killer cells stimulated with tumor cells

The effect of transforming growth factor-beta (TGF-beta) secreted by glioblastoma (T98G) cells on the secretion of interferon-gamma (IFN-gamma) by lymphokine-activated killer (LAK) cells stimulated with tumor cells was investigated in cocultures of LAK and Daudi cells supplemented with T98G culture supernatant, T98G culture supernatant preincubated with anti-TGF-beta 1 and anti-TGF-beta 2 neutralizing antibodies, anti-TGF-beta 1 and anti-TGF-beta 2 antibodies, or natural human TGF-beta 1 or recombinant human TGF-beta 2. LAK cells were incubated with anti-TGF-beta 1 and anti-TGF-beta 2 antibodies, and with T98G cells of which the supernatant contained both active and latent forms of TGF-beta 1 and TGF-beta 2, with or without neutralizing antibodies. Addition of the supernatant from T98G cells to LAK/Daudi culture caused inhibition of IFN-gamma secretion by LAK cells. The inhibition was abolished by pretreatment of the supernatants with anti-TGF-beta antibodies. Addition of TGF-beta 1 and TGF-beta 2 to the LAK/Daudi culture inhibited IFN-gamma secretion by LAK cells in a dose-dependent manner. Addition of anti-TGF-beta antibodies to the LAK culture resulted in increased IFN-gamma secretion. T98G cells failed to stimulate LAK cells to secrete more IFN-gamma. Addition of anti-TGF-beta antibodies to the LAK-T98G culture resulted in increased IFN-gamma secretion by LAK cells. These results suggest that most malignant glioma cells which secrete high levels of TGF-beta can inhibit IFN-gamma secretion by LAK cells even after tumor cell stimulation.     

Immunization of mice with allogeneic fibroblasts genetically modified for interleukin-2-secretion and expression of melanoma-associated antigens stimulate predetermined classes of anti-melanoma effector cells

Immunization of C57BL/6 mice (H-2b) with a mouse fibroblast cell-line of C3H origin (H-2k) genetically modified for interleukin-2 (IL-2)-secretion and the expression of melanoma-associated antigens (MAAs) (RLBA-IL-2 cells) resulted in a systemic anti-melanoma cellular immune response that led to a prolongation of survival of mice with established melanoma. Here we report certain of the effector cell-types activated for anti-melanoma immunity in mice immunized with the modified cells and, for comparison, the anti-melanoma cell-types activated following immunization with IL-2-secreting, MAA-negative fibroblasts (LM-IL-2 cells) or with non-IL-2-secreting, MAA-positive fibroblasts (RLBA-ZipNeo cells). The data indicate that both Lyt-2.2+ (CD8+) and natural killer/lymphokine-activated killer (NK/LAK) cells with anti-melanoma cytotoxicity were predominant in mice immunized with RLBA-IL-2 cells. NK/LAK cells alone were predominant in mice immunized with LM-IL-2 cells, and Lyt-2.2+ cells were predominant in mice immunized with RLBA-ZipNeo cells. The involvement of L3T4+ (CD4+) cells in the effector phase of the response was not detected in mice immunized with the genetically modified cells. Immunization of mice with both LM-IL-2 cells and RLBA-ZipNeo cells resulted in an anti-melanoma response of greater magnitude than was present in mice immunized with either cell-construct alone. It was equivalent to the melanoma immunity in mice immunized with RLBA-IL-2 cells. These data indicate that the immunogenic properties of the modified cells determined the anti-melanoma effector cell-types and suggest that combination immunotherapy with cell-constructs that stimulate different classes of effector cells may be more effective in immune-mediated tumor regression than immunization with a construct that activates a single effector cell-type alone.     

