Differential activation of lymphokine-activated killer cells with different surface phenotypes by cultivation with recombinant interleukin 2 or T-cell growth factor in gastric cancer patients

Fourteen days' culture of human peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL 2) or T cell growth factor (TCGF) results in the generation of lymphokine-activated killer (LAK) effector cells which have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, as well as NK-sensitive, K562 cells. LAK cells were generated from both normal and gastric cancer patients' PBL. However, LAK cell activities induced by rIL 2 or TCGF decreased with the progress of the tumor growth. In addition, TCGF-induced LAK cell activities were found to be lower than the rIL 2-induced LAK cell activities. Different mechanisms may be involved in the decreases of the rIL 2-induced and TCGF-induced LAK cell activities. This study further demonstrates that the cell types involved are also heterogeneous, as determined by phenotypic characteristics. The LAK-effector cell type was analyzed by two-color flow cytometry. RIL 2-induced LAK cells showed increased proportions of CD4+Leu 8- and Leu 7+CD16-, and a decreased proportion of CD8+CD11- cells, which are believed to be associated with killer T cell functions. In contrast, TCGF-induced LAK cells revealed significantly increased proportions of CD8+CD11- and CD4+Leu8- cells, and a decreased proportion of Leu 7+CD16- cells. Thus, LAK cells with different surface phenotypes were induced by the cultivations with rIL 2 and with TCGF.     

Differential Activation of Lymphokine-activated Killer Cells with Different Surface Phenotypes by Cultivation with Recombinant Interleukin 2 or T-cell Growth Factor in Gastric Cancer Patients

Fourteen days' culture of human peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL 2) or T cell growth factor (TCGF) results in the generation of lymphoklne-activated killer (LAK) effector cells which have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, as well as NK-sensitive, K562 cells. LAK cells were generated from both normal and gastric cancer patients' PBL. However, LAK cell activities induced by rIL 2 or TCGF decreased with the progress of the tumor growth. In addition, TCGF-induced LAK cell activities were found to be lower than the rIL 2-indnced LAK cell activities. Different mechanisms may be involved in the decreases of the rIL 2-induced and TCGF-induced LAK cell activities. This study further demonstrates that the cell types involved are also heterogeneous, as determined by phenotypic characteristics. The LAK-effector cell type was analyzed by two-color flow cytometry. RIL 2-induced LAK cells showed increased proportions of CD4^±Leu 8^- and Leu 7^±CD16^-, and a decreased proportion of CD8^±CD11^- cells, which are believed to be associated with killer T cell functions. In contrast, TCGF-induced LAK cells revealed significantly increased proportions of CD8^±CD11^-and CD4^±Leu 8^- cells, and a decreased proportion of Leu 7^±CD16^- cells. Thus, LAK cells with different surface phenotypes were induced by the cultivations with rIL 2 and with TCGF.     

In vitro induction of cytotoxic activity against carcinomatous pleural or peritoneal lymphoid cells cultivated with cytokines, and an immunological phenotypic analysis of the effector cells

The functional and phenotypic characteristics of carcinomatous pleural or peritoneal lymphoid cells cultivated with either rIL 2 or TCGF have been investigated. The cultivation of the lymphoid cells with cytokines was initiated by a mixture of coexisting, viable carcinoma cells for 14 days. Results have indicated that cytokine-activated lymphoid cells from malignant pleural and peritoneal effusions showed considerable cytolytic activity against K562 and Daudi cells. The cell population responsible for LAK and/or CTL effector cells of TCGF-activated lymphoid cells were CD8+ CD11- cells. Further, in rIL 2-expanded cultures from pleural and peritoneal lymphoid cells, the CD4+ Leu 8- population was found to contain effector cells of cytotoxic activity against the tumor cells. It further was seen that the TCGF-activated CD8+ CD11- T cells possessed a more potent killing activity, in comparison to the rIL 2-activated CD4+ Leu8- T cells. However, rIL 2-activated lymphoid cells from ascites in liver cirrhosis (used for a control) showed a higher tumoricidal activity.     

