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We studied the safety, tolerance, and clinical effects of the combined administration of subcutaneous recombinant human interleukin-2 and interferon alfa-2b in 54 patients with advanced cancer, for whom no effective standard therapy was available. Treatment courses consisted of a 2-day interleukin-2 pulse (14.4-18 million units (MU) m2/day), followed by 3.6 up to 4.8 MU/m2/day, 5 days per week, over 6 consecutive weeks and interferon alfa-2b at 3 up to 6 MU/m2, administered two-three times weekly for 6 weeks. Overall, patients received more than 90% of the projected dose of interleukin-2 and interferon alfa-2b, respectively. Of 54 evaluable patients (32 renal cell cancer, 12 melanoma, eight colorectal cancer, one B-cell lymphoma, one Hodgkin's disease), four complete responses occurred in patients with renal cell carcinoma, and a greater than 50% reduction in tumour size (partial response) in six renal cell carcinoma patients and one melanoma patient. Moreover, 21 patients (13 renal carcinoma) had stable disease. The median duration of response was 19 months (range 16-22 months) in complete responders. Clinical responses were associated with a mean peripheral blood eosinophil count of more than 1,000/microL (P less than 0.05 versus non-responders). Systemic toxicities included fever, chills, nausea, anorexia, and hypotension limited to WHO grades I and II in more than 80% of patients treated. No treatment-related deaths occurred. This combination of subcutaneously administered recombinant interleukin-2 and interferon alfa-2b has significantly diminished the side effects normally observed with high-dose intravenous recombinant interleukin-2, which requires admission to hospital. It has been shown to induce objective tumour regression in out-patients with progressive metastatic renal cell carcinoma and malignant melanoma.

Based on the granulocyte-macrophage colony-stimulating factor (GM-CSF) dependency of a newly established human myeloid cell line GM/SO, we developed a highly specific and sensitive bioassay for human GM-CSF. The presence of bioactive GM-CSF could be determined by measuring the formazan concentration produced from MTT by the cells that survived and proliferated in the presence of either natural or recombinant human GM-CSF. With this assay we were able to quantify the level of GM-CSF in two human sera as well as in conditioned media from human bladder cell carcinoma cell line 5637, a human fibroblast line, and phytohemagglutinin-stimulated peripheral blood mononuclear cells. The sensitivity of the assay allows measurement of concentrations of GM-CSF as low as 0.1 U/ml.

We treated 17 patients who had progressive metastatic renal carcinoma with a combination of subcutaneous recombinant human interleukin-2 (administered every 12 hours, at 9.0 million IU/m2 on days one and two, followed by 1.8 million IU/m2, five days per week, over six consecutive weeks) and interferon-alpha 2b (given at 5 million U/m2 three times weekly, for six consecutive weeks). Treatment courses were repeated in patients presenting with stable or regressive disease after the six weeks of combination therapy (11 of 14 evaluable). Two and three of 14 evaluable patients achieved complete and partial remissions, respectively. Toxicity of this regimen was moderate, with local inflammation of the injection sites, grade I-II (WHO) fevers, chills, malaise, nausea/vomiting, and anorexia in more than two-thirds of the patients treated.

