Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Clinical studies on cell-mediated immunity in patients with renal cell carcinoma: interleukin-2 and interferon-γ production of lymphocytes

Summary The authors examined interleukin-2 (IL-2) production and interferon (IFN) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, 5 cm and >5 cm. The production of IL-2 and IFN was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN production was impaired. However, in the patients with tumors 5 cm, IFN production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN production.     

Antitumor effect of combination of murine recombinant interferon β, murine recombinant interferon γ and human recombinant interleukin-2 in MethA-bearing mice

Summary We have previously reported that the combination of murine recombinant interferon (Mu-rIFN) with murine recombinant interferon (Mu-rIFN) provided greater inhibition of tumor growth than did each one alone in MethA-bearing mice. In the present study the effect of addition of human recombinant interleukin-2 (Hu-rIL-2) to the combination of Mu-rIFN with Mu-rIFN on tumor growth in BALB/c mice bearing syngeneic MethA fibrosarcoma was examined. Low doses of Hu-rIL-2 (5 × 10³ U or 5 × 10⁴ U at 3-day intervals) showed no antitumor activity, while a high dose of Hu-rIL-2 (5 × 10⁵ U) showed profound growth inhibition. The administration of IL-2 (ranging between 5 × 10³ U and 5 × 10⁵ U) in addition to the combination of IFN and IFN showed more augmented antitumor effects in a dose-dependent manner. Furthermore, the simultaneous administration of IL-2, IFN and IFN had more effective therapeutic activity, compared with the sequential administration of interferons and IL-2. These findings indicated that IL-2 in combination with IFN and was effective for cancer treatment.     

Complete response of metastatic renal cell carcinoma to low-dose interferon-γ treatment

The course of metastatic renal cell carcinoma may be positively influenced by immunotherapeutic agents. We report a case of renal cell carcinoma showing a complete response to once-weekly low-dose s. c. interferon- (INF) treatment in multiple metastatic sites (lung, chest wall, abdomen, vertebral body), but concomitantly developing a solitary brain metastasis. High initial interleukin-6 (IL-6) levels returned to normal during IFN treatment suggesting that IFN may have interrupted an autocrine IL-6/IL-6-receptor loop of the tumor cells. The duration of complete remission in the extracerebral sites is now 46+ months. IFN may be less active beyond the blood/brain barrier.     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.     

Phenotypic analyses of lymphokine-activated killer cells that release interferon γ and tumor necrosis factor α

Summary We recently reported that interleukin-2(IL-2)-activated peripheral blood lymphocytes and CD3⁺, lymphokine-activated killer (LAK) cell clones release tumor necrosis factor (TNF) and interferon (IFN) when stimulated with K562 erythroleukemia cells. We examined the phenotype of IL-2-activated peripheral blood leukocytes that secrete TNF and IFN when stimulated with K562 cells and demonstrated that TNF secretion is not due to the presence of contaminating mononuclear phagocytes. Further, we demonstrate that IL-2-activated natural killer (NK) cells release only IFN when stimulated with K562 cells while T lymphocytes exposed to monoclonal anti-CD3 and K562 cells secrete both TNF and IFN. However, T cells stimulated only with K562 cells did not release IFN or TNF while the admixture of these T cells with NK cells, when stimulated with K562 cells, released levels of TNF comparable to those produced by the unseparated cells. At present it is unclear whether only one or both effector cell types respond to K562 by releasing TNF or why the presence both cell types is needed.This work was supported by grants from the national Institutes of Health (CA 23074 and CA 17094) and the Arizona Disease Commission (8277-000000-1-0-YR-9301)     

Increasing infiltration and activation of CD8+ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois     

Interactions between interferon γ and retinoic acid with transforming growth factor β in the induction of immune recognition molecules

