Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice

The adoptive transfer of lymphokine-activated killer (LAK) cells combined with low dose interleukin 2 (IL-2) mediates the regression of established pulmonary metastases in mice and has efficacy in the treatment of human cancer. Systemic administration of high dose IL-2 alone can mediate tumor regression. Cortisone acetate (CA), 25-75 mg/kg, was administered daily to mice receiving high dose IL-2 for 10 days. CA significantly reduced the toxicity induced by IL-2; 38 of 48 mice receiving CA survived compared to 0 of 30 controls (P less than 0.0001). In addition, CA administration caused a decrease in IL-2-induced 125I-labeled albumin leakage in mouse organs. However, CA abrogated the in vivo antitumor effect of high dose IL-2, and to a lesser extent the therapeutic effect of exogenous LAK cells plus lower dose IL-2. Mice treated with 100,000 units of IL-2 showed 98, 63, and 33% reductions of pulmonary metastases in Hanks' balanced salt solution, 25 mg Ca/kg, and 75 mg Ca/kg groups, respectively; treatment with LAK and 7,500 units of IL-2 resulted in reductions of 94, 77, and 57% in these same groups. CA treatment of animals did not affect LAK generation, although the absolute number of LAK precursors was greatly reduced. These results show that although CA can reduce the toxic effect(s) of IL-2, it can be detrimental to successful immunotherapy using this approach.     

Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant interleukin 2

Lymphokine-activated killer (LAK) cells are generated in vitro by the incubation of normal murine splenocytes in interleukin 2. We have shown previously that the systemic injection of LAK cells in conjunction with recombinant interleukin 2 can reduce the number of established pulmonary metastases in mice. In an attempt to study this approach in the treatment of hepatic metastases, we developed a technique for the induction of hepatic metastases in mice based on the intrasplenic injection of tumor cells and have tested the effects of LAK cells and recombinant interleukin 2 produced in Escherichia coli (RIL-2) therapy on these metastases. Treatment with LAK cells alone in 14 consecutive experiments rarely produced significant reduction in metastases over control (mean percentage reduction, 12%). Therapy with RIL-2 alone produced a dose-dependent reduction in the number of liver metastases. In 20 consecutive experiments when RIL-2 was administered i.p. three times a day at doses varying from 1,000 to 5,000, 10,000 to 15,000, and 25,000 units, a statistically significant (P less than 0.05) reduction in liver metastases was seen in 2 of 12, 2 of 4, and 8 of 12 determinations, respectively (percentage reduction, 0 to 97; mean, 42%). At doses greater than 25,000 units, the reduction in metastases was highly reproducible (percentage reduction, 66 to 95; mean, 83%) and was statistically significant in 14 of 14 determinations. When LAK cells were given i.v. in addition to RIL-2 administration in 16 consecutive experiments, the percentage reduction in liver metastases was markedly increased over that seen with RIL-2 alone (mean percentage reduction, 77% at doses of 5,000 to 25,000 units of RIL-2 and mean reduction, 97% for doses greater than 25,000 units of RIL-2). At doses of 5,000, 10,000, 25,000, and greater than 25,000 units of RIL-2 plus LAK cells, significant reduction of liver metastases (P less than 0.05) was achieved in 3 of 7, 2 of 2, 8 of 8, and 6 of 6 determinations, respectively. When animals were given fresh splenocytes or splenocytes cultured in complete medium without RIL-2 instead of LAK cells, no reduction in liver metastases was seen except for that attributable to the administration of RIL-2 alone. Sublethal total body irradiation of the mice prior to therapy abrogated the therapeutic effects of RIL-2, but the effects of treatment with LAK cells plus RIL-2 were maintained. Thus, treatment with RIL-2 alone or in combination with LAK cells is effective in reducing the number of established hepatic micrometastases in a murine model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergy of Nocardia rubra cell wall skeleton and interleukin 2 in the in vivo induction of murine lymphokine-activated killer cell activity

