Corticosteroids inhibit the generation of lymphokine-activated killer activity in vitro

Summary In phase-I clinical trials of adoptive immunotherapy using lymphokine-activated killer (LAK) cells plus recombinant interleukin-2 (rIL-2) (Cetus) for the treatment of malignant glioma, we observed that blood mononuclear cells (MNC) from patients dependent on dexamethasone for management of cerebral edema produced substantially less LAK activity as compared to MNC of normal blood donors or glioma patients not receiving steroid therapy. Therefore, we examined the in vitro effects, brought about by therapeutically attainable concentrations of various corticosteroids, on the proliferative response, production of interferon (IFN-), and induction of LAK activity from blood MNC of normal donors. Incubation in media containing rIL-2 (1000 U/ml) with either dexamethasone, hydrocortisone, methylprednisolone, or prednisolone profoundly affected all of these parameters. First, while 0.01 g/ml of either dexamethasone or hydrocortisone caused a slight enhancement of the mitogenic response of lymphocytes to phytohemagglutinin, a dose-dependent decline occurred as concentrations increased to 10 g/ml. The addition of prednisolone and methylprednisolone elicited a dose-dependent inhibition of lymphocyte proliferation over the entire concentration range tested. At 0.1 g/ml or higher, dexamethasone, hydrocortisone, methylprednisolone and prednisolone significantly (P<0.02) inhibited the production of IFN-: respectively 18.9%, 4.4%, 2.2%, and 12.3% of the IFN- produced by MNC in the absence of steroids. All four corticosteroids inhibited the induction of LAK activity. Compared to MNC that had been incubated with 1000 U/ml rIL-2 alone, MNC cultured with rIL-2 and 10 g/ml either dexamethasone or prednisolone demonstrated significantly lower cytotoxicity (P<0.05) for the natural-killer-cell-resistant cell line, Daudi. Culturing MNC with hydrocortisone had a more dramatic result, causing a significant decline (P<0.01) in lytic activity at both 1.0 g/ml and 10 g/ml, while incubation with methylprednisolone produced a significant drop (P<0.02) in LAK-mediated cytotoxicity at 0.1 g/ml as well as 1.0 g/ml and 10 g/ml. When cytotoxicity was expressed as lytic units per million effectors, a dose-response decline in lytic activity was once again apparent, with hydrocortisone, methylprednisolone and prednisolone showing significant inhibition (P<0.05) at both 1.0 g/ml and 10 g/ml and dexamethasone at 10 g/ml (P<0.01). These results indicate that corticosteroids commonly used in the management of cerebral tumors and other malignancies inhibit induction of LAK activity in vitro, and this may explain why it is often difficult to generate LAK activity from blood MNC of patients who are receiving chronic steroid therapy.     

Soluble factors produced by macrophages/monocytes inhibit lymphokine-activated killer activity in rat splenocyte cultures

In the present study we investigated the inhibition of interleukin-2(IL-2)-induced lymphokine-activated killer (LAK) activity in rat splenocyte cultures in relation to the presence of 2-mercaptoethanol and macrophages/monocytes. The presence of 2-mercaptoethanol is necessary for induction of LAK activity in rat splenocyte cultures. Removal of macrophages/monocytes from rat splenocytes by plastic or nylon-wool adherence, or iron ingestion resulted in LAK induction by IL-2 in the absence of 2-mercaptoethanol. The effect of macrophages/monocytes on LAK activity was also studied in transwell co-cultures. In the absence of 2-mercaptoethanol, the induction of LAK activity was very low in macrophage/monocyte-depleted splenocytes with macrophages/monocytes in the upper compartment of a transwell culture. In contrast, in the presence of 2-mercaptoethanol a high level of LAK activity was induced in these transwell cultures, showing that 2-mercaptoethanol abolished the LAK-inhibiting capacity of macrophages/monocytes. In addition, established LAK activity was strongly inhibited when, after LAK induction, splenocytes were cultured with supernatant of unfractionated splenocytes, which were cultured with IL-2 but in the absence of 2-mercaptoethanol. Addition of 2-mercaptoethanol abrogated the inhibiting effect of the supernatant completely. These experiments demonstrate that rat macrophages/monocytes produce 2-mercaptoethanolsensitive soluble LAK-inhibiting factors. Ultrafiltration of conditioned culture medium of macrophages/monocytes revealed the presence of LAK-inhibiting factors larger than 10 kDa. We concluded that 2-mercaptoethanol-sensitive soluble factors produced by macrophages/monocytes determine the level of LAK induction in rat splenocyte cultures.     