The cell-surface expression of major histocompatibility (MHC) antigens and the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) is essential for target cell recognition by T lymphocytes. The expression of both classes of molecule is induced by various cytokines, notably interferon (IFN). Since transforming growth factor (TGF) has been recently reported to antagonise HLA-DR induction by IFN we have examined, using a number of murine and human cell lines, the effect of TGF on IFN-induced MHC class I and class II and ICAM-1 expression. All of the cell lines tested expressed elevated class I MHC following IFN treatment. Class II MHC induction was seen on most but not all of the cells, the exceptions being among a panel of human colorectal carcinoma cell lines. A striking difference between cells of different origin was noted in the response to TGF. TGF was found to antagonise IFN-induced class I and class II MHC expression on C3H 10T1/2 murine fibroblasts, early-passage BALB/c mouse embryo fibroblasts, a murine oligodendroglioma cell line, and on MRC5 human fibroblasts and two human glioblastoma cell lines. Class II MHC was much more strongly inhibited (sometimes completely) than class I MHC. TGF also inhibited induction of class I MHC expression by IFN. However, TGF did not inhibit class I or class II MHC induction by IFN in any of the nine colorectal carcinoma cell lines, although two of five of the lines tested were growth-inhibited by TGF. On the other hand, human ICAM-1 induction by IFN was not affected by simultaneous treatment with TGF in any of the cell lines. The down-regulation of IFN-induced MHC antigens by TGF is not, therefore, the result of a general antagonism of IFN. Retinoic acid has recently been reported to induce ICAM-1 expression on human tumour cells. We have confirmed this observation on MRC5, and the two human glioblastoma cell lines, however six colorectal carcinoma cell lines tested did not respond. In contrast to IFN-induced ICAM-1 expression, retinoic-acid-induced ICAM-1 expression was inhibited by TGF on two of the three responsive lines.     

Cytokine production in whole blood cell cultures of patients undergoing therapy with biological response modifiers or 5-fluorouracil

We have measured the levels of interferon (IFN), tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-1, and IL-2 in the whole blood cell culture supernatants of 43 tumor patients undergoing a treatment with biological response modifiers or a conventional therapy with 5-fluorouracil and leucovorin. In the blood cell cultures of the 16 patients who received 5-fluorouracil and leucovorin IFN levels decreased (P0.01) and TNF levels rose (P0.05) during each therapy cycle. However, in the blood samples a declining number of total leukocytes and lymphocytes was measured (P0.05). Progressive disease could be correlated to a tendency towards lower IFN levels in the pretherapeutic cultures of these patients. The second group analyzed consisted of 8 patients receiving a low-dose IL-1 therapy. In this group we found either an unchanged or an augmented IFN production of the blood cells during treatment. In the group of 13 patients receiving low-dose recombinant IL-2 (4.5×10⁶IU m^–2 day^–1) significantly increasing IFN levels were seen in the blood cell cultures during the therapy (P0.05), although total leukocyte counts decreased. In this group, 4 had stable disease for at least 2 months and 9 patients had tumor progression under therapy. In the cultures of the latter a tendency towards lower IFN values was found. Finally, the cytokine production in the blood cell cultures of 6 patients receiving a combination therapy of IFN and high-dose IL-2 was studied. During this therapy a dramatically reduced production not only of IFN but also of all other measured cytokines was found. In this group all patients had tumor progression under therapy. It is concluded that the measurements of cytokine production in a reproducible whole blood culture system may be useful for monitoring immunological therapies and may help us to find out which doses of biological response modifiers have enhancing or suppressive effects on the functions of the immune cells.     

Production of tumor necrosis factor α and interferon γ in interleukin-2-treated melanoma patients: Correlation with clinical toxicity

Summary Interleukin-2 (IL-2)-based immunotherapy regimens are accompanied by dose-limiting toxicity consisting of fever, tachycardia, chills and capillary leak syndrome. We hypothesized that the toxicity was caused by the induction and release of endogenous cytokines such as tumor necrosis factor (TNF) and interferon (IFN). We measured the serum levels of TNF and IFN in IL-2-treated melanoma patients and attempted a correlation with clinical toxicity. A total of 23 patients received either 6 × 10⁶ IU or 12 × 10⁶ IU Cetus IL-2/m² by i. v. bolus daily for 5 consecutive days on weeks 1, 3 and 5. Serum TNF and IFN levels were measured by enzyme-linked immunosorbent assay. Clinical toxicity was scored each day by objective measurements of hypotension, tachycardia, fever and chills/rigors. Clinical toxicity and IFN levels correlated nicely, peaking on the 5th day of each treatment cycle. The kinetics and magnitude of TNF production, however, were not predictable and did not correlate with either IFN or toxicity. Some patients had modest increases in TNF production while others had markedly increased levels during the second and third treatment weeks. Remarkably, these high levels persisted during nontreatment weeks and after completion of therapy. This clinical study demonstrates novel kinetics for immunoreactive TNF in IL-2 cancer patients, which do not correlate well with toxicity.This work was supported by NIH Grants CA 50 780 (J. E.) and CA 29 605, CA 12 582 (D. L. M.) and the U. C. Tobacco-Related Disease Research Program RT-62 (J. E.). J. E. is the recipient of an NCI Clinical Investigator Award (KO8-01360) and is a Dorothy and Leonard Straus Scholar at UCLA     