Combination of an ip injection of Nocardia rubra cell wall skeleton (N-CWS) and 3 daily sc injections of human recombinant interleukin 2 (rIL 2) into C3H/HeN mice resulted not only in a significant increase in the number of peritoneal cells (PC) but also in a potent induction of their lymphokine-activated killer (LAK) activity, compared with results obtained with N-CWS or rIL 2 alone. The augmented LAK activity of PC was mediated by nonadherent, nonphagocytic, Thy-1.2+(-)- and asialo GM1+ cells. Nonadherent PC induced by an ip injection of N-CWS bound more 125I-labeled rIL 2 than did normal, nonadherent PC, and generated high LAK activity when cultured overnight with rIL 2. In contrast, normal, nonadherent PC responded only weakly to the overnight stimulation with rIL 2. The phenotype of N-CWS-induced PC with an elevated IL 2 responsiveness was Thy-1.2+(-)-, Lyt-1.1-, Lyt-2.1- and asialo GM1+, suggesting that the N-CWS-stimulated LAK precursors were derived mainly from the NK cell lineage. However, mature T cells may also be involved in this mechanism, because N-CWS failed to augment the IL 2 responsiveness of nonadherent PC in BALB/c nu/nu mice. Treatment of C57BL/6N mice bearing solid Lewis lung carcinoma (3LL) tumors with an intratumoral injection of N-CWS followed by 6 daily sc injections of rIL 2 resulted in the apparent suppression of tumor growth, while N-CWS or rIL 2 alone produced no such suppression. These results suggest that N-CWS augments the antitumor effect of rIL 2 by accumulating LAK precursors and elevating their responsiveness to rIL 2 at the injection site.     

Interleukin 2 and lymphokine-activated killer cell therapy: analysis of a bolus interleukin 2 and a continuous infusion interleukin 2 regimen

Several groups have described the efficacy of interleukin 2 (IL-2) plus lymphokine-activated killer (LAK) cells in the treatment of cancer patients with significant response rates noted in patients with renal cell cancer and malignant melanoma; however, the optimum regimen remains undefined. The Biological Response Modifiers Program of the National Cancer Institute conducted two consecutive Phase I/II studies evaluating the toxicity and clinical efficacy of different methods of IL-2 and LAK cell therapy. In the first trial, we modified the standard Rosenberg regimen by decreasing the duration of priming in an attempt to reduce the toxicity related to this phase of the therapy and thereby administer more IL-2 doses with the LAK cells. In the second trial, we used a continuous i.v. infusion IL-2 regimen and altered both the leukapheresis procedure and the LAK cell culture techniques based on our in vitro and preclinical studies suggesting that 2-day LAK cells were superior. Thirty cancer patients received i.v. bolus IL-2 at 100,000 units/kg every 8 h for 3 days during priming and for 5 days during LAK cell administration. A second group of 22 cancer patients received IL-2 by continuous i.v. infusion at 3 x 10(6) units/m2 for 5 days during priming and an additional 5 days of IL-2 with the LAK cell phase of the treatment. The timing of the start of the leukapheresis procedures, their duration and number, and the LAK cell culture techniques differed in the two trials. Overall, 52 patients with various cancers were treated. The toxicities associated with each regimen were similar to those seen in other IL-2 plus LAK cell trials. Four patients (one each with melanoma and diffuse large cell lymphoma and two with renal cell cancer) exhibited partial responses lasting 2, 4, 10, and 15+ mo. Serial tumor biopsies from treated patients demonstrated that therapy can produce a marked mononuclear cell infiltrate and an increase in HLA-DR expression on tumor cells. There was no difference in the overall response rate between the two regimens, but toxicity was less with continuous i.v. infusion IL-2. The 5-day continuous i.v. infusion regimen resulted in significantly higher rebound lymphocytosis, cell yield from leukapheresis, and number of LAK cells harvested from culture.     