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.     

Synergistic effect of Nocardia rubra cell wall skeleton and recombinant interleukin 2 for in vivo induction of lymphokine-activated killer cells

C57BL/6 mice inoculated i.p. with 3LL tumor cells were treated by local combination therapy with Nocardia rubra cell wall skeleton (N-CWS) and recombinant interleukin 2 (rIL-2). The combination treatment significantly prolonged their survival and augmented lymphokine-activated killer (LAK) activity of peritoneal cavity lymphocytes (PCL), compared with treatments with rIL-2 alone or N-CWS alone. After in vitro culture of peritoneal exudate mononuclear cells with rIL-2, the nonadherent population derived from N-CWS-injected tumor-bearing mice showed a significantly higher LAK activity than did that population derived from saline solution-injected mice. When N-CWS-induced PCL were cocultured with either N-CWS-induced macrophages or control macrophages in the presence of rIL-2, their LAK activity was higher than that of control PCL. Therefore, it was suggested that N-CWS-induced PCL themselves have a more potent ability as precursors of LAK cells. Phenotypic analysis on PCL populations revealed that N-CWS-induced PCL contained increased proportions of CD3+CD4-CD8- cells and asialo GM-1+ cells compared with control PCL. These results suggest that N-CWS selectively accumulates potent LAK precursors, namely, CD3+CD4-CD8- T-cells and asialo GM-1+ natural killer cells, at the injection site and that LAK cells are efficiently induced by subsequent administration of rIL-2.     

In vivo and in vitro activation of natural killer cells in advanced cancer patients undergoing combined recombinant interleukin-2 and LAK cell therapy

Patients with advanced metastatic cancer were given combined autologous lymphokine activated killer (LAK) cell and recombinant interleukin-2 (rIL-2) therapy on a National Cancer Institute extramural phase II trial. Systemic administration of rIL-2 resulted in pronounced lymphocytopenia. Within two days after completion of in vivo rIL-2 therapy, there was a dramatic increase in absolute numbers of circulating lymphocytes, and cytotoxic activity against tumor cell targets was mediated by peripheral blood lymphocytes, indicating in vivo generation of LAK activity. Patients were leukapheresed and cells cultured for three to four days in rIL-2. rIL-2 cultured cells from all patients demonstrated cytotoxic activity. In order to characterize the effector cell, T cells and natural killer (NK) cells were isolated to greater than 95% purity by flow cytometry. Cytotoxic activity was mediated by rIL-2--activated NK cells, whereas T cells demonstrated no substantial activity. The circulating in vivo cytotoxic effectors detected after in vivo rIL-2 therapy were also shown to be rIL-2--activated NK cells. Results from these studies demonstrate that all patients were capable of generating a cytotoxic response, and that the cytotoxic effector cells were rIL-2--activated NK cells, identified by the phenotype CD3--, Leu 19+.     

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.     

Lymphokine-activated killer cells in rats: analysis of progenitor and effector cell phenotype and relationship to natural killer cells