Ixodid tick salivary gland extracts inhibit production of lipopolysaccharide-induced mRNA of several different human cytokines

Extracts prepared from the salivary glands (SGE) of partially fed adult female Rhipicephalus appendiculatus ticks reduced the expression by human peripheral blood leukocytes (PBLs) of lipopolysaccharide (LPS)-stimulated cytokine mRNA. Treatment with SGE had no obvious effect on cytokine mRNA production when compared with untreated PBLs. LPS treatment induced or increased mRNA production for IFN, IFN, TNF-, IL-1, IL-1, IL-5, IL-6, IL-7 and IL-8. All the LPS-stimulated cytokine mRNAs were reduced when treated with a mixture of LPS and SGE. The results indicate the potential of ticks in modulating the cytokine network of their vertebrate hosts, possibly to facilitate blood feeding.     

Retroviral transduction of interferon-γ cDNA into a nonimmunogenic murinefibrosarcoma: generation of T cells in draining lymph nodes capable of treating established parental metastatic tumor

Gene modification of tumor cells with the cDNA for interferon (IFN) has been shown to increase the immunogenicity of some tumor cells. In order to explore further the possible therapeutic relevance of these previous findings, two clones of the nonimmunogenic MCA-102 fibrosarcoma of C57BL/6 origin were retrovirally transduced with the cDNA encoding murine IFN: 102.4JK (4JK), a clone with relatively high major histocompatibility complex (MHC) class I expression, and 102.24JK (24JK), a clone with low expression of surface MHC class I molecules. Retroviral transduction of tumor cells with the cDNA encoding for IFN resulted in a substantial up-regulation of MHC class I surface expression in the 24JK clone but little change of class I in the 4JK clone. In an attempt to generate antitumor lymphocytes, these gene-modified cells were inoculated into mouse footpads and draining lymph nodes (DLN) were removed, dispersed, and cultured in vitro for 10 days with irradiated tumor cells and interleukin-2. DLN from mice bearing either unmodified tumor or tumor transduced with cDNA encoding neomycin resistance (Neo ^R) or IFN, were used to treat recipients harboring 3-day pulmonary metastases induced by the parental, unmodified tumor. Treatment with DLN cells obtained following the injection of 24JK tumor cells modified with the gene for IFN significantly reduced the number of pulmonary metastases in four separate experiments, compared to groups treated by DLN cells generated from inoculation of either the unmodified, parental 24JK clone or the same clone transduced with theNeo ^R gene only. In contrast, DLN cells induced either by IFN-transduced 4JK (high expression of MHC class I) or an unmodified 4JK tumor (moderate expression of MHC class I) had significant but equal therapeutic efficacy. Although the in vitro growth rate of tumor cell lines was unaffected by the insertion of the mouse IFN cDNA, their in vivo (s.c.) growth rates were significantly slower than those of the nontransduced tumors. Thus, after retroviral transduction of the murine IFN cDNA into a nonimmunogenic tumor with a very low level of surface expression of MHC class I, modified tumor cells could elicit therapeutic T cells from DLN capable of successfully treating established pulmonary metastases upon adoptive transfer. This strategy significantly confirms previous observations on the potential therapeutic effects of gene modification of tumor cells with IFN and extends the realm of therapeutic possibilities to include the use of DLN cells for the development of T-cell based immunotherapies against nonimmunogenic human tumors.     

Human lymphokine preparations which generate tumoricidal properties of human monocytes in vitro may be distinct from gamma interferon

Summary The purpose of these studies was to determine whether stimulated human lymphocytes produce lymphokines distinct from IFN, that can activate human blood monocytes to lyse tumor cells. We undertook this investigation because of the controversy concerning whether MAF and IFN are the same molecule. Crude lymphokine preparations prepared from normal human mononuclear cells incubated with Con A and rich in MAF activity also contained 1000 U/ml IFN as measured by the virus neutralization assay. However, the induction of tumoridical activity in monocytes by the lymphokine preparation could be dissociated from the IFN activity, based on the following data: (1) Heat treatment (100 °C for 2 min) removed the antiviral activity of the lymphokine yet did not diminish its MAF-like activity when measured in a 72 h cytotoxicity assay against ¹²⁵I IUdR-labeled human A375 melanoma cells. (2) Likewise, treatment of this lymphokine preparation with a twofold excess of anti-IFN antibody neutralized antiviral activity but once again had no effect on its ability to activate monocyte tumoricidal function. In contrast, both heat treatment and anti-IFN antibody abolished monocyte activation by equivalent units of human recombinant IFN. Taken together, these data suggest that there is a molecule(s) distinct from IFN which can activate human monocytes for tumoricidal function. Furthermore, this dissociation of MAF and IFN activity was dependent on the use of a long-term (72 h) assay, since activation of tumoricidal activity in an 18–24 h assay appeared to be attributable solely to IFN.     