Autocytotoxic activity of lymphokine-activated killer cells: characterization of effector cells and susceptible targets

The specificities and surface markers of murine autocytotoxic cells induced by in vitro culture with interleukin 2 (IL2) were studied. Culturing murine spleen cells with recombinant human IL2 resulted in the generation of cytotoxic cells which killed syngeneic lymphoblasts and syngeneic activated macrophages (M phi). Both lectins and protein antigens were capable of inducing lymphoblasts recognized by lymphokine-activated killer (LAK) cells. B-lymphoblasts as well as T-lymphoblasts were sensitive to lysis by these effector cells. In addition, peritoneal M phi activated in vivo with Bacille Calmette-Guérin (BCB), Corynebacterium parvum (C. parvum), thioglycollate (TG) or lipopolysaccharide (LPS) were shown to be susceptible to lysis by LAK cells. In contrast, neither unstimulated T cells nor resident peritoneal M phi were sensitive to lysis by LAK cells, suggesting that normal cells have to be activated in order to be sensitive to lysis by these effector cells. Surface marker analysis indicated that majority of effector cells which killed syngeneic lymphoblasts and activated M phi were Thy1+, asialo GM1+, L3T4-, Ly2-.     

Immunotherapy of murine hepatic metastases with lymphokine-activated killer cells expanded in serum-free media and recombinant interleukin 2

It has been shown that the systemic administration of lymphokine-activated killer (LAK) cells with recombinant interleukin 2 (RIL-2) is effective in reducing the number of established pulmonary and hepatic metastases in murine models. Similarly, this modality of therapy has been proven effective against certain selected human tumors as well. In view of the rising concern with transmission of virally related communicable diseases such as hepatitis and AIDS, we have undertaken the evaluation of a serum-free medium (AIM V) for the generation and expansion of murine LAK cells for use in in vivo tumor immunotherapy against murine hepatic metastases. Day 3 LAK cells generated in AIM V medium demonstrated a greater percentage of viable cells than cells generated in serum containing complete medium (CM) (mean percentage of yield, 59 versus 25%, AIM V medium versus CM, respectively, P less than 0.001, N = 6 consecutive experiments). When day 3 LAK cells were transferred to new medium (CM to CM and AIM V to AIM V), a highly reproducible expansion of these cells was demonstrated which was significantly better for cells expanded in AIM V medium versus cells expanded in CM (mean fold expansion on day 21 of culture; 201 versus 54, AIM V medium versus CM, respectively, P less than 0.005, N = 4 consecutive experiments). When day 3 LAK cells, day 5 expanded LAK cells, and day 13 expanded LAK cells grown in CM or in AIM V medium were given in vivo with RIL-2 to mice harboring hepatic metastases, cells grown in AIM V medium demonstrated an increased antitumor activity compared to cells grown in CM. As an example in experiment 1, the mean number of metastases with day 5 expanded LAK cells grown in CM and given with RIL-2 was 47 while the mean number of metastases with day 5 expanded LAK cells grown in AIM V medium and given with RIL-2 was 5 (P less than 0.002). These experiments demonstrate that AIM V medium can be utilized to generate greater numbers of murine LAK cells with enhanced in vivo antitumor activity compared to cells generated in CM. These findings could be applied to the expansion of cytotoxic cells for human antitumor therapy.     

Monoclonal antibody therapy of murine lymphoma: enhanced efficacy by concurrent administration of interleukin 2 or lymphokine-activated killer cells

Lymphokine-activated killer (LAK) cells have recently been shown to be very efficient effector cells for antibody-dependent cellular cytotoxicity. Thus, we explored, in a murine lymphoma model, administration of LAK-inducing doses of interleukin 2 (IL-2) or adoptive transfer of LAK cells as a means of enhancing therapy with tumor-specific monoclonal antibody (mAb). AKR/Cum (Thy-1.2+) hosts were inoculated on day 1 s.c. with the SL-2 thymoma of AKR/J origin (Thy-1.1+) and developed palpable tumor on day 4. Tumor-specific anti-Thy-1.1 IgG2a mAb, 1A14, was given on days 4 and 8 with 50,000 units/day IL-2 i.p. divided in two doses on days 4-12. Therapy with IL-2 or mAb alone had minimal activity, prolonging control median survival of 22 days to 25 and 29 days, respectively, whereas therapy with IL-2 plus mAb significantly prolonged median survival to 40 days. However, combined therapy did not result in cures and long term survival. The efficacy of combined therapy did not result from alterations in the biodistribution of mAb by concurrent IL-2 infusions, as determined by studies with radiolabeled mAb. The combined effect of in vitro generated LAK (10(8) cells) adoptively transferred i.v. with 1A14 on days 4 and 8 following SL-2 inoculation was also evaluated. This regimen had no detectable toxicity, and treatment of mice with LAK and mAb resulted in 60% long term survival compared with 17% or 0% for mice treated with mAb or LAK alone. Thus, the therapeutic effects of tumor-specific mAb was enhanced by in vivo administration of IL-2 or by adoptively transferred LAK, which may represent means to provide the host with increased antibody-dependent cellular cytotoxicity effector cells. Adoptively transferred LAK has the additional benefit of augmenting mAb therapy of tumor without the toxicity associated with the induction of such cells in vivo with high dose IL-2.     