Recombinant interleukin-2 (rIL-2; EuroCetus, Amsterdam, Netherlands) was studied in an outpatient phase II trial in 14 patients with progressive metastatic renal carcinoma, malignant melanoma, and colorectal cancer. Escalating doses of rIL-2 were administered as subcutaneous bolus every 12 hours, starting at 0.3 million U/m2/d. A 100% dose increase occurred at weekly intervals, up to a maximum of 2.4 million U/m2/d. Responding patients or patients with stable disease after 4 weeks of rIL-2 (n = 9) were continued on maintenance therapy at 1.8 million U/m2 of rIL-2 administered once weekly. After 12 weeks of therapy, one renal cell cancer patient had a partial regression in lung metastases. Bolus injection of rIL-2 (1.2 million U/m2) resulted in peak serum levels of 25 to 30 U/ml. Toxicity of this regimen was moderate, with local inflammation at the injection sites, grade I-II (World Health Organization) malaise, nausea and/or vomiting, and fevers in 70% to 100% of patients treated. Thyroid dysfunction was observed in 10 patients receiving subcutaneous rIL-2; four of these patients had laboratory evidence of hyperthyroidism, and one had hypothyroidism. rIL-2-induced toxicity reversed spontaneously after cessation of treatment. In all patients receiving rIL-2, a dose-dependent increase in peripheral blood lymphocyte and eosinophil counts was noted, with a mean of 2.6 and 3.8 x 1,000/microliters after 4 weeks of therapy; mean lymphocyte and eosinophil counts were measured at 2.0 and 2.4 x 1,000/microliters in patients who received prior high-dose chemotherapy, compared with 3.2 and 5.1 x 1,000/microliters in those who did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Modern immunotherapy of human cancer has evolved as a rapidly expanding field of clinical and experimental research. Employing the systemic application of recombinant interleukin-2 (IL-2) in humans, Rosenberg and colleagues from the National Cancer Institute reported the regression of advanced metastatic tumors in approximately 10%-30% of patients treated. The additional adoptive transfer of autologous patient-derived activated lymphocytes was performed to enhance therapeutic efficacy. While the exact mechanisms of IL-2 based immunotherapy in cancer remain unclear, it has been hypothesized that both the IL-2 activated lymphocyte and its secretory products such as interferon-gamma or tumor-necrosis factor beta may contribute to the lysis of tumor cells in vivo. Accordingly, research has been directed toward enhancing both the activation state and the specificity of IL-2 induced killer cells in humans. Based on in vitro and animal data, the retransfusion of tumor-infiltrating lymphocytes has been shown to mediate the regression of metastatic neoplasms in up to 50% of patients receiving systemic IL-2. Considerable toxicity from the use of high-dose IL-2 has prompted attempts to develop low-dose regimens which allow for the outpatient treatment of patients presenting poor prognosis. While in most clinical trials involving IL-2, patient follow-up has been short, and no or only limited data have become available from controlled prospective and randomized clinical studies, IL-2 has shown some promise in patients with metastatic renal cell cancer or malignant melanoma. Novel approaches toward the improvement of clinical efficacy of IL-2 include local (e.g., intracavitary) application or combinations with other cytokines such as interferon-alpha or cytostatic drugs.

The immunological phenotypes of lymphocytes and myeloma cells in 48 patients with multiple myeloma (MM) were analyzed using a panel of monoclonal antibodies (mAbs). Myeloma cells were positive for OKT10, BL3, PCA1 and BA2. In a few cases, they were also positive for the B cell-associated antigens J5, B1 and I2. Eight of 48 cases had more than 15% J5-positive lymphocytes, and some lymphocytes in MM expressed plasma cell-associated antigens (PCA1, BL3, OKT10), suggesting a possible clonal involvement. These observations demonstrate the heterogeneity of surface antigen expression of myeloma cells and suggest that BL3, PCA1, BA2 and J5 may be useful mAbs for purging myeloma cells from bone marrow for autologous transplantation.

The potential of various photoradiation therapy for the in vitro purging of residual tumor cells from autologous bone marrow (BM) transplants is discussed in this paper. The results with fluorescent dyes, Dihematoporphyrin Ether (DHE) and Merocyanine-540 (MC-540) are detailed. Following photoradiation of cells with white light, both DHE and MC-540 showed high cytocidal activity towards lymphoid and myeloid neoplastic cells, but had significantly less effect on normal granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and mixed colony-forming (CFU-GEMM) progenitor cells. Acute promyelocytic leukemia (HL-60), non-B, non-T, cALLa positive acute lymphoblastic leukemia (Reh), and diffuse histiocytic B-cell lymphoma (SK-DHL-2) cell lines were exposed to different drug concentrations in combination with white light at a constant illumination rate of 50,000 lux. With DHE doses varying from 2.0 to 2.5 ug/ml and MC-540 concentrations of 15 to 20 ug/ml, clonogenic tumor cells could be reduced by more than 4 logs, when treated alone or in mixtures with normal irradiated human marrow cells. However, preferential cytotoxicity towards neoplastic cells was highly dependent on the mode of light activation. MC-540 had no substantial effect on malignant lymphoid (SK-DHL-2) and myeloid (HL-60) cells, and on normal marrow myeloid (CFU-GM) precursors, when the drug incubation was performed in the dark and followed by light exposure of washed cells. Equal doses of MC-540 (15-20 ug/ml) could preferentially eliminate tumor cells under conditions of simultaneous light and drug treatment (30 minutes at 37 degrees C). Using DHE (2.5 ug/ml), 29.3%, 46.8%, and 27.5% of normal marrow CFU-GM, BFU-E, and CFU-GEMM, respectively, were spared, following sequential drug and light exposure of cells, while simultaneous treatment reduced both normal (CFU-GM) and neoplastic cells below the limits of detection. The data from various centers is briefly discussed with special emphasis on clinical trials. Our results provide a useful model for leukemia and lymphoma cells and suggest that these phototherapy experiments can be implemented into clinical trials.