The progenitor and effector cell phenotype of lymphokine-activated killer (LAK) cells generated in F344 rats by recombinant human interleukin 2 (IL-2) (rIL-2) were analyzed. Highly purified populations of peripheral blood large granular lymphocytes (LGL) exhaustively depleted of T-cells were fully capable of generating high levels of LAK activity by 3 to 5 days in culture while purified populations of resting T-cells devoid of LGL could not generate LAK activity. This pure population of LGL expressed surface markers characteristic of rat natural killer (NK) cells [i.e., OX8+, asialomonoganglioside (asialo-GM1+), laminin+, OX19-, R1-3B3-, W3/25-, Ia-, surface immunoglobulin negative (SIg-)]. Further evidence that NK cells were the progenitors of cells with LAK activity was obtained by treatment of spleen or peripheral blood lymphocytes with anti-laminin or anti-asialo-GM1 antibodies plus complement or with the lysosomotropic agent L-leucine methyl ester. These treatments effectively depleted LGL/NK cell activity and the subsequent generation of rIL-2-induced LAK activity. Analysis of the LAK effector phenotype by cell sorting demonstrated that the majority of cells with LAK activity were OX8+, asialo-GM1+, laminin+, OX6+, OX19-, R1-3B3-, W3/25-, and SIg-. Furthermore, treatment of LAK cells with L-leucine methyl ester also significantly reduced their cytolytic activity. Thus, the LAK effector cells were also LGL and expressed surface marker characteristic of activated NK cells and not those of mature T- or B-cells. The proliferative response of rat spleen or blood lymphocytes to rIL-2 appeared to be primarily associated with LGL/NK cells since depletion of NK cells by anti-asialo-GM1 or anti-laminin antibody plus complement or by L-leucine methyl ester significantly (P less than 0.001) reduced the incorporation of [3H]thymidine into DNA. In contrast, depletion of T-cells (by anti-T-cell antibody plus complement) did not significantly affect rIL-2-induced proliferation. Similarly, T-cell-depleted, highly purified populations of LGL gave substantial proliferative responses to rIL-2. These studies clearly indicate that in the rat, the major cell population activated by rIL-2 is the LGL/NK cell and these cells appear to represent the major population of cells in blood or spleen which generate broad antitumor (LAK) cytotoxicity.     

Postoperative adjuvant adoptive immunotherapy with lymph node-LAK cells and IL-2 for pathologic stage I non-small cell lung cancer

We conducted a clinical trial of adoptive immunotherapy with lymph node-lymphokine-activated killer (LN-LAK) cells and recombinant interleukin 2 (rIL-2) for a surgical adjuvant therapy of pathologic stage I non-small cell lung cancer. The regimen consisted of the subcutaneous administration of low-dose rIL-2 for 6 consecutive days and the transfer of ex vivo generated LAK cells from regional lymph node lymphocytes, obtained at the time of surgical operation. A group of 19 patients with primary lung cancer received the immunotherapy about 2 weeks after surgery (pulmonary lobectomy). The regimen was postoperatively well tolerated by the patients. In peripheral blood lymphocytes (PBL) obtained after the treatment, the proportion of CD3+ T cells predominantly increased with the increase of CD4+ T cell subsets. On the other hand, the proportion of CD20+ B cells decreased. Both NK and LAK activity of PBL significantly increased. However, the immunomodulatory effects did not result in a prolongation of the postoperative survival time in comparison to the postoperative survival of patients (n = 21) with surgery alone during the same period. These results suggested that the treatment with low-dose LN-LAK cells and concurrent low-dose IL-2 could, therefore, neither reduce nor eradicate minimal micrometastatic diseases.     

Immunomodulatory properties and toxicity of interleukin 2 in patients with cancer

We performed a phase Ia/Ib study of interleukin 2 (IL2) in patients with cancer. Single doses of IL2 from 10(3) units/m2 to 10(7) units/m2 were well tolerated but failed to induce significant immunological changes. Chronic IL2 treatment for 5 days out of 7 for 3 weeks was well tolerated at doses below 10(7) units/m2 and was accompanied by significant immunological changes. Following chronic treatment with intramuscular injections of IL2 at 1 x 10(6) units/m2, we observed augmentation of peripheral blood natural killer activity and induction of peripheral blood LAK activity. Induction of LAK activity was most evident when IL2 was included in the cytotoxicity assay. There was a marked increase in the number of peripheral blood mononuclear cells bearing the Leu-19 marker in association with the observed increases in natural killer and LAK activity. A small percentage of Leu-19+ cells coexpressed CD3. There was heterogeneous expression of the low affinity Fc receptor (CD16). In vivo induced Leu-19+ cells could be divided into two populations, dim and bright, based on the intensity of fluorescent staining with antibodies to Leu-19. The majority of Leu-19 bright cells were CD16- while the majority of Leu-19 dim cells were CD16+. In addition, the intensity of CD16 staining was higher for Leu-19 dim cells than for Leu-19 bright cells. Increases in the amounts of CD38 and CD8 antigens were also observed. Significant increases in serum levels of the soluble IL2 receptor were observed during treatment. One partial remission was noted in a woman with non-Hodgkin's lymphoma.     