Synergy of human recombinant interleukin 1 with interleukin 2 in the generation of lymphokine-activated killer cells

Peripheral blood mononuclear cells cultured in vitro with interleukin 2 (IL-2) become cytolytic towards both autologous and allogeneic tumor cells. We report here that IL-1 synergizes with IL-2 in serum-free conditions to produce increased (1.3-286-fold) lymphokine-activated killer (LAK) activity. The most dramatic synergy is seen with low IL-2 concentrations (10 U/ml, 222 pM) and 50-250 U/ml IL-1 alpha or beta. Kinetics of addition experiments demonstrate a specific requirement for IL-1 at or before addition of IL-2 to the culture. We postulate that one of the mechanisms whereby IL-1 augments LAK activity is by rendering LAK-precursors more responsive to IL-2. Up-regulation of the IL-2 receptor beta chain (Tac) and increased [3H]thymidine incorporation in cultures containing IL-1 and IL-2 support this view. In some instances, IL-1 alone is capable of maintaining/generating a small degree of cytolytic activity. Collectively, our data demonstrate that IL-1 is capable of interacting with low dose IL-2 to significantly augment LAK activity, potentially playing an important role in the early stages of LAK activation and differentiation. Because synergy is observed with dramatically reduced IL-2 concentrations, this system may offer an alternative approach to high dose IL-2 therapy for the treatment of neoplastic disease.     

Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2.

Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.     

Adoptive immunotherapy of a rat glioma using lymphokine-activated killer cells and interleukin 2

The aim of the present study was to develop an animal model to test the therapeutic potential of purified adherent lymphokine-activated killer (A-LAK) cells against an intracerebrally implanted rat glioma, designated F98. Highly purified A-LAK cells demonstrated greater activity against F98 tumor cells than conventional lymphokine-activated killer cells, as determined by means of 51Cr-release and clonogenic assays. Therapeutic efficacy was evaluated by means of a Winn neutralization assay, in which F98 targets and A-LAK cells or control nonadherent mononuclear cells were incubated for 18 h in vitro and then implanted stereotactically into the right caudate nuclei of Fischer rats. Animals given injections of 4000 F98 cells alone or control nonadherent mononuclear cells had a mean survival time of 22.3 days, compared to 46.1 days (P less than 0.001) for rats treated with A-LAK cells. Increasing the tumor inoculum to 12,500 cells reduced the survival time of A-LAK-treated animals to 27.8 days, compared to 20.8 days for untreated controls. Systemic administration of 50,000 units/kg of interleukin 2 every 12 h for 5 days failed to improve survival. The mean survival time of rats implanted with the F98 tumor ranged from 16 days for 10(5) cells to 29 days for 10(2) cells. Extrapolating from these survival data, treatment with A-LAK cells may have decreased the number of F98 cells to less than 10, but even this small number was still lethal. Supernatants from F98 cells had immunoinhibitory activity that, further, may have modulated the antitumor effects of A-LAK cells. Our results indicate that curative, adoptive immunotherapy of the F98 glioma by means of A-LAK/interleukin 2 is impossible to achieve and provide some explanation for the clinical failures that have been observed in the adoptive immunotherapy of malignant gliomas.     