Blocking of MHC class I antigens on leukemic B-cells enhances their conjugate formation with cytotoxic lymphocytes and their susceptibility to lysis

The role of major histocompatibility complex (MHC) class I antigens and adhesion molecules (AM) in the resistance of leukemic B-cells to cell-mediated cytotoxicity was investigated using cells from eight patients with B-chronic lymphocytic leukemia (B-CLL) and six patients with immunocytoma (IC). Both CLL and IC cells were completely resistant to natural killer (NK) and lymphokine activated killer (LAK) cytotoxicity and no binding to effector cells was observed, irrespectively of AM expression. Blocking of MHC class I antigens with monoclonal antibodies or their temporary elimination from leukemic B-cell surface by acid treatment resulted in a significant (p < 0.005) increase in both conjugate formation and susceptibility to lysis, thus suggesting the relevance of MHC class I expression on leukemic B-cells for the NK/LAK resistance phenomenon.     

Reinforced cytotoxicity of lymphokine-activated killer cells toward glioma cells by transfection with the tumor necrosis factor-alpha gene

Lymphokine-activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin-2 were transfected with the human tumor necrosis factor (TNF)-alpha gene by means of novel liposomes with a positive change on their surface. The cells secreted significant amounts of TNF-alpha into the culture medium and exhibited reinforcement of cytotoxicity toward a human glioma cell line (U251-SP), being three times more cytotoxic than nontransfected LAK cells. The mechanism for the reinforcement of cytotoxicity is considered to involve not only an increase in TNF-alpha secretion from LAK cells but also its expression on their surface. Intratumoral or intrathecal injection of LAK cells transfected with the TNF-alpha gene may be useful for the treatment of patients with malignant gliomas.     

Epidural perfusion cooling protection against protracted spinal cord ischemia in rabbits

In in vivo allogeneic bone marrow transplantation studies with the Brown Norway (BN) rat as recipient and the WAG/Rij rat as allogeneic donor a significant graft-versus-leukemia (GVL) effect is observed. Studies were performed to investigate whether lymphokine-activated killer (LAK) cells play a role in this GVL effect. Splenocytes from WAG/Rij and BN rats were activated in vitro by recombinant human interleukin-2 (rhIL-2) for 5-6 days. The cytolytic activity of these LAK cells was tested on four rat solid tumor cell lines, i.e. an ureter carcinoma, a rhabdomyosarcoma, and two lung tumors, and on leukemic cells derived from the BN rat acute myelocytic leukemia (BNML) and the WAG/Rij acute lymphocytic leukemia (L4415). The panel of target cells also included the murine cell lines P815 and YAC. Both WAG/Rij and BN LAK cells were not capable of lysing the leukemic cells in contrast to significant cytolytic activity on the rat solid tumor cell lines and P815 and YAC. BNML cells showed to be resistant to lysis by human NK cells. Phenotypical analysis of the rat LAK population revealed a decrease in the CD4/CD8 ratio compared to the unstimulated splenocyte population. Rat LAK cells displayed no antibody-dependent cellular cytotoxicity (ADCC) on the leukemic cells, whereas IL-2-stimulated human peripheral blood cells showed moderate ADCC activity on the leukemic cells. To investigate whether cytokines play a role in lysis of leukemic target cells, graded numbers of LAK cells and leukemic cells were co-cultivated for seven days in an agar-based colony culture system. This resulted in moderate suppression of leukemic colony formation. From the current in vitro studies it appears that the graft-versus-leukemia observed in in vivo allogeneic bone marrow transplantation studies is probably not due to a direct leukemic cell kill by LAK cells.     