Various chemical compounds have been described to induce photosensitization of tumor cells resulting in cell death. We studied the effect of merocyanine-540 (MC-540) on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) and common acute lymphoblastic leukemia antigen-positive non-T, non-B acute lymphoblastic leukemia (Reh) cell lines were incubated with MC-540 and simultaneously exposed to white light. Normal human BM and mixtures of leukemic cells with BM cells were treated under similar conditions. At constant illumination rates of 50,000 lx, significant (at least 4 to 5 logs) tumor cell destruction was obtained with MC-540 concentrations of 20 micrograms/ml or more for HL-60, and 10 micrograms/ml or more for Reh cells. Incubation of BM under equivalent conditions preserved 18.0% of granulocyte-macrophage colony-forming units and 14.2% of erythroid burst-forming units. Similar results were obtained when tumor cells were mixed with irradiated BM and then treated with MC-540. In summary, cell photosensitization with MC-540 has a selective cytotoxic effect towards leukemic cells and therefore may be useful for purging tumor cells from autologous BM.

We studied the ex vivo sensitivity of continuously cultured neuroblastoma cells from 3 different patients towards interleukin-2-induced cell-mediated cytotoxicity. A mean (+/- SD) target cell lysis (4 h 51Cr release) of 49 +/- 11, 46 +/- 8, and 32 +/- 11% in SMS-SAN, LA-N-1, and SK-N-BE2 cell lines, respectively, was achieved when neuroblastoma cells were co-cultured at an effector-to-target (E:T) ratio of 50:1 with peripheral blood mononuclear cells (PBMC) that had been preincubated for 4 days in the presence of recombinant interleukin-2 (rIL-2; 100 U/ml). Under identical conditions, 93 +/- 9% of Daudi cells (a standard target for rIL-2-activated killer cells) were lysed. Preincubation of rIL-2-induced PBMC cultures in the presence of irradiated neuroblastoma targets (LA-N-1, SK-N-BE2) resulted in a significant cytolytic augmentation. At E:T ratios of 50:1 and 10:1, day-4 rIL-2/LA-N-1-stimulated PBMC produced 69 +/- 7 and 41 +/- 11% lysis of LA-N-1 cells, as compared to 46 +/- 8 and 22 +/- (mean +/- SD) 7% lysis by untargeted PBMC that were preincubated with rIL-2 (100 U/ml) in the absence of LA-N-1 target cells (p less than 0.05). Co-incubation of rIL-2-induced PBMC preparations with irradiated LA-N-1 and SK-N-BE2 cells, respectively, did not significantly enhance the cytolytic activity against other neuroblastoma targets and the standard Daudi cell line (p greater than or equal to 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)

Intensive chemotherapy requiring rescue with autologous bone marrow may be an encouraging mode of treatment for poor prognosis Ewing's sarcoma (ES). In vitro chemosensitivity test may be useful for establishing an effective purging condition. We studied the in vitro effects of a variety of chemotherapeutic agents on two established ES cell lines (ES-5838 and ES-A4573) and marrow colony-forming unit-granulocyte macrophage (CFU-GM). 4-Hydroperoxycyclophosphamide (4-HC), at 100 microM produced complete inhibition (greater than 5 log) of clonogenic growth of both ES cell lines and spared 6.9% of normal CFU-GM growth. Etoposide (VP-16), at 100 microM produced 3-3.5 log inhibition of ES cell lines and complete inhibition of CFU-GM growth. Adriamycin (ADR) and vincristine (VCR) were more cytotoxic to ES-5838 cells than ES-A4573 cells. ADR at 1 microM produced 99.7% inhibition of ES-5838 cells, 92.2% of ES-A4573 cells, and 86% inhibition of CFU-GM. VCR at 1 microM produced 98.6% inhibition of ES-5838 cells, only 43.7% of ES-A4573 cells, and 75% inhibition of CFU-GM growth. Addition of verapamil did not enhance VCR cytotoxicity of ES cell lines. These studies indicate that 4-HC may be a useful agent for purging metastatic ES cells from the bone marrow for autologous marrow transplantation.