Ewing's sarcoma: ex vivo sensitivity towards natural and lymphokine-activated killing

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.     

Lymphoid irradiation results in long-term increases in natural killer cells in patients treated for Hodgkin's disease.

Therapeutic lymphoid irradiation has been shown to produce profound long-term alterations in lymphocyte subpopulations and immunologic responsiveness. Dual immunofluorescence flow cytometry and functional cytolytic assays were used to investigate the effects of lymphoid irradiation either alone or in combination with chemotherapy on T-cell and natural killer (NK) cell populations in the blood of patients treated for Hodgkin's disease. Patients treated with mantle and paraaortic lymphoid irradiation show significant increases in the proportion of cells bearing the NK cell phenotypic marker Leu-11 (CD16). These patients also display proportionately increased cytotoxicity against K562 tumor targets in vitro. A sizable number of these NK cells label dimly with Leu-2 (CD8) although they lack the pan-T-cell marker Leu-4 (CD3). The emergence after lymphoid irradiation of this population of Leu-11+2+ NK cells may lead to an apparent decrease in the ratio of helper to suppressor T-cells, although the actual ratio of these T-cell subsets generally is normal. These changes persist for years after the completion of radiation therapy. It was concluded that lymphoid irradiation may produce profound changes in NK cell populations in patients treated for Hodgkin's disease; the clinical significance of these changes is unclear.     

Two-color flow cytometry analyses of peripheral blood lymphocytes (PBL) and lymphokine-activated PBL in gastric cancer patients

A two-color flow cytometric analyses of the PBL of 51 patients (24 resectable, and 27 nonresectable), and of 30 healthy controls has been completed. The proportion of antigens, on the PBLs, defined by combinations of anti-Leu 2a and anti-Leu 15, anti-Leu 3a and anti-Leu 8, anti-Leu 3a and anti-HLA-DR, anti-HLA-DR and anti-Leu 2a and, anti-HLA-DR and anti-IL 2R, was not significantly different among the three groups. TCGF increased the proportion of Leu 2a+ Leu 15-, Leu 3a+ Leu 8-, HLA-DR+ Leu 2a+, Leu 3a+ HLA-DR+ and, HLA-DR+ IL 2R+ cells and reduced the proportion of Leu 2a+ Leu 15+ cells in gastric cancer patients, while the cultivation by IL 2 (2 weeks) increased the proportion of Leu 3a+ Leu 8-, HLA-DR+ IL 2R+ and Leu 3a+ HLA-DR+ cells, and did not change the proportion of Leu 2a+ Leu 15- and Leu 3a+ Leu 8+ cells, and decreased the proportion of Leu 2a+ Leu 15+ cells. These results, taken together with our previously published studies, suggest that the TCGF-activated Leu 2a+ Leu 15- subset but not the Leu 2a+ Leu 15+ subset serves as a phenotypic marker of suppressor-effector T cells, and that the IL 2-activated Leu 3a+ Leu 8- subset contained killer T cells against tumor cells in our experimental systems.     

Antitumour effect of intratumoral injection of human recombinant interleukin-2 in patients with hepatocellular carcinoma: a preliminary report

Five hepatoma patients with small resectable tumour received an intratumoral injection of recombinant interleukin-2 (rIL-2) once weekly over 2–4 weeks (1.05 × 10⁶−3.6 × 10⁶ U in total per patient). Tumour regressions of 32% and 57% were observed in two patients at day 42 after the first rIL-2 injection. No response was observed in two patients and disease progressed in one. Lymphokine-activated killer (LAK) activity was enhanced and Leu-11⁺ cells increased in the peripheral blood in the patients with 32% and 57% tumour regression after rIL-2 therapy. However, LAK activity, Leu-7⁺ cells were reduced in the patient who progressed. No consistent changes in Leu-2a⁺ cells and Leu-3a⁺ cells were demonstrated. In the three patients showing no response or 32% tumour regression, hepatic segments containing tumour were resected; histologically the tumour showed severe necrosis and lymphocytic infiltration in the patient with 32% tumour regression but mild or moderate changes in the other two. IL-2 mediated tumour killing can be induced in tumours by intratumoral injection of rIL-2, leading to tumour regression.     