Elevated prostaglandin E2 production by monocytes is responsible for the depressed levels of natural killer and lymphokine-activated killer cell function in patients with breast cancer

BACKGROUND: Human natural killer (NK) cells mediate spontaneous cytotoxicity against tumor cells and represent the main precursors of lymphokine-activated killer (LAK) cell activity. A comparison of some aspects of NK and LAK cell activity was undertaken in 85 preoperative patients with breast cancer and 75 healthy donors. METHODS: NK cell activity (tested in 18-hour cultures of effector peripheral blood mononuclear cells [PBMC] with K562 or MOLT-4 tumor target cells) was significantly diminished in these patients as it was the fully mature LAK cell activity (i.e., interleukin-2 (IL-2)-induced cytotoxicity in PBMC) against NK resistant target cells. Using immunoenzymatic methods we showed that the reduced NK cell activity was due to abnormally high levels of prostaglandin E2 (PGE2) produced by monocytes in culture. RESULTS: PGE2 was found to suppress the production of IL-2 in these cultures. Removal of monocytes from PBMC restored to almost normal levels the deficient NK and LAK cell activity in patients with breast cancer and was also associated with a normalization in the levels of PGE2 and IL-2. Indomethacin and gamma-interferon (IFN-gamma) increased the NK and LAK cell activity in these patients up to the levels of healthy donors. When highly purified CD56+ cells (obtained by an immunomagnetic isolation technique) were used as effector cells, no differences in LAK cell activity could be noticed between healthy donors and patients with cancer. FACS and northern blot analyses demonstrated a PGE2-mediated down-regulation of IL-2 receptor (IL-2R) expression on CD56+ cells that correlated with reduced LAK cell activity. This inhibitory effect of PGE2 was noticeable in long-term LAK cultures and was abrogated in the presence of IFN-gamma or indomethacin. CONCLUSION: This study may have important implications in the potentiation of NK and LAK cell activity for immunotherapeutic protocols in patients with breast cancer.     

Induction of lymphocytes cytotoxic to oral epithelial cells by Streptococcus mitis superantigen

The preparation of a superantigenic fraction F-2 from the culture supernatant of Streptococcus mitis 108, a fresh isolate from human tooth surfaces, was reported previously. Now, to determine the possible pathogenic role of the superantigen in oral mucosal diseases, we examined the cytotoxic effects of human peripheral blood T-cells activated with F-2 on human oral epithelial cells. T-cells activated with F-2 were cytotoxic to the human squamous carcinoma HO-1-N-1 cells derived from the oral mucosa, similar to those activated with Staphylococcus aureus enterotoxin B (SEB). This cytotoxic effect was increased in a dose-dependent manner by the addition of the respective stimulant, F-2 or SEB, to the cytotoxic assay system. F-2 endowed mainly CD8+ T-cells with cytotoxic activity. Pretreatment with human interferon gamma increased the sensitivity of the HO-1-N-1 cells to the cytotoxic effects of F-2-activated T-cells. The F-2-activated T-cells were also cytotoxic to human keratinocytes derived from gingiva. There was no correlation between the degree of cytotoxicity and the levels of tumor necrosis factor alpha in co-cultures of F-2-activated T-cells and HO-1-N-1 cells. A double-chamber plate experiment revealed no cytotoxic effects when the F-2-activated T-cells were separated from the HO-1-N-1 cells. Supernatants of the co-cultures of target and effector cells were not cytotoxic to HO-1-N-1 cells. These findings suggest that the cytotoxic effects of the F-2-activated T-cells on HO-1-N-1 cells were mediated not by soluble factors but by the direct interaction between the activated T-cells and the target cells. The cytotoxicity of the F-2-activated T-cells against HO-1-N-1 cells was markedly inhibited by monoclonal antibodies (MAbs) against CD11a and CD54, but was only slightly inhibited by MAbs against human leukocyte antigen (HLA)-DR and CD2. Thus, the interaction between lymphocyte-function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) was crucial for the F-2-dependent T-cell-mediated cytotoxicity against oral epithelial cells, while HLA-DR and CD2 molecules are not necessarily involved in the cytotoxicity observed.     