To develop an effective neuroblastoma (NB) purging condition, we have compared in vitro cytotoxicity of 6-hydroxydopamine (6-OHDA) and ascorbic acid, with 4-hydroperoxycyclophosphamide (4-HC) on three NB cell lines (SK-N-BE2, SMS-SAN, and LA-N-1) and also upon human hematopoietic stem cells. Our study included mixing NB cells with 20-fold excess of irradiated bone marrow buffy coat cells to simulate the borderline remission marrow. When NB cells were treated without marrow cells, all three NB cell lines were very sensitive to 6-OHDA; complete inhibition of SK-N-BE2 and SMS-SAN cells was achieved at 10 micrograms/ml, and greater than 4 log inhibition of LA-N-1 was observed at 100 micrograms/ml of 6-OHDA. Addition of marrow cells caused marked reduction of the 6-OHDA-induced cytotoxicity of NB cells, and under similar conditions, colony-forming units-granulocyte-macrophage (CFU-GM) growth was not inhibited significantly. In the absence of normal marrow cells, 60 minutes of treatment with 100 microM of 4-HC produced complete inhibition (greater than 4.5 log) of SK-N-BE2 and SMS-SAN cells, greater than 4 log inhibition of LA-N-1 cells, and 97% of CFU-GM. Addition of marrow cells reduced the cytotoxicity of 4-HC, and 100 microM of 4-HC produced 99.8% inhibition of LA-N-1 colony growth. Shortening incubation duration to 30 minutes resulted in further reduction of 4-HC cytotoxicity; 100 microM of 4-HC caused 98.3%, 45%, and 33% inhibition of LA-N-1 cells, marrow CFU-GM, and burst-forming units-erythrocytes (BFU-E), respectively. At 200 microM, complete inhibition (greater than 4 log) of LA-N-1 colony growth was noted, and 9.9% of CFU-GM and 9.3% of BFU-E growth was observed. These data favor the use of 4-HC for purging marrow of NB, cells in the clinical autologous marrow transplantation.

We studied the sensitivity of human myeloma (plasma cell leukemia) toward autologous and allogeneic lymphokine-activated killer (LAK) cells. Fresh plasma cell leukemia (PCL)-derived peripheral blood mononuclear cells (PBMC) and PBMC from 3 normal donors were cultured in the presence of recombinant interleukin-2 (rIL2; 1,000 U/ml) for subsequent use as cytotoxic effectors against fresh and continuously cultured myeloma cells. Target cell lysis was measured in a 4-hour 51Cr radioisotope release assay. At an effector to target (E:T) ratio of 50:1, rIL2-induced PCL-PBMC lysed 48 +/- 19% (mean +/- 1 SD) of autologous myeloma targets, as compared to 89 +/- 5, 95 +/- 15, and 100 +/- 9% lysis of standard LAK-sensitive Daudi cells and allogeneic myeloma cell lines SKO-007, and RPMI-8226, respectively. Normal PBMC-derived rIL2-induced (LAK) cells exhibited a slightly lower cytotoxic reactivity against allogeneic targets (61 +/- 9, 60 +/- 6, and 81 +/- 8% cytolysis of SKO-007, RPMI-8226, and Daudi cells, respectively, at a 50:1 E:T ratio). Cytotoxicity against myeloma (PCL) of autologous PCL-derived killer cells could be significantly (at least 2-fold) enhanced when rIL-2-induced effector cells were preincubated for 18 h in the presence of recombinant Interferon-alpha rIFN-alpha; 1,000 U/ml). In summary, our results indicate the potential antitumor efficacy of rIL2- and rIL2 + rIFN-alpha-activated killer cells in human myeloma (PCL).