A randomized phase II trial of continuous infusion interleukin-2 or bolus injection interleukin-2 plus lymphokine-activated killer cells for advanced renal cell carcinoma.

PURPOSE: Since 1985, multiple centers have demonstrated that interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells produce durable anticancer responses in patients with metastatic renal cell carcinoma. High-dose recombinant IL-2 (rIL-2) has been administered by intravenous bolus injection (Rosenberg SA, et al: N Engl J Med 313:1485-1492, 1985) and by continuous intravenous infusion (West WH, et al: N Engl J Med 316:898-905, 1987) combined with lymphokine-activated killer (LAK) cells, with both methods producing responses in patients with advanced renal cell carcinoma. The Extramural IL-2/LAK Working Group has conducted a randomized phase II trial of two intravenous high-dose rIL-2 regimens (bolus three times daily or 24-hour continuous infusion) to determine if either one manifests greater anticancer activity or a more acceptable toxicity profile. PATIENTS AND METHODS: Ninety-four patients with measurable advanced renal cell carcinoma were enrolled on this study: 46 to the bolus injection arm and 48 to the continuous infusion arm. On both arms, patients underwent a priming phase of rIL-2 administration, four daily lymphocytaphereses to harvest mononuclear cells that were placed in 3- to 4-day culture for generation of LAK cells, and an rIL-2/LAK coadministration phase. Patients were then observed monthly for evidence of response to this therapy and were offered up to two additional courses of treatment every 3 months if evidence of response was detected. RESULTS: Twenty percent of patients on the bolus injection arm experienced objective responses (three complete responses and six partial responses); 15% of patients on the continuous infusion arm responded (two complete responses and five partial responses). Complete responses were durable, persisting for 310+ to 700+ days. The incidence of severe life-threatening toxicities typical of high-dose rIL-2 therapy was similar in both arms (eg, patients with hypotension requiring pressors: bolus 71%, continuous 63%; oliguria less than or equal to 200 mL/8 hours: bolus 65%, continuous 71%). More episodes of fever, infection, and serum alkaline phosphatase elevation were associated with the continuous infusion arm, while more thrombocytopenia occurred on the bolus injection arm. Four patients (three bolus injection, one continuous infusion) died of respiratory and circulatory failure while under treatment. No clinical or laboratory parameter accompanying treatment on either arm was, by univariate or multivariate analysis, associated with an increased likelihood of response. CONCLUSIONS: Both methods of high-dose rIL-2/LAK cell administration produce nearly equivalent anticancer activity and toxicity in the treatment of renal cell carcinoma. The ability to predict responding patients based on patient or treatment characteristics is not possible.     

Appearance and phenotypic characterization of circulating Leu 19+ cells in cancer patients receiving recombinant interleukin 2