Induction of TH1- and TH2-associated cytokine mRNA in mouse bladder following intravesical growth of the murine bladder tumor MB49 and BCG immunotherapy

Productive immunity to murine and human parasites is associated with the development of a type I T cell response (interferon--producing) while type II responses (interleukin-4-producing) suppress the development of delayed-type hypersensitivity (DTH) and the elimination of the parasite. To determine if a similar regulatory pathway might exist in tumor systems and may be effected by immunotherapeutic manipulation, we have studied the localized cytokine response to the murine bladder tumor MB49 growing intravesically in syngeneic mice. Intravesical growth of MB49 results in the host-derived expression of mRNA for both interleukin-4 (IL-4) (TH2) and interferon (IFN) (TH1), as well as tumor necrosis factor (TNF) expression of indeterminate origin. Intravesical instillation of bacillus Calmette-Guérin (BCG), highly effective in eliminating bladder tumors clinically and in experimental systems, results in IFN and TNF mRNa production in the bladder wall, but no IL-4. Following BCG treatment of intravesical MB49, the number bladders expressing IL-4 mRNA decreases, while IFN and TNF expression remains constant. These results are consistent with the mechanism of action of BCG involving the generation of an enhanced TH1 immune milieu in the bladder wall, which may contribute to the generation of productive tumor-specific immunity.Supported by USPHS grant CA-42908. K.M.M. is the recipient of Foerderer and N. S. E. Predoctoral Fellowships. Presented in part at the American Association for Cancer Research, April 1993 and May 1994     

Recombinant interferon γ up-regulates in vivo and down-regulates in vitro monocyte CD14 antigen expression in cancer patients

Summary The expression of the monocyte membrane glycoprotein CD14 was measured and related to the serum interferon (IFN) concentration in thirteen patients with disseminated cancer during treatment with human recombinant interferon (rIFN). The drug was administered by continuous subcutaneous infusion using an escalating dose schedule, starting at 50 µg/day or 100 µg/day and increasing weekly up to 600 µg/day, if tolerated. Treatment was continued at a mean maximal tolerated dose of 200 µg/day for a median duration of 43 days. Serum IFN concentration and monocyte CD14 antigen expression (immunofluorescence with the monoclonal antibody LeuM3 and fluorescence-activated cell sorting analysis) were determined weekly. The serum IFN concentration was positively correlated with the rIFN dose (P <0.05). Therapy induced a dose-dependant enhancement of CD14 antigen expression. The increase in mean CD14 fluorescence intensity was on average 60% after 3 weeks of treatment at a mean dose of 220 µg rIFN/day and was reversed after withdrawal of therapy. Patients with a rapidly rising serum IFN concentration (starting dose 100 µg/day) showed a smaller increment in CD14 fluorescence intensity than those with slowly rising serum IFN levels (starting dose 50 µg/day). Since rIFN is known to down-regulate CD14 antigen expression in vitro, monocytes from patients off therapy and from healthy volunteers were cultured with this cytokine. A similar decrease of CD14 fluorescence was observed in both groups. In patients several factors, such as IFN concentration, duration of drug effect and type of serum, were evaluated and could not explain the discrepant in vivo and in vitro findings. In conclusion, the monocyte marker CD14 was found to be differentially regulated by rIFN in vivo and in vitro. In vivo, secondary mediators, induced by rIFN and acting on a constantly renewed cell population, may contribute to the enhanced CD14 expression.     