Effect of macrophages on interleukin-2 (IL-2)- and IL-4-induced murine lymphokine-activated killer activity

The enhancing effect of macrophages on interleukin-2 (IL-2)- and IL-4-induced murine lymphokine-activated killer (LAK) activity was investigated in this study. Peritoneal macrophages significantly enhanced LAK activity generated from accessory cell-depleted splenic lymphocytes in both IL-2 and IL-4 cultures. This effect was dependent on the number of macrophages and was not replaced by a factor derived from macrophages or lymphocytes. Macrophages enhanced IL-2- and IL-4-induced LAK activity against both natural killer (NK)-sensitive (YAC-I, P388D1) and NK-resistant (P815) tumor cells. Negative selection of cells with antibodies and complement showed no differences in surface markers between IL-2 LAK effectors and IL-4 LAK effectors generated in the presence of macrophages. These results suggest that the same LAK effector subsets can be enhanced by macrophages in either IL-2 or IL-4 cultures.     

Prolongation of serum half-life of interleukin 2 and augmentation of lymphokine-activated killer cell activity by pepstatin in mice

We reported previously using a murine model that the kidney is the organ involved in catabolism of exogenous human recombinant interleukin 2 (IL-2) and that cathepsin D, a major renal acid protease, is responsible for the degradation of IL-2. In the present report also using BALB/c mice we have investigated the effect of in vivo pepstatin, an acid protease inhibitor, treatment on serum half-life of IL-2, and generation of lymphokine-activated killer (LAK) cell activity. The in vivo pepstatin treatment by i.p. injection resulted in a significant reduction in the accumulation of 125I-IL-2 by the kidney in a reverse dose-response manner. Pepstatin treatment prolonged the serum half-life of 125I-IL-2, and the increase in serum half-life of 125I-IL-2 was pepstatin dose dependent. A significant reduction in renal cathepsin D activity, as monitored by the degradation of 125I-IL-2, was detected. In vivo pepstatin (0.6 mg/kg) treatment along with IL-2 (300,000 IU/mouse) daily for 3 or 6 days resulted in an augmentation of natural killer activity exhibited by freshly prepared and uncultured splenocytes against YAC-1 cells. An additional culturing of the splenocytes with IL-2 (3,000 IU/ml) in vitro for 1 day significantly enhanced the effect of in vivo pepstatin treatment; i.e., LAK cell activity generated from the splenocytes of animals treated with IL-2 plus pepstatin was greatly augmented in comparison with that treated with IL-2 alone. Phenotypic assessment by cell surface markers (Thy-1.2, Lyt-2, L3T4, and asialo-GM1) on the fresh splenocytes prepared from animals treated in vivo with pepstatin plus IL-2 revealed a decrease in the percentage of cells expressing Thy-1.2 and Lyt-2 and an increase in those carrying asialo-GM1. These results demonstrated that, as a result of in vivo pepstatin treatment, renal cathepsin D activity was greatly inhibited, which in turn reduced the degradation of circulating IL-2, then prolonged serum half-life of IL-2, and subsequently augmented natural killer and LAK cell activity. The in vivo pepstatin and IL-2 treatment decreased the T-cells and increased the natural killer-like LAK precursor cells, possibly also with an increase in its activity, which were further induced by in vitro IL-2 culture to generate an augmented LAK cell activity. This study also suggests the clinical potential of pepstatin in IL-2-related immunotherapy.     

Prostaglandin E2 from macrophages of murine splenocyte cultures inhibits the generation of lymphokine-activated killer cell activity