Lysis of human monocytes by lymphokine-activated killer cells

Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.     

Induction of lymphokine-activated killer cell against human leukemia cells in vitro

The induction of lymphokine-activated killer (LAK) cells against fresh human leukemia cells was investigated. Two thirds of the 62 leukemias examined were susceptible to the lytic effect of allogeneic IL-2 induced LAK cells in vitro. No substantial differences could be detected between myeloid or lymphoid leukemias or with regard to the FAB subtype or the immunophenotype. Culturing mononuclear cells from peripheral blood or bone marrow of leukemia patients with IL-2 resulted in an expansion of residual large granular lymphocytes and development of cytotoxic activity. The combination of IL-2 with IFN-gamma or the presence of tumor cells during the activation process led to an enhancement of LAK cell cytotoxicity. These results suggest that LAK cells may be useful in the treatment of leukemia.     

Inhibition of lymphokine-activated killer cell generation by cultured tumor cell lines in vitro

Summary The co-culture of human peripheral blood mononuclear cells (PBMC) with high concentrations of interleukin 2 normally generates lymphokine-activated killer (LAK) cells capable of indiscriminate lysis of tumor targets. However, the addition of certain cell-line-derived tumor cells to the LAK generation cultures within the first 48 h of culture initiation resulted in the suppression of the LAK cytotoxicity measured after 3–4 days of culture. Suppression could be achieved with tumor cell:PBMC ratios as low as 1:50 when tumor cells were derived from melanoma and colorectal cancer (G361, COLO320, HT-29), but suppression was not observed with cells from the breast cancer cell line SKBr3. No suppression of LAK generation was observed with normal epithelial cells from colon or breast, with autologous or allogeneic lymphoblasts, or with allogeneic vascular endothelial cells. Suppression was independent of the removal of adherent cells from PBMC, could not be prevented by indomethacin and was not attributable to interleukin 2 absorption/adsorption by tumor cells. The suppressive activity of some tumor cells could be augmented by preculture in recombinant gamma interferon. Serum-free supernatants from G361, COLO320 and HT-29 (but not SKBr3 or endothelial cells) were also highly suppressive towards the generation of LAK cells. The elaboration by tumor cells of fractors capable of inhibiting LAK generation may partially explain the failure of LAK/interleukin 2 therapy in some experimental and clinical protocols.     

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 (IFN) and tumour necrosis factor . A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN 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 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, IFN 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.     

Inert particles inhibit natural killer cell function in vitro

Aqueous suspensions of inert particles were found to inhibit the baseline and interferon-enhanced natural killer (NK) cell activity of peripheral blood mononuclear cells (PBMC) and large granular lymphocytes (LGLs). This inhibition was induced with latex, silica, and Sephadex particles. The suppression of NK activity was not related to effector cell death as determined by trypan blue exclusion. The inhibition of NK cell function was more pronounced with prolonged incubation and could be partially reversed with monocyte depletion or the addition of indomethacin, a prostaglandin synthesis inhibitor, but not with the addition of the lipoxygenase inhibitors nordihydroguaiaretic acid and BW755C. Similarly, particle exposure inhibited the NK cell function of monocyte-depleted large granular lymphocytes with and without the add-back of glass adherent cells, implying that monocyte-independent NK suppressive mechanisms were also present. These data demonstrate that inert particles are immunosuppressive in vitro and can inhibit baseline and interferon-stimulated NK cell function of LGLs and PBMC through monocyte-dependent and independent pathways.     