To assess the usefulness of 4-hydroperoxycyclophosphamide (4-HC) and VP-16-213 (VP-16) as a purging agent for myeloma cells in bone marrow (BM) ex vivo, myeloma cell lines (SK-RCS-1, RPMI-8226), lymphoma cell line (SK-DHL-2) and normal BM cells were treated at different concentrations of 4-HC or VP-16. Clonogenic tumor cells from all three cell lines could be reduced by more than 4 logs, when treated alone or as a mixture with irradiated normal BM cells at a 4-HC concentration of 60 microM. Under similar conditions, approximately 1% of the normal BM myeloid progenitor granulocyte-macrophage colony-forming cells survived. These observations support the use of 4-HC for purging myeloma cells for autologous BM transplantation.

Previous studies using unseparated normal human bone marrow cells have indicated that recombinant tumor necrosis factor alpha (rTNF-alpha) can inhibit the in vitro colony growth by normal granulocyte/macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells in a dose-dependent manner. In the present studies, by using very low numbers of highly enriched normal bone marrow progenitor cell populations as target cells, we have extended these previous findings to provide convincing evidence that erythroid and myeloid colony growth suppression by rTNF-alpha is manifested by a direct interaction between rTNF-alpha and CFU-GM and BFU-E progenitor cells. In addition, the sensitivity of normal peripheral blood and chronic myeloid leukemia bone marrow CFU-GM and BFU-E colony growth to inhibition by rTNF-alpha was examined and found to be comparable with that of normal bone marrow CFU-GM and BFU-E. Although the continuous presence of high doses of rTNF-alpha (5000 units/ml) was required in methylcellulose cultures for maximal CFU-GM (90%) and BFU-E (70%) colony suppression, short-term exposure (24 to 72 hr) of normal bone marrow-enriched progenitor cells to rTNF-alpha, in the absence of hematopoietic growth factors, was sufficient to irreversibly suppress up to 50 to 65% of CFU-GM colony growth. In contrast, the number of BFU-E colonies was increased under these conditions. If, however, hematopoietic growth factors (Mo-T-cell-conditioned medium and erythropoietin) were present during preincubation of the cells with rTNF-alpha, BFU-E were then slightly suppressed while the extent of CFU-GM inhibition remained essentially the same. The suppressive effect of rTNF-alpha on erythroid and myeloid progenitor cell growth appears to be most pronounced on the more primative stages of committed progenitor cell development, since inhibition of CFU-GM- and BFU-E-derived colony growth progressively decreased with the delayed addition of rTNF-alpha to methylcellulose cultures. [3H]Thymidine incorporation was also inhibited by rTNF-alpha in normal bone marrow-enriched progenitor cell populations stimulated to proliferate in liquid culture by colony-stimulating factors. This effect was transient, however, since the activity of rTNF-alpha declined after the first 24 h of culture at 37 degrees C, particularly at low doses of rTNF-alpha where the activity was completely lost after 48 h of culture. This loss of activity appeared to be due to a decreased sensitivity of progenitor cells to the antiproliferative effects of tumor necrosis factor (TNF) after an initial exposure rather than a lack of available TNF.(ABSTRACT TRUNCATED AT 400 WORDS)

Using a modified alkaline-phosphatase/antialkaline-phosphatase method for phenotyping fresh human leukemias, we could demonstrate peripheral blood and bone marrow-derived blast cells to specifically react with two monoclonal antibodies (MoAbs), H25 and H366, previously shown to recognize natural killer cells, activated T lymphocytes and a proportion of normal hematopoietic precursor cells. MoAbs H25 and H366 were found to identify the majority of leukemic cells in patients presenting with T-ALL, LGL leukemia, pre-B-ALL, CML, and AML, respectively.

The success of autologous bone marrow transplantation for B cell lymphoma may depend on the efficacy of in vitro purification of patients' tumor cell-contaminated marrow. In this study, we tested the toxicity of seven different chemotherapeutic agents against two B cell lymphoma lines (LY-16 and SK-DHL-2) as compared to normal human bone marrow granulocyte-macrophage progenitor cells (CFU-GM). 4-Hydroperoxycyclophosphamide (4-HC), VP-16-213 (VP-16), nitrogen mustard, and vincristine showed a highly selective toxicity against cultured lymphoma cells; i.e., at doses sufficient to induce a 4-log clonogenic tumor cell reduction (4-HC 21 micrograms/ml, VP-16 50 micrograms/ml, nitrogen mustard and vincristine 5 micrograms/ml), 10.0 +/- 6.7, 3.0 +/- 3.2, 23.2 +/- 22.7 and 24.0 +/- 17.0% (mean +/- 1 SD), respectively, of normal bone marrow CFU-GM were preserved. The differential sensitivity of tumor cells and normal hematopoietic precursors was less prominent after exposure of cells to cis-diamminechloroplatinum II (cis-platinum); thus, at a drug dose of 100 micrograms/ml, all detectable lymphoma cells could be eradicated (i.e., greater than or equal to 4 log reduction) while a CFU-GM recovery of only 0.2 +/- 0.2% was observed. In contrast, adriamycin and bleomycin, at the highest tumoricidal concentrations tested (5 and 100 micrograms/ml, respectively) did not exhibit a selective toxicity toward lymphoma cell lines. In summary, our results suggest that nitrogen mustard and vincristine, as well as 4-HC and VP-16, may be useful agents for the ex vivo treatment of bone marrow grafts form B cell lymphoma patients.