Increase in tumor necrosis factor-alpha mRNA but not perforin mRNA expression in response to two newly characterized anti-LFA-1 monoclonal antibodies

We have generated two monoclonal antibodies (mAb), designated anti-1B11 and anti-4F9, directed to the human lymphocyte-function-associated antigen-1 (LFA-1). Indirect immunofluorescence with both mAb showed a bimodal distribution of antigen on the surface of T, natural killer (NK), and lymphokine-activated killer (LAK) cells. Neither mAb reacted with the epitopes recognized by TA1 and Mo-1 mAb on the alpha-chain of the heterodimer. Anti-1B11 and anti-4F9 immunoprecipitated polypeptide chains with molecular weights of 177 and 95 kD. Both mAb inhibited cytolytic T lymphocytes (CTL), NK, and LAK cell-mediated cytotoxicity without affecting antibody-dependent cellular cytotoxicity (ADCC). The proliferative responses of T cells to allogeneic cells were inhibited by anti-1B11 and anti-4F9, whereas the responses to phytohemagglutinin P and concanavalin A were not affected. Anti-1B11 and anti-4F9 blocked effector cell (EC)-target cell (TC) conjugate formation by 50%. Only anti-4F9 cross-reacted with LFA-1 on porcine peripheral blood lymphocytes and inhibited porcine NK, LAK, and ADCC activities. Because LFA-1 also functions at the level of signal transduction during T cell activation and we previously showed that CTL rapidly degraded perforin and tumor necrosis factor-alpha (TNF alpha) mRNA after interaction with sensitive TC, we examined the effects of the mAb on the messages for perforin and TNF alpha. Treatment of CTL with anti-1B11 and anti-4F9 induced TNF alpha message and protein levels of TNF alpha, but did not alter perforin mRNA levels.     

Resistance of HER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared to HER2/neu-nonexpressing lines. HER2/neu+ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in "cold" target inhibition experiments. HER2/neu+ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and beta 1 integrins was comparable to that of HER2/neu- targets. In contrast, the HER2/neu+ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis of HER2/neu- carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression on HER2/neu+ cells contributes to their resistance. The modest binding and lysis of HER2/neu+ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 on HER2/neu+ targets. The following also supported deficiencies in post-binding events when HER2/neu+ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin. HER2/neu+ cell lines were still resistant to LAK cytolysis, and (b) HER2/neu+ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance of HER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.     

Anti-fungal and cytokine producing activities of CD8 + T lymphocytes from HIV-1 infected individuals

Lymphokine activated killer (LAK) cells are capable of killing not only malignant cells but also hyphal form of Candida albicans in vitro. When peripheral blood mononuclear cells (PBMC) from normal healthy donors were cultured for 72-96 hrs with 1,500 international unit (IU)/ml interleukin-2 (IL-2), marked LAK activity was induced. However, even prior to IL-2 activation, PBMC isolated from some normal subjects and those from almost all individuals who are infected by human immunodeficiency virus type 1 (HIV-1) exhibited significant levels of anti-fungal activity. Such pre-activation ("in situ") antifungal activity of PBMC decreased during the initial 48 hrs of IL-2 activation. PBMC from HIV-1 seropositive subjects showed higher levels of "in situ" anti-fungal activity than normal PBMC did. After a decline of "in situ" activity during the initial 48 hours, LAK activity gradually increased and reached near maximal levels by day 4 and remained more or less constant until day 6. No significant difference was observed between the LAK activity of normal and HIV-1(+) PBMCs on days 4-6. In IL-2 activated normal and HIV-1(+) PBMC cultures, both CD4 and CD8 T cells produced IL-2, INF-gamma as well as TNF-alpha. Production of IL-2 by both CD4 and CD8 T cells was suppressed in HIV-1(+) PBMC cultures, but no significant suppression of INF-gamma production was noted. Meanwhile, TNF-alpha production by CD4 was very much suppressed but no significant changes in TNF-alpha production by CD8 T cells was noted in HIV-1(+) PBMC cultures.     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon gamma (IFN gamma) and tumour necrosis factor alpha. A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 beta treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN gamma in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN gamma or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN gamma, IFN gamma treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.     

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.     