Human lymphokine-activated killer (LAK) cells

Summary Pretreatment of peripheral blood mononuclear cells (PBMC) with 5 mMl-phenylalanine methyl ester (PheOMe) provides an efficient means to deplete monocytes. PheOMe does not affect the number of large granular lymphocytes after the pretreatment, but does inhibit natural killer cell cytotoxicity temporarily after the pretreatment. However, depletion of monocytes by PheOMe allows lymphokine-activated killer (LAK) cell generation with recombinant interleukin-2 (rIL-2) at high cell density (> 5 × 10⁶ cells/ml). The time of the PheOMe pretreatment is 40–60 min, though some effect could be observed within 15 min, and the pretreatment could be performed at room temperature. Pretreatment density of PBMC with 5 mM PheOMe could be achieved at cell density up to 3 × 10⁷ cells/ml. PheOMe-pretreated cells could be activated by rIL-2 in serumless media at high cell density. Pretreatment of PBMC with 5 mM PheOMe provides an efficient means to deplete monocytes, as compared to plastic and nylonwool adherence. LAK cell generation is similar in both methods of monocyte depletion; therefore, depletion of monocytes allows, LAK cell generation at high cell density. The PheOMe procedure provides an improved and convenient process for preparing LAK cells for adoptive immunotherapy.     

Heterogeneous lymphokine-activated killer cell precursor populations

Summary We developed a monoclonal antibody (mAb) 211, which recognizes the precursors in peripheral blood of lymphokine-activated killer cells (LAK) induced by recombinant interleukin-2 (rIL-2). In conjunction with complement mAb 211 also eliminates natural killer cells (NK) and a majority of the cytotoxic T lymphocytes. B cells and monocytes do not express the 211 antigen. Since mAb 211 recognized such a large percentage of peripheral blood lymphocytes we examined which 211⁺ subpopulation was the predominant precursor of rIL-2-induced LAK cells using two-color fluoresence-activated cell sorting (fluorescein-conjugated 211 mAb plus phycoerythrin-CD11b). This method identified the 211⁺/ CD11b⁺ population as the predominant phenotype of the rIL-2-induced LAK precursor. In addition, we directly compared the phenotype of the LAK precursor induced by delectinated T-cell growth factor (TCGF) to that induced by rIL-2. The 211-depleted population, which was devoid of NK cells and LAK precursors (inducible by rIL-2), was capable of generating LAK activity when TCGF was used as the source of lymphokine. LAK cells induced by TCGF from the 211-depleted population lysed a fresh sarcoma and an NK-resistant cultured melanoma tumor target but not the Daudi cell line, which was lysed by rIL-2-induced LAK cells. Lymphoid subpopulations, depleted using NKH1a mAb, behaved similarly, generating high levels of lysis against the two solid tumor targets when cultured with TCGF but not with rIL-2. CD 3-depleted populations showed enrichment for LAK precursors using either rIL-2 or TCGF. These results indicate that while rIL-2-induced LAK precursors cannot be separated from cells with NK activity, TCGF-induced LAK cells can be generated from populations of peripheral blood mononuclear cells without NK activity.     

IL-4 regulation of murine lymphokine-activated killer activity in vitro. Effects on the IL-2-induced expansion, cytotoxicity, and phenotype of lymphokine-activated killer effectors

The in vitro incubation of B6 splenocytes with purified, mouse rIL-4 for 4 to 5 days was sufficient to generate lymphokine-activated killer (LAK) activity. In addition, rIL-4 augmented LAK cytotoxic activity when combined with rIL-2, as measured in a 4 h 51Cr-release assay against fresh, syngeneic MCA-sarcoma (MCA-102 and MCA-105) cells. Interestingly, this augmentation was not observed against the cultured YAC-1 target. LAK generation and augmentation of cytotoxicity by rIL-4 was species-specific, because human rIL-4 (up to 20,000 U/ml) failed to elicit these effects in the mouse splenocyte cultures. When 5-day B6 LAK cells (splenocytes incubated in rIL-2 at 1000 U/ml for 5 days) were split and recultured in the combination of rIL-2 plus rIL-4 for 4 additional days at least a twofold greater expansion in cell number resulted compared to similar cells cultured in either rIL-2 or rIL-4 alone. Moreover, LAK cells expanded in rIL-2 plus rIL-4 exhibited substantial increases in in vitro cytolytic activity (on a per cell basis) against MCA-102 and MCA-105 sarcoma cells, but not against YAC-1 targets. FACS analysis or negative selection using Lyt-2 or NK-1.1 mAb plus C revealed no differences in effector phenotype(s) of LAK cells expanded in rIL-2 alone compared to rIL-2 plus rIL-4 to account for the differences observed in both expansion and cytolytic activity by rIL-4. The majority of cells was Thy-1+, Lyt-2+, T3+, and ASGM-1+. However, a marked increase in the granule-associated serine esterase, BLT-E, was found only in LAK cells expanded in the combination of both lymphokines. Collectively, these studies show that rIL-4 has potent regulatory activities on splenic LAK generation, expansion, and cytotoxic function in the mouse.     