The present work investigated the association between prostaglandin E2 (PGE2) from macrophages and its inhibition of murine lymphokine-activated killer (LAK) cell generation. The coculture of indomethacin with interleukin-2 (IL-2) augmented LAK cell activity in an indomethacin dose-response manner, and diminished PGE2 content in the corresponding culture supernatant in a reverse dose-response manner. The correlation between the increase in LAK cell activity and the decrease in PGE2 content was highly significant. Identical results were obtained with diclofenac. A profound inhibition of LAK cell activity by exogenous PGE2 in a dose-response manner was detected. Polyclonal anti-PGE2 antiserum augmented in a dose-dependent manner the LAK cell activity, by neutralizing PGE2 in the medium. A reduction of PGE2 content in the culture supernatant was also detected when the macrophage subpopulations were cultured and was indomethacin dose-dependent. In comparison with that of normal mouse splenocytes, the incubation of whole splenocytes of tumor-bearing mice, which contained a greater subpopulation of macrophages (24% vs. 12%), produced a greater PGE2 content and a correspondingly depressed LAK cell activity. Additionally, PGE2 reduced protein kinase C (PKC) activity along with LAK cell activity generated from macrophage-depleted T cells and natural-killer-like cells. These results overall indicate that PGE2 from macrophages in murine splenocyte cultures inhibits the LAK cell generation, and PKC may be involved in the inhibition mechanism.     

Adoptive immunotherapy of human cancer using low-dose recombinant interleukin 2 and lymphokine-activated killer cells

The adoptive transfer of recombinant-methionyl human interleukin 2 (rIL-2)-activated autologous peripheral blood mononuclear lymphokine-activated killer (LAK) cells to cancer patients is being evaluated as an alternative to conventional cancer therapy. We have independently developed an alternative regimen to previously reported adoptive immunotherapy protocols using rIL-2 and LAK cells which features the prolonged administration of low-dose rIL-2 (30,000 units/kg) and an automated, entirely enclosed system of peripheral blood cell procurement, culture, harvest, and reinfusion of activated cells. The cell culture system was tested with a murine tumor model in which LAK cells generated in plastic culture bags were reinfused into tumor-bearing mice. Tumor regression was as effective with cells activated in the bags as in conventional culture flasks. Twenty-eight cancer patients were treated for 5 consecutive days with low-dose rIL-2, followed by leukapheresis, infusion of LAK cells, and prolonged IL-2 administration. At least 50% tumor regression was observed in 46% of all patients treated. These data imply that human peripheral blood mononuclear cells retain fully their capacity for rIL-2-induced activation and effector cell function under this alternative approach, and further, that a low-dose rIL-2 regimen with markedly reduced toxicities can be as effective as high-dose rIL-2 regimens if low-dose rIL-2 is given for a prolonged period of time following LAK cell infusion.     

Augmentation of interleukin-2 immunotherapeutic effects by lymphokine-activated killer cells and allogeneic stimulation in murine tumor cells

Interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells were used in intraperitoneal and pulmonary tumor models in C57BL/6 mice. To maintain the immunotherapeutic effects of IL-2 plus LAK treatment but reduce its toxicity, ways were sought to augment IL-2 effects. The investigation showed that the adoptive transfer of LAK cells was a prerequisite for successful therapy of intraperitoneal cancer. When LAK cells were given on consecutive days within one course of immunotherapy, antitumor efficacy was augmented with additional doses of LAK cells. However, with the reduction of 1 complete cycle of IL-2 + LAK cells, no further reduction in intraperitoneal tumor was observed as compared to the reduction after 2 or 4 cycles. LAK cells generated from splenocytes of mice that had received an allogeneic tumor challenge 1 week earlier exerted a highly increased cytotoxicity as compared to normal LAK cells. Furthermore, the potentiation effect of an allogeneic response of the host at the tumor site was demonstrated by decreased numbers of lung implants and improved survival in mice given mixtures of syngeneic and allogeneic tumor cell suspensions. An alloimmune response within the microenvironment of tumor tissue markedly enhanced the antitumor effect of IL-2 against the syngeneic tumor. It was concluded that there is a fundamental need to improve the recruitment of adoptively transferred LAK cells or LAK precursors into tumor tissue. This may be the next step required in the further development of IL-2 and LAK immunotherapy.     