Effects of interferon-alpha and gamma on development of LAK activity from mononuclear cells in breast cancer patients

We examined the effect of recombinant IFN-alpha and IFN-gamma on induction of LAK cells from peripheral blood mononuclear cells (PBMNCs) in 7 pre-operative breast cancer patients and 4 healthy volunteers. Significant LAK activity was developed from PBMNCs of pre-operative breast cancer patients and healthy volunteers after incubation for 4 days with IL-2 (presence of IL-2 vs. absence of IL-2). Incubation of PBMNCs of pre-operative breast cancer patients with 1000 U/ml of IFN-alpha for 4 days suppressed the LAK activity significantly (P < 0.05). By contrast, incubation of PBMNCs of pre-operative patients with 1000 U/ml of IFN-gamma for 4 days increased the LAK activity significantly (P < 0.05). Significant cytotoxicity against MCF-7 cells (estrogen receptor positive human breast cancer cell line) was developed from PBMNCs of pre-operative breast cancer patients at 20:1 and 40:1 E/T ratios after incubation for 4 days with IL-2 (absence of IL-2 vs. 20:1 or 40:1, P < 0.05, P < 0.05), whereas PBMNCs of healthy volunteers did not. Stimulation of LAK cells with IFN-gamma produced a significant augmentation of cytotoxic activity against MCF-7 (P < 0.05), while IFN-alpha suppressed the cytotoxicity significantly (P < 0.05). These findings suggested that combined stimulation by IFN-gamma and IL-2 might be a reasonable treatment for breast cancer patients.     

Differential expression of natural killer cell markers: human versus baboon

The use of baboons as a model for the study of allo- and xenotransplantation has become increasingly important, but there are few studies on the basic immunological responses in baboons that might be relevant for a rejection reaction. In present study, the cell-surface phenotype, cytokine-induced activation and growth, and cytotoxicity of baboon and human natural killer (NK) and lymphokine-activated killer (LAK) cells were compared. A panel of murine monoclonal antibodies specific for human cell-surface markers expressed on lymphocytes was used to compare relevant baboon and human peripheral blood lymphocytes (PBL). Baboon PBL were 52.1+/-2.9% CD8+, 18.5+/-2.2% CD16+, 3.0+/-0.5% CD25+, and 5.5+/-1.8% CD69+. The corresponding proportions in humans were 23.8+/-7.1%, 12.8+/-3.2%, 4.5+/-1.0%, and 2.3+/-1.1%. In contrast to human PBL, less than 1% of baboon lymphocytes expressed CD56, CD57, and CD122 (interleukin [IL]-2Rbeta). Baboon lymphocytes showed NK cytotoxic activity against the human K562 and CEM cell lines, which was comparable to human NK activity. Depletion of baboon CD16+ or CD8+ cells led to dramatic decreases in NK cytotoxicity, and removal of both subsets completely abrogated NK activity. Incubation of baboon lymphocytes with human recombinant IL-2 for 1 week led to the appearance of CD56+ cells (11.3+/-2.8%). Most of the baboon CD56+ cells induced in culture were in S and G2 phases of cell cycle. Both baboon and human IL-2-activated lymphocytes were highly cytotoxic against the human LAK-sensitive cell line Daudi. Depletion of baboon CD8+ but not CD56+ cells significantly decreased LAK activity. These studies revealed differences in the NK system of humans and baboons that should be taken into consideration when analyzing immune responses to allo- and xenotransplantation in baboons.     

