A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry.

A flow cytometric assay for the combined measurement of cell-mediated cytotoxicity and conjugate formation has been developed. Cytolysis is detected by propidium iodide uptake. Target cells, effector cells and conjugates between targets and effectors are separated by post-culture immunophenotyping and their scatter profiles. Pre-assay staining of cells is thus not required. Each cluster of cells can be further examined at the single-cell level by simultaneously performed additional immunophenotyping. Two applications were established: the assessment of NK cell activity against K562 cells and the evaluation of LAK cell cytotoxicity against both K562 and Daudi cells. A comparison with the standard 51Cr release assay for the detection of NK cytotoxicity showed that the two assays were strongly correlated, but the sensitivity of the flow cytometric assay was significantly higher.

Assessment of the therapeutic potential of cytokines, cytotoxic drugs and effector cell populations for the treatment of multiple myeloma using the 5T33 murine myeloma model.

The therapeutic potential of six cytokines, eight cytotoxic drugs and two effector cell populations for the treatment of multiple myeloma was assessed in vitro using the 5T33 murine myeloma model. The efficacy of combination IFN-alpha and melphalan therapy was also evaluated in vitro and in vivo. Of the cytokines tested in vitro using the MTT assay, only IFN-alpha demonstrated significant inhibition of myeloma cell growth at non-toxic concentrations (ED50 = 1508.3 +/- 181.3 U/mL and 2617.9 +/- 334.0 U/mL for murine IFN-alpha [mIFN-alpha] and human IFN-alpha hybrid B/D [hIFN-alpha B/D], respectively). The ED50 for the eight cytotoxic drugs tested ranged from 2.3 x 10(-9) to 4.3 x 10(-13) mol/L and all were within the therapeutic range for humans. Combination hIFN-alpha B/D and melphalan were found to be additive in their inhibitory effects on myeloma cell growth in vitro and this finding was confirmed in vivo in C57BL/KaLwRij mice bearing disseminated 5T33 myeloma. Control animals demonstrated a median survival duration of 25.3 days whereas hIFN-alpha B/D or melphalan treatment alone increased survival to 30.5 and 33.3 days, respectively (P < 0.001). Combination IFN-alpha/melphalan therapy increased median survival duration to 38.5 days (P < 0.001) which was also significantly greater than that obtained with single agent therapy (P < 0.01). The murine myeloma cells were found to be resistant to NK cell lysis but susceptible to lysis by LAK cells (49.3 +/- 6.3% lysis at an effector to target ratio of 100:1).(ABSTRACT TRUNCATED AT 250 WORDS)

Super-sensitive epithelial cell line and colorimetric assay to replace the conventional K562 target and chromium release assay for assessment of non-MHC-restricted cytotoxicity.

Using colorimetric MTT assay the susceptibility of a newly established bladder epithelial cell line, Fen cells was compared with conventional target cells, i.e., K562 and Daudi and other epithelial lines for investigation of non-specific killing activity (NK/LAK) of effector cells previously activated with interleukin-2 (IL-2). The results showed that Fen cell line was more sensitive than K562 and Daudi targets and this was seen whether the effector cells were IL-2-activated or not. The percent killing of effector cells from nine normal donors against Fen, K562 and Daudi targets at effector/target (E/T) ratio of 10/1 after IL-2 activation were 63.4 +/- 7.3, 42.6 +/- 4.3 (p = 0.0001) and 38.6 +/- 5.1 (p = 0.0001) respectively. The corresponding values for inactivated effector cells at 50/1, E/T ratio were 44.8 +/- 9.0, 25.1 +/- 8.3 (p = 0.0001) and 24.4 +/- 9.4 (p = 0.0001) indicating exquisite sensitivity of Fen cells to NK/LAK killing. The susceptibility of Fen cells was found to increase by pre-treatment of target cells with interferons (IFN). Thus the percent killing of untreated, IFN-alpha (1000 U/ml), beta (2000 U/ml) and gamma (100 U/ml) treated cells were 52%, 64% (p = 0.005), 70% (p = 0.001) and 67% (p = 0.001) respectively. These results indicated that Fen cells were more susceptible to NK/LAK killing than the conventional K562 and Daudi target cells. These results and the epithelial origin of Fen cells indicate that this cell line might prove to be a more realistic system to replace the conventional approach for assessment of NK/LAK activity in patients with cancer of epithelial origin.