Flow cytometric separation was performed on the normal human bone marrow (BM) by using the low-angle (0 degrees) or high-angle (90 degrees) light scatter. Four distinct subpopulations of cells can be enriched from normal human BM and these fractions were subsequently evaluated for their morphological properties as well as their clonogenic capacity in various progenitor cell assays. Our results indicate that human erythroid and granulocyte-macrophage progenitor cells can be separated from BM low-density cells by cell sorting, and these cells show similar 0 degrees and 90 degrees light scatter properties to those observed with murine bone marrow studies. Flow cytometric analysis also suggests that the majority of sorted BFU-E and CFU-GM resides in the blast cell subset of human BM mononuclear cells.

To assess the usefulness of 4-hydroperoxycyclophosphamide (4-HC) and Etoposide (VP-16) as a purging agent for myeloma cells in bone marrow ex-vivo, myeloma cell lines (SK-RCS-1, RPMI-8226), lymphoma cell line (SK-DHL-2) and normal bone marrow (BM) cells were treated at different concentrations of 4-HC, VP-16. In separate experiments, LAK cells or antibodies were also used to treat the above cell lines. Clonogenic tumor cells from all three cell lines could be reduced by more than 4 logs, when treated alone or as a mixture with irradiated normal bone marrow cells at a 4-HC concentration of 60 mumol/l. Under similar conditions, approximately 1% of normal BM myeloid progenitor granulocyte-macrophage colony forming cells (CFU-GM) survived. The results with LAK cells and antibodies were also encouraging. These observations support the use of various purging methods for myeloma cells for autologous bone marrow transplantation.

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.

We studied the ex vivo cell-mediated cytotoxicity of natural killer (NK) and lymphokine-activated killer (LAK) cells against continuously cultured Ewing's sarcoma cells from 3 different patients. Target cell lysis was measured in a 4-hour 51Cr radioisotope release assay. At an effector to target (E:T) ratio of 50:1, the mean (+/- 1 SD) cytolysis by fresh purified large granular lymphocytes (NK cells) was 20 +/- 8, 25 +/- 2, and 21 +/- 3% in Ewing's sarcoma cell lines 6647, 5838, and A4573, respectively. Under identical conditions, NK cells lysed 56 +/- 7% of K562 (a standard NK target), and 3 +/- 3% of Daudi (a standard NK-resistant LAK target). When compared to fresh unseparated peripheral blood mononuclear cells (PBMC), purified NK cells did not exhibit an enhanced cytotoxic reactivity against either Ewing's sarcoma target. In contrast, LAK cells (i.e., PBMC that were preincubated for 4 days in the presence of rIL-2) were highly cytotoxic against all three Ewing's sarcoma targets. LAK activity was dependent on the concentration of rIL-2 used in PBMC cultures. Optimum cell-mediated toxicity against the standard LAK target Daudi (99 +/- 10% cytolysis at 50:1 E:T ratio) was achieved at rIL-2 concentrations of 1,000 u/ml. LAK cells grown under these conditions were also effective against Ewing's sarcoma cells. At an E:T ratio of 50:1, 86 +/- 16, 85 +/- 16, and 67 +/- 13% inhibition was observed in 6647, 5838, and A4573 cells, respectively, as compared to 17 +/- 10, 19 +/- 15, and 29 +/- 11% cytolysis by fresh uninduced PBMC. In summary, our results suggest that rIL-2-induced LAK-type immune effector cells may be of some therapeutic value in the treatment of poor prognosis Ewing's sarcoma.