Combination treatment with irritant and recombinant interleukin 2 in the peritoneal cavity for evoking effective anti-tumor activity: generation of lymphokine-activated killer cells and tumor-specific killer cells

Meth A sarcoma, growing in the subcutaneous tissue of syngeneic BALB/c mice, regressed completely after an intraperitoneal (ip) injection of proteose peptone (PP) (on day 6) followed by 2 ip administrations (on days 7 and 8) of human recombinant interleukin-2 (IL-2, 25 micrograms/day), whereas one such treatment alone had little effect on the tumor growth. While this combination treatment was effective in anti-asialo GM1 antibody-treated mice, no such effect was noted in T cell-depleted ATXFL (thymectomized, irradiated and fetal liver cell-reconstituted) mice. These results show that T cells are mainly responsible for this antitumor effect. Treatment with a combination of PP and IL-2, but not with either PP or IL-2 alone, resulted in a marked increase in the T cell population in the peritoneal cavity after the treatment. At an early stage after the combination treatment, both peritoneal exudate cells (PEC) and spleen cells exhibited killing activity with a promiscuous specificity. However, at a later stage, 7 days after the treatment, Meth A-specific killer activity was observed in both PEC and the spleen. Meth A rechallenge was rejected by the mice in which the tumor had regressed, but the antigenically different Meth 1 was accepted by them. A similar result was obtained in Winn's neutralization test. These results suggest that this combination treatment, which is effective in the generation of lymphokine-activated killer cells in the peritoneal cavity, finally resulted in the induction of tumor-specific killer cells in the periphery. These results clearly show the anti-tumor efficacy of combination treatment with PP and rIL-2.     

Lymphokine-activated killer activity in long-term cultures with anti-CD3 plus interleukin 2: identification and isolation of effector subsets

Peripheral blood lymphocytes cultured in recombinant interleukin 2 during 3 to 5 days (short-term cultures) develop the ability to lyse natural killer-resistant tumor lines and fresh tumor cells, i.e., express lymphokine-activated killer (LAK) function. Phenotypic analysis has shown these cells to be natural killer cells, i.e., CD16+ and/or Leu 19+ cells. CD3+,CD16- T-cells, instead, develop very low LAK function in these cultures. We recently reported the development of long-term (up to 21 days) cultured cells with LAK activity by stimulation with OKT3 + interleukin 2(IL2). These culture conditions repeatedly resulted in a several hundred-fold expansion in cell number. Specific LAK activity on Day 14 of culture was comparable to that of 3-day LAK cultures and could be further enhanced by the addition of interleukin 1 beta, beta-, or gamma-interferon. Total LAK activity was greatly increased in OKT3 + IL2 cultures over that found in short-term cultures. Isolation of effectors mediating LAK function in long-term cultures stimulated with OKT3 + IL2 showed that both CD3+,CD16- cells and CD16+,CD3- cells tested on Day 14 of culture expressed equivalent levels of LAK activity as shown by lysis of natural killer-resistant targets, HL60 and Daudi. Further dissection of the subpopulations developing LAK activity demonstrated that, in addition to CD16+,CD3- cells, CD3+, CD4-,CD8- cells and Leu 19+,CD3-,CD16- cells also developed high LAK activity in long-term cultures with OKT3 + IL2. Further, long-term culture with OKT3 + IL2 induced increases in the numbers not only of CD3+,CD4-,CD8- cells but also of CD16+,CD3- and Leu 19+,CD3-,CD16- cells. Although there is a significant increase in the number of CD3+,CD8+ cells, neither these, nor the CD3+,CD4+ cells, mediate LAK activity to the same extent as the populations mentioned above.     

Phase I clinical trial of interleukin 2 and alpha-interferon: toxicity and immunologic effects