Effect of interferon γ on the sensitivity of bovine-papilloma-virus(BPV1)-transformed cell lines to cell-mediated cytotoxicity

Summary The effect of interferon (IFN) on the immunogenicity and immunosensitivity of mouse cell lines transformed by bovine papillomavirus type 1 (BPV1) DNA was examined in a syngeneic mouse model. The overnight incubation of BPV1-transformed cell lines with 100 IU/ml IFN did not affect their ability to induce the generation of cytotoxic effector cells but it clearly increased their sensitivity to lysis by interleukin-2-induced lymphokine-activated killer (LAK) cells and by nonspecific LAK-type effector cells induced by BPV-1-transformed cell lines. The treatment of two allogeneic lymphoid tumour cell lines, P815X2 and YAC-1, with IFN either decreased or had no effect on their sensitivity to LAK-cell-mediated lysis.     

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.     

Interleukin 2-activated killer cells: generation in collaboration with interferonγ and its suppression in cancer patients

Summary The generation of lymphokine activated killer (LAK) cells by recombinant IL2 (rIL2) in collaboration with interferon (IFN) was examined in peripheral blood mononuclear cells (PBMC) from patients with malignant tumors of the digestive organs and breast cancer. LAK cytotoxicity could be induced by rIL2 at 10 units/ml in 10 of 12 patients and 20 of 37 using fresh autologous tumor cells and PK-1, an established solid tumor cell line as a target, respectively. Among 34 patients, in which titers of IFN produced were assayed, 12 showed no IFN production. All of these 12 patients had no or extremely low LAK activity, suggesting the correlation of LAK generation with the production of IFN in response to rIL2. LAK induction by rIL2 in PBMC of cancer patients was almost completely inhibited by addition of anti-IFN serum. Depressed LAK generation, which was accompanied by no or low levels of IFN production, was partially restored by addition of exogenous recombinant IFN. These results indicate that LAK induction by rIL2 in cancer patients involves the production of IFN and its interaction with rIL2.The results also suggested the presence of a factor(s) suppressing LAK induction by rIL2 in the serum of cancer patients. Based on these results, the cancer patients could be divided into the following three groups. Group 1, in which the serum suppressor activity was undetectable, had the same level of LAK cytotoxicity in PBMC as healthy controls. Group 2 showed the serum suppressor factor and had the lower level of cytotoxicity in PBMC when cultivated in autologous serum (AS) compared to healthy controls. The depressed LAK induction in AS medium was restored to a normal level in culture with fetal calf serum (FCS) plus rIL2, or by addition of rIFN, or high concentrations of rIL2 in AS medium. The last group (group 3), in which the serum suppressor factor was also found, had the lowest level of cytotoxicity compared to healthy controls. The LAK induction in these patients could not be restored to a normal level by culture in FCS medium, addition of exogenous rIFN or high concentrations of rIL2, suggesting the possibility that the deficit of LAK generation in this group might involve the dysfunction or the lack of IL2 responder cells, in addition to the presence of a serum suppressor factor(s).     

Induction of bacillus-Calmette-Guérin-activated killer cells from human peripheral blood mononuclear cells against human bladder carcinoma cell lines in vitro

Cytotoxicity against two human bladder carcinoma cell lines (BT-A and BT-B) was investigated using human peripheral blood mononuclear cells (PBMC) stimulated with viable bacillus Calmette-Guérin (BCG) or sonicated BCG (s-BCG). We applied a cytotoxicity assay based on radioactive labelling of tumour cells by incorporation ofl[³H]methionine. The results were compared with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interleukin-2 (IL-2) and interferon (IFN). BCG-stimulated PBMC showed a cytotoxic potential against BT-A and BT-B comparable to that of IFN-generated LAK cells, but this did not reach the level of IL-2-generated LAK cells. We termed these cytotoxic effectors BCG-activated killer (BAK) cells. In contrast to their cytotoxicity against bladder tumour cells. BAK cells did not differ from unstimulated PBMC in the killing of K562 cells. Only viable but not sonicated BCG was able to induce cytotoxicity against BT-A and BT-B. We could demonstrate the presence of the cytokines IFN, IL-2, tumour necrosis factor (TNF) and TNFß in the supernatants harvested during the generation of BAK cells. Monoclonal antibodies neutralizing IFN were able to inhibit BCG-mediated cytotoxicity, giving evidence of the involvement of IFN in the induction of BAK cells. Furthermore, we performed experiments to investigate the cytotoxic potential of distinct cell populations. The cells effective in BCG-activated killing of bladder tumour cells could be localized within the CD8+/CD56+ lymphocyte subset. CD4+ cells and macrophages did not exhibit cytolytic activity. Our findings imply that the activation by BCG of CD8+/CD56+ killer cells might be an important antitumoral mechanism during BCG therapy against superficial urothelial bladder cancer.