Natural killer and lymphokine-activated killer cell functions in chronic myeloid leukemia

Summary The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors — 2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.     

Heterogeneity of the lymphokine-activated killer cell phenotype.

Lymphokine-activated killer cells (LAK) are functionally defined by their ability to mediate the MHC-unrestricted lysis of a range of tumor targets, while sparing normal cells. They can also lyse TNP-modified normal syngeneic lymphoblasts. We show here that lysis of TNP-modified targets is mediated by CD8+ LAK in a self-MHC-restricted manner, whereas lysis of tumor targets is largely by CD8- LAK and is MHC-unrestricted. LAK generated from the immune-deficient strains Balb/c nude and C.B-17 scid lyse tumor targets as effectively as LAK from normal mice but do not lyse TNP-modified normal targets. Further, lysis of TNP-modified targets, but not tumors, can be inhibited by antibody to the T cell receptor complex. These experiments strongly suggest that recognition of TNP-modified targets is not accomplished by the same mechanism as that of tumors. Rather, they are consistent with recognition of TNP-modified targets by CD8+ LAK cells being mediated via recognition through the T cell receptor.     

Leukoregulin up-regulation of tumor cell sensitivity to natural killer and lymphokine-activated killer cell cytotoxicity

The present investigation demonstrates that leukoregulin, a cytokine secreted by natural killer (NK) lymphocytes up-regulates the sensitivity of tumor cells to lymphokine-activated killer (LAK) cell cytotoxicity. It has been previously established that leukoregulin increases the sensitivity of sarcoma, carcinoma and leukemia cells to natural killer (NK) cell cytotoxicity. Tumor cells were treated with leukoregulin for 1 h at 37 degrees C and tested for sensitivity to NK and LAK cytotoxicity in a 4-h chromium-release assay. NK-resistant Daudi, QGU and C4-1 human cervical carcinoma cells became sensitive to NK cytotoxicity after leukoregulin treatment, and their sensitivity to LAK was increased two- to sixfold. Y-79 retinoblastoma cells, which are moderately sensitive to NK and very sensitive to LAK, became increasingly sensitive (two- to four-fold) to both NK and LAK cell cytotoxicity. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant interleukin-1 (alpha and beta), recombinant interferon gamma, recombinant tumor necrosis factor or combinations of the latter two failed to up-regulate tumor cell sensitivity to NK and LAK cell cytotoxicity. However, treatment with recombinant interferon gamma for 16-18 h, GM-CSF and interleukin-1 beta for 1 h induced a state of target cell resistance to both NK and LAK cell cytotoxicity. Leukoregulin may have an important physiological function in modulating NK and LAK cell cytotoxicity by increasing the sensitivity of target cells to these natural cellular immunocytotoxicity mechanisms.     

Cytostatic and cytotoxic activity of lymphokine-activated killer cell supernatants

Summary Incubation of peripheral blood mononuclear cells with interleukin-2 (IL-2) results in the release of a factor which is cytostatic and cytotoxic both to tumor cell lines (A375M, A375P, C480, MCF-7, Hey) and fresh tumor cells (in the human tumor cloning assay), including breast cancer, colon cancer, melanoma, myeloma and ovarian cancer. The factor cannot be detected in a 4-h chromium-release assay, but is best demonstrated after tumor cells have been to it for exposed 3 days. The factor is not cytotoxic to normal peripheral blood leukocytes or normal fibroblasts, and is not toxic to certain targets sensitive to lymphokine-activated killer (LAK) cells, such as K562 and Daudi cells. The factor is diffusible, non-dialyzable, relatively stable to heat and acid and does not contain appreciable amounts of targets resistant to interferon- and , tumor necrosis factor and interleukin-1. The data suggest that there are several mechanisms of LAK cell activity against tumor cells including one which requires direct interaction of LAK and tumor cells and one which is mediated by LAK cell supernatant. The former is detected by 4-h chromium release while the latter is not.Supported by a grant from the Arizona Disease Research Commission and by Grants CA-17094 and CA-23074 from the National Institute of Health     