The effects of recombinant interleukin 2 (rIL-2) therapy on peripheral blood mononuclear cells expressing the Leu 19 surface marker were evaluated in 20 cancer patients. Leu 19 is a protein with a molecular weight of 220,000 expressed on 15% of normal peripheral blood mononuclear cells and is found on a majority of cells that mediate non-major histocompatibility complex-restricted cytotoxicity. Increased relative and absolute numbers of circulating Leu 19+ cells were observed in all patients receiving rIL-2. Increases in Leu 19+ cells were due in part to the development of a subpopulation of "bright" Leu 19+ cells (Leu 19b+) that possessed a higher density of membrane Leu 19 antigen than Leu 19+ cells assayed prior to therapy. Further characterization of rIL-2 induced Leu 19+ cells by dual immunofluorescence revealed considerable phenotypic heterogeneity within this population based on the coexpression of "dim" CD8 (CD8d+), CD16, and CD2 markers. The percentage of Leu 19+ CD8d+ cells was increased during rIL-2 therapy and comprised up to 60% of all circulating Leu 19+ cells. CD16+ and CD16- subsets of Leu 19+ cells were also increased by rIL-2. The density of CD16 antigen coexpression varied inversely with the density of Leu 19. Conversely, whereas the percentage of Leu 19 cells coexpressing CD2 was also increased by rIL-2 administration, the density of CD2 antigen expression was higher on the Leu 19b+ subset of cells. The development of circulating lymphokine-activated killer activity in three patients was temporally associated with the development of increased levels of circulating Leu 19+ cells. These studies demonstrate that rIL-2 administration induces preferential increases in cells expressing the natural killer and lymphokine-activated killer cell-associated marker Leu 19 and that these increases are associated with the development of circulating lymphokine-activated killer activity. Furthermore, Leu 19+ cells are comprised of phenotypically heterogeneous subsets which undergo characteristic changes during rIL-2 administration.     

Lymphokine-activated killer function following autologous bone marrow transplantation for refractory hematological malignancies

Disease recurrence remains the major factor which limits the success of autologous bone marrow transplantation (ABMT) for refractory hematological malignancies. The administration of interleukin 2 (IL2) with or without ex vivo generated lymphokine-activated killer (LAK) cells represents a potential approach to eradicating residual disease after ABMT. However, since LAK precursor activity is radiosensitive, high dose chemoradiotherapy may abrogate LAK function and preclude clinical responsiveness to IL2 after ABMT. Furthermore, since lymphocyte subsets which mediate LAK activity may recover at different rates after ABMT, LAK cells may be phenotypically and/or functionally altered after ABMT. To determine whether IL2 responsive LAK precursor cells are present in the circulation after ABMT, peripheral blood mononuclear cells (PBMC) from 21 patients with acute leukemia or lymphoma were tested for IL2-inducible LAK activity 17-83 days after ABMT. Cells were cultured with IL2 (1000-2000 units/ml) for 4 or 5 days and then tested for cytolytic activity and/or cell phenotype. LAK activity against the Daudi cell line was detected in every PBMC sample from every patient at every time point tested. The Raji cell line and a fresh allogeneic ovarian carcinoma were also lysed by LAK cells generated after ABMT. In the subgroup of patients transplanted for non-Hodgkin's lymphoma, LAK precursor activity appeared comparable to that of healthy controls. Culture with IL2 resulted in increased mean IL2 receptor expression in lymphocytes from patients after ABMT (3.1-9.9%) and from healthy controls (3.1-12.0%). After culture with IL2, the percentage of cells bearing the natural killer cell-associated Leu-19 determinant was significantly higher in patient PBMC than in normal control PBMC (28.3 versus 8.7%). Positive and negative cell selection by fluorescence sorting after culture with IL2 revealed that most of the LAK activity after ABMT was mediated by the Leu-19+ cells. Although CD5+ T-cells were devoid of LAK activity, a subset LAK effectors was CD8+. Thus, LAK activity is rapidly reconstituted after ABMT and is mediated by cells phenotypically similar to those in normal controls. These results support the feasibility of IL2 +/- LAK as consolidative immunotherapy after ABMT.     