Role of spontaneous and interleukin-2-induced natural killer cell activity in the cytotoxicity and rejection of Fas+ and Fas- tumor cells

In the current study, we investigated whether the naive, poly I:C or interleukin-2 (IL-2)-induced natural killer (NK)/lymphokine-activated killer (LAK) cells use perforin and/or Fas ligand (FasL) to mediated cytotoxicity. We correlated these findings with the ability of mice to reject syngeneic Fas+ and Fas- tumor cells either spontaneously or after IL-2 treatment. The spontaneous NK-cell-mediated cytotoxicity was primarily perforin based, whereas the poly I:C and IL-2-induced NK/LAK activity was both FasL and perforin dependent. L1210 Fas+ tumor targets were more sensitive than L1210 Fas- targets to poly I:C and IL-2-induced cytotoxicity in wild-type, gld/gld, and perforin knockout mice. When L1210 Fas+ and Fas- tumor cells were injected subcutaneously (sc) or intraperitoneally into syngeneic mice, Fas- tumor cells caused mortality earlier than Fas+ tumor cells. Also, approximately 20% of the mice injected sc with L1210 Fas+ tumor cells survived the challenge(>60 days), whereas all mice injected similarly with L1210 Fas- tumor cells died. When immunotherapy using IL-2 (10,000 U, three times/d for a week, followed by once/d for an additional week) was attempted in mice injected sc with tumor cells, IL-2 treatment was very effective against mice bearing L1210 Fas+ (40% survival) but not L1210 Fas- (0% survival) tumors. These data correlated with the finding that the LAK cells from IL-2-injected mice caused increased cytotoxicity against L1210 Fas+ when compared with L1210 Fas- targets. Also, L1210 Fas+ tumor-bearing mice showed increased tumor-specific cytotoxic T lymphocyte (CTL) activity when compared with those bearing L1210 Fas- tumor cells. Together our studies show for the first time that expression of Fas on tumor targets makes them more immunogenic as well as susceptible to CTL- and IL-2-induced LAK activity. The Fas+ tumor cells are also more responsive to immunotherapy with IL-2.     

The lack of NK cytotoxicity associated with fresh HUCB may be due to the presence of soluble HLA in the serum

Human bone marrow transplantation is becoming more common in the treatment of certain forms of cancer despite the scarcity of HLA matched donors. Because human umbilical cord blood (HUCB) has been used as a source for stem cells in bone marrow transplantation, and because NK cells appear to be important in graft versus leukemia response, we investigated the lytic activity of freshly isolated HUCB NK cells (HUCB-NK) against tumor targets and their ability to differentiate into LAK cells following stimulation with various cytokines. Although cytotoxicity mediated by fresh HUCB-NK was low compared to that of adult peripheral blood lymphocyte-derived NK cells (PBL-NK), the ability of HUCB-NK to bind to K562 target cells (TC) was similar to PBL-NK. In addition, the PBL-NK cytotoxicity of postpartum mothers was also low compared to that of normal adult PBL-NK. When we incubated HUCB for 18 hr in either IL-2 or IL-12, we boosted the level of HUCB-NK cytotoxicity to approximately the level observed in PBL-NK and increased the level of perforin, granzyme A, and granzyme B mRNA expression. In addition, when we incubated HUCB in IL-2, IL-4, IL-7, IL-12, TNF-alpha, IFN-alpha, IFN-gamma, or TGF-beta for 5 days, we observed that HUCB was capable of generating LAK cells only when incubated with either IL-2 or IL-12. In contrast, IL-2, IL-7, IL-12, TNF-alpha, and IFN-gamma all generated LAK cells from adult PBL. When we added to the medium low-dose IL-2 and irradiated K562 as feeder cells (mini-LAK), we were unable to generate LAK activity from HUCB-NK, whereas we could generate it with PBL-NK cells under the same conditions. Addition of serum derived from HUCB in a 4-hr 51Cr release assay with PBL-NK as the effector cells (EC) and K562 as the TC resulted in a 42% decrease in PBL-NK-mediated cytotoxicity. Although we detected no TGF-beta in HUCB serum, we did detect high concentrations of soluble class I MHC (sHLA). To our knowledge, sHLA has not previously been shown to inhibit NK cytotoxicity, although the expression of class I HLA on the surface of TC has been shown to inhibit NK cytotoxicity. To study further the effect of sHLA on cell-mediated cytotoxicity, we added various concentrations of sHLA to EC mediating NK, ADCC, and CTL activities. All were inhibited in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)