Accessory cells, dendritic cells, or monocytes, are required for the lymphokine-activated killer cell induction from resting T cell but not from natural killer cell precursors

In this study we have investigated the role of accessory cells in the development of lymphokine-activated killer cells (LAK) from highly purified human NK and small resting T cell progenitors. As accessory cells we used autologous, as well as allogeneic, monocytes, and dendritic cell enriched cells. Both NK and T cells were able to generate LAK activity, but their activation requirements were different. NK cells were activated merely by IL-2, and accessory cells did not enhance their lytic activity in the presence or absence of IL-2. Conversely, T cells were practically unresponsive to even high concentrations of IL-2 having a strict requirement for accessory cells for the development of lytic activity and proliferation. Accessory cells differed in their ability to activate T cells presumably depending on their ability to induce IL-2 synthesis, allogeneic dendritic cells being the most effective accessory cells and IL-2 synthesis stimulators. Allogeneic accessory cells could induce lytic activity in T cells even in the absence of exogenous IL-2. Thus, accessory cells play a central role in expanding the LAK effector cell population.     

Human lymphokine-activated killer cells: further isolation and characterization of the precursor and effector cell

In a series of experiments we have demonstrated the progressive enrichment (5- to 40-fold) in lymphokine-activated killer (LAK) precursor activity by adherence depletion, sheep red cell rosetting, and depletion of CD3- and DR-positive lymphocytes. The LAK precursor cell thus appears to fall within the 'null' cell population. CD16 and CD11 are cell surface antigens expressed on the surface of the LAK precursor as demonstrated in sorting experiments. A 6- to 100-fold enrichment compared to unseparated peripheral blood was noted when sorted cells positive for CD16 and CD11 were tested. The LAK effector has been identified as being primarily CD3- and CD2+. Similar sorting equipment demonstrated a 7- to 500-fold difference in lytic activity for fresh tumor when comparing CD2+/CD3- and CD2+/CD3+ cells. The CD16+/CD11+ lymphocyte can proliferate in response to interleukin-2 (IL-2) alone in the absence of accessory cells and can be expanded in IL-2 alone with maintenance of lytic activity.     

Characteristics of interleukin-6-enhanced lymphokine-activated killer cell function

To study the effect of IL-6 on the development of cytotoxic cells, we examined lymphokine-activated killer (LAK) activity generated from human nonadherent PBL. Addition of rIL-6 at the initiation of 5-day PBL cultures significantly increases LAK activity in the presence of low concentrations (between 5 and 25 u/ml) of rIL-2. RIL-6 alone induces no PBL LAK activity but at doses as low as 0.8 u/ml rIL-6 enhances LAK activity with optimal enhancement of LAK at 5.0 u/ml of rIL-6. This enhancement is independent of effects on cells growth as rIL-6 did not affect the cell recovery of PBL cultured in rIL-2. RIL-6-enhanced LAK is mediated by the same type of effector cells as those of LAK from rIL-2 alone with effector cells primarily generated from large granular CD3-negative E rosetting lymphocytes. RIL-6 does not change the time course of LAK development and pretreatment of PBL with rIL-6 has no effect on the PBL response to subsequent rIL-2 induction of LAK. Addition of rIL-6 to LAK cultures 2 hr before the cytotoxicity assay shows equal enhancement as addition at the initiation of the culture. However, rIL-6 requires the presence of both rIL-2 and another factor in the supernatant from LAK cultures in order to enhance LAK. Our results indicate that IL-6 can modulate LAK activity at a very late stage of LAK development, and that the enhancement by IL-6 is dependent on the presence of IL-2 and another soluble factor generated during rIL-2 culture.     