Because recombinant interleukin 2 (rIL-2) and recombinant alpha-interferon (rIFN-alpha) exhibit synergistic antitumor activity in C3HMT1820 T-cell lymphoma and B16 melanoma tumor systems, we have performed a Phase I study of this combination in 55 patients with advanced malignancies for whom no standard therapy exists. Successive groups of greater than or equal to 4 patients have been entered into 12 dose levels (1A-3D), with dose levels 1-3 referring to doses of rIL-2 of 0.1, 0.5, and 2.0 x 10(6) units/m2, respectively, and dose levels A-D referring to doses of recombinant human alpha 2a-interferon (rHuIFN-alpha 2a) of 0, 0.1, 1.0, and 10.0 x 10(6) units/m2. Both agents were given on Mondays, Wednesdays, and Fridays, with rIL-2 being given as i.v. bolus injections and rHuIFN-alpha 2a being given intramuscularly. Myelosuppression was dose-limiting and was related primarily to the dose of rHuIFN-alpha 2a. The maximum-tolerated dose level was reached at a dose of rIL-2 of 2.0 x 10(6) units/m2 and of rHuIFN-alpha 2a of 10.0 x 10(6) units/m2 (dose level 3D). At this dose level, 3/6 patients developed grade 3 neutropenia (absolute granulocyte count less than 1 x 10(9)/liter). Myelosuppression was transient, with no documented infections being associated with neutropenia. Hypotension was mild; a single patient was treated with a vasopressor, but all other cases of hypotension responded to fluid administration. No significant pulmonary toxicity was produced. Fever, chills, and malaise were universal but not dose-limiting. Three partial responses and one minor response were observed in patients with malignant melanoma, renal cell carcinoma, and breast cancer. Immunological studies suggested that natural killer activity was related to both the dose of rIL-2 and the dose of rHuIFN-alpha 2a, with natural killer activity being positively related to the dose of rIL-2 and maximal at the lowest dose of rHuIFN-alpha 2a of 0.1 x 10(6) units/m2.     

Cytokine gene expression during the generation of human lymphokine-activated killer cells: early induction of interleukin 1 beta by interleukin 2

Culture of human peripheral blood leukocytes with interleukin 2 (IL-2) stimulates their differentiation into lymphokine-activated killer (LAK) cells, with a broad range of cytotoxicity against fresh tumor cells and tumor cell lines (Grimm et al., J. Exp. Med., 155: 1823-1841, 1982). We chose to utilize a molecular approach to determine whether IL-2 stimulates the expression of cytokine genes by the mixed cell population which may be involved in the generation or regulation of lytic activity. Northern blot analysis performed with total cellular RNA from LAK cells cultured for varying periods of time with IL-2 revealed that the genes which code for cytokines [interleukin 1 (IL-1)alpha and beta, gamma-interferon, tumor necrosis factor alpha, and lymphotoxin] were not spontaneously expressed. As soon as 2 h after IL-2 treatment, IL-1 alpha and IL-1 beta mRNAs were expressed. Both nonadherent and adherent populations of LAK cells express IL-1 beta mRNA; however, the adherent population produced more IL-1 beta mRNA and maintained its expression for a prolonged period of time. Other cytokine mRNAs (gamma-interferon, tumor necrosis factor alpha, and lymphotoxin) were expressed later than the IL-1 mRNAs with maximal levels between Days 2 through 7. Our results indicate that LAK cell populations can generate a variety of cytokines which may be involved in the generation of lytic activity.     

The effects of interleukin 2 and alpha-interferon administration on hepatic drug metabolism in mice.

We have administered the cytokines interleukin 2 (IL-2), alpha-interferon (IFN-alpha), and gamma-interferon (IFN-gamma) to mice and measured the alterations in hepatic drug-metabolizing enzyme activities. For comparative purposes and to understand the mechanism of diphtheria and tetanus toxoids and pertussis (DTP) vaccine-induced inhibition of drug metabolism, we also studied the effects of vaccine administration in mice. The administration of IL-2 alone or in combination with IFN-alpha or IFN-gamma causes dose-dependent increases in hexobarbital-induced sleep times. These increases correlate well with the inhibition of specific microsomal mixed-function oxidase activities. Sublethally irradiated mice and athymic nude mice receiving injections of IL-2 or IL-2 plus IFN-alpha do not show the inhibition of drug metabolism seen in normal mice. However, the inhibition of drug metabolism in DTP vaccine-treated mice was similar in all three groups. These observations indicate a possible role for immune cells (probably T-lymphocytes) in the inhibition of drug metabolism caused by administration of these cytokines, which is different from the inhibition of drug metabolism caused by DTP vaccine.