Lymphokine-activated killer-cell function of lymphocytes from peripheral blood, regional lymph nodes and tumor tissues of patients with oral cancer

Lymphokine-activated killer (LAK) cells, generated from peripheral blood lymphocytes (PBL) from patients with oral cancer or oral leukoplakia and from healthy donors showed comparable lysis of 6 target tumor cell lines, including 3 derived from head and neck and oral cancers. The tumor burden of the host did not appear to influence the systemic LAK activity. LAK activity of lymphocytes infiltrating the tumor tissues (TIL) was also comparable to that of the PBL. Both TIL and PBL showed a parallel increase in proportion of HNK-I+ and CD-25+ cells upon activation with IL-2. The lymph-node lymphocytes (LNL) from metastatic (met) and non-metastatic (non-met) draining lymph nodes, however, showed reduced LAK activity and an increase in CD8+ cells, in addition to CD25+ and HNK-I+ cells, when cultured with IL-2. When IL-2-activated LNL were co-cultured with autologous PBL during IL-2 activation of the latter, a strong suppressive effect was exerted by LNL. In contrast, IL-2-activated PBL did not suppress autologous LAK generation in spite of an increase in CD8+ cells seen after activation with IL-2. Frequency distribution of LAK precursors was significantly lower in LNL than in PBL from oral cancer patients. LAK precursor frequency in TIL was comparable to that of PBL. The results show that, in oral cancer, regional lymph nodes may not have adequate IL-2-inducible cytotoxic potential, due to a reduced number of LAK progenitors and possible activation of suppressor cells. Alternatively, TIL can be a potential source for LAK cell function.     

Interleukin-2-stimulated natural killer activity against malignant and benign endometrium

Natural cell-mediated immunity against autologous tumor cells, autologous endometrial epithelium, and allogeneic epidermoid carcinoma cell line HeLa was tested in 8 patients with endometrial carcinoma and one patient with endometrial stromal sarcoma. The average cytotoxicity of unstimulated peripheral blood lymphocytes against autologous tumor and HeLa cells was weak but significant. Pretreatment of effector cells for 3-5 days with 300 U/ml recombinant interleukin-2 (rIL-2) resulted in increased cytotoxicity against malignant target cells in 7 out of 9 cases. The 2 patients' effector cells which were refractory to rIL-2 could be stimulated to appreciable lytic activity against the malignant target cells with a recently described cytokine which induces morphological differentiation of natural killer cells. Benign endometrial cells were weakly sensitive to rIL-2-activated lysis in 2 cases. The precursors of the rIL-2-activated killer cells were mostly CD16-positive and CD3-negative, and co-sedimented with endogenous natural killer cells in discontinuous density gradient centrifugations. These results indicate the rIL-2-activated killer cells have a capacity to distinguish between normal and malignant endometrial cells, and that the precursors of the lytic cells in this system belong to the same subpopulation of lymphocytes as endogenous natural killer cells. In addition, rIL-2 alone may not in all cases be sufficient for optimal generation of cytotoxicity against malignant cells.     

Interleukin-2-activated lymphocytes from brain tumor patients. A comparison of two preparations generated in vitro

Two preparations of human recombinant interleukin-2 (rIL-2)-activated lymphocytes from patients harboring malignant brain tumors were characterized as autologous-stimulated lymphocytes (ASL) and lymphokine-activated killer (LAK) cells. ASL were generated from Ficoll-Paque-isolated, nonadherent, defibrinated peripheral blood lymphocytes (PBL) that were stimulated overnight with phytohemagglutinin (PHA) and cultured with rIL-2 (100 U/ml) for 10 days. LAK cells were produced by culturing all PBL in rIL-2 (500 U/ml) for 4 days. In 4-hour chromium release assays, LAK cells showed greater cytotoxicity than ASL against natural killer (NK)-sensitive and NK-resistant tumor cell lines; by 18 hours, the effectiveness of ASL equaled that of LAK cells. By electron microscopic study, PBL, LAK cells, and ASL showed differences. The helper/inducer to suppressor/cytotoxic ratio (T4+/T8+) of PBL, LAK cells, and ASL was 1.1:1, 1.0:1, and 0.4:1, respectively. ASL, when compared with PBL or LAK cells, have a significantly higher percentage of MO1+/DR+ and T8+/9.3+ subpopulations. ASL and LAK cells, used for the therapy of gliomas, are distinct.