Generation of lymphokine-activated killer cell activity from human thymocytes

We have generated lymphokine-activated killer (LAK) cells from human thymocytes in order to assess the relationship between LAK cells and T cells. Fresh thymocytes lack natural cytotoxic activity, and cytotoxicity cannot be stimulated by short term (1 hr) incubation with interferon or recombinant interleukin 2 (rIL-2). In addition, thymocytes are phenotypically devoid of cells bearing the natural killer (NK)-associated markers cluster designation (CD) 16 and NKH-1. After culture for 5 to 8 days with rIL-2, thymocytes display high levels of cytotoxic activity against both NK-sensitive and NK-resistant targets. Thymocytes require slightly more IL-2 than do peripheral blood lymphocytes to generate LAK activity. We have examined the phenotype of the thymocyte LAK precursor and effector cells. Thymocyte LAK precursors are of low to medium density, CD1-negative, and predominantly CD3-negative. Although CD3-positive cells proliferate in response to rIL-2, they are low in cytolytic capabilities. The effector cells, like the LAK precursors, are low to medium density lymphocytes. The cytotoxic cells are predominantly CD3-negative, and cytotoxic activity cannot be blocked with the use of anti-CD3 monoclonal antibodies. The effector cells also lack most NK-associated markers (HNK-1, and the CD16 markers Leu-11b and B73.1) but possess the NK-associated marker NKH-1 (N901). The responsive cell appears to be at a very early stage of thymic development, and it does not appear to either require or express the CD3-T cell receptor complex.     

Chemotherapy-induced modulation of natural killer and lymphokine-activated killer cell activity in euthymic and athymic mice

Combinations of chemotherapy and interleukin-2 (IL-2) aimed at improving therapeutic efficacy in cancer patients have generally proved disappointing. Although chemotherapy blocks tumor growth and sometimes boosts immune functions, most drugs are immunosuppressive, at least transiently. Therefore, it is reasonable to assume that maximal exploitation of the immunostimulatory and antitumor activity of both modalities requires careful coordination of chemotherapy and IL-2 timing. We analyzed the temporal effect of 5-fluorouracil (5-FU, 100–120 mg/kg), cyclophosphamide (CY, 100 mg/kg), Adriamycin (8 mg/kg) and dacarbazine (100 mg/kg) on the activation of natural killer/lymphokine-activated killer (NK/LAK) cells by IL-2 in several strains of euthymic mice and in athymic nude mice. Following in vivo or in vitro exposure to IL-2 1–15 days after chemotherapy, the total lytic activity of the spleen and the number of LAK precursors (LAK-p) were measured. In euthymic mice injected with IL-2 (5×10⁴ Cetus units twice daily for 4–5 days), 5-FU augmented (up to 37-fold, days 1–9) and CY reduced (up to day 6) LAK activity, as compared with that in the IL-2 control. In bulk cultures containing IL-2 (1000 CU/ml, 3–4 days), both 5-FU and CY reduced LAK activity of euthymic mice splenocytes for up to 6 days after chemotherapy, which was followed on day 9 by full recovery. In splenocytes of nude mice, 5-FU increased and CY diminished LAK activation in bulk cultures, starting 3 days after chemotherapy. In athymic mice, 5-FU markedly augmented the total number of LAK-p/spleen (up to 30-fold, days 3–9), as determined by limiting-dilution cultures with IL-2 (for 7–8 days). In euthymic mice, in contrast, LAK-p levels decreased for up to 6–9 days after treatment with 5-FU, Adriamycin or dacarbazine, later recovering to pretreatment levels, whereas CY markedly increased LAK-p (up to 15-fold) when administered 6–12 days before limiting-dilution culture initiation. The effect of chemotherapy on LAK and NK activity was essentially similar. In other experiments, a subset of asialoGM1-LAK-p was found in the spleens of 5-FU-treated mice, but not in untreated mice. Our results suggest that the immunomodulatory effect of chemotherapy on NK/LAK activity in mice is variable and largely depends on the drug itself, the interval between chemotherapy and IL-2 administration, the strain of mice and the assay used.