In vitro and in vivo assessment of the graft-versus-leukemia activity of cord blood.

Allogeneic bone marrow transplantation (BMT) frequently is accompanied by the occurrence of graft-versus-host disease (GVHD). GVHD is thought to mediate a beneficial graft-versus-leukemia (GVL) effect believed to be important for disease-free survival in cancer patients. However, it is uncertain if GVHD and GVL are mediated by unique effector cell populations in the graft. The lack of bone marrow donors for individuals needing HLA-matched, unrelated BMT has recently led to the use of cord blood for transplantation. Cord blood transplantation has generated much enthusiasm because of its very low incidence of GVHD, even in HLA-mismatched situations, owing to intrinsic defects in mature T cell functions. Concerns have arisen, however, as to whether cord blood would mediate a significant GVL activity in vivo in the absence of GVHD, and thus prevent relapse in patients treated for malignancies. In this study in vitro and in vivo assessments have been made of the ability of cord blood to mediate GVL activity, focusing on non-specific effector cell mechanisms. Although minimal non-specific cytotoxic activity is found in freshly isolated cord blood (both NK and LAK cell activity), it is rapidly induced and displays a spectrum of lytic activity similar to adult peripheral blood. The kinetics of LAK cell induction in cord blood as well as the responsiveness to IL-2 stimulation was identical to adult peripheral blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell cycle assessment of adoptive transferred lymphokine activated killer cells.

The use of lymphokine activated killer (LAK) cells for adoptive transfer therapy has been reported from number of clinical trials. To our knowledge, however, there has been no report concerning the cell cycle progression of LAK cells. Thus, for the present study we have attempted to examine the LAK cell cycle either before or after transfer. In vitro and in vivo analyses of LAK cells labeled with bromodeoxyuridine (BrdU) were carried out using two-parameter flow cytometries of their nuclear staining using fluorescein isothiocyanate(FITC)-conjugated anti-BrdU antibody and propidium iodide (PI). The in vitro growth of BrdU-positive cells showed the cells to divide once in the S phase, continue to the G2-M phase and return to the G0-G1 phase with a similar pattern after 48 or 72 h culture. They formed a definite subpopulation and were of phenotypes, thy-1.2 (+), Lyt-1.1 (-), Lyt-2.1 (+), L3T4 (-) and AGM1 (+). The percentage of BrdU-positive cells decreased significantly (P < 0.05) when treated with complement plus anti-thy-1.2, anti-Lyt-2.1 or anti-AGM1 anti-bodies, demonstrating BrdU-labeled LAK cells to have the same phenotype as control LAK cells. As in vitro, the in vivo cell cycle of LAK cells 48 and 72 h after transfer had a similar pattern, and the LAK cells also continued on to the S phase after the first division. The in vivo growth of the LAK cells treated with interleukin-2 (IL-2) was promoted 24 and 48 h after transfer. In conclusion, the present study showed LAK cells to be capable of dividing following transfer and to keep their own phenotypic characteristics; also, that treatment with IL-2 may promote their division. Adoptive transfer therapy seems to be a viable therapeutic method.

Assessment of therapeutic potential of interleukin 2 for myelodysplastic syndromes.

The therapeutic potential of interleukin 2 (IL-2) for myelodsplastic syndromes (MDS) was evaluated in vitro. IL-2-induced lymphokine-activated killer (LAK) cells were prepared from 38 MDS patients and 20 normal subjects. The cytotoxicity of LAK cells against K562 and Raji cell lines and MDS blasts was significantly reduced in high-risk MDS (refractory anaemia with excess blasts (RAEB), RAEB in transformation, and leukaemic transformation of MDS), but was relatively well-preserved in low-risk MDS (refractory anaemia (RA) and RA with ringed sideroblasts). Examination of the immunophenotypes of freshly-isolated lymphocytes showed that the percentage of CD4+ cells in low-risk MDS and the percentage of CD3+, CD4+ and CD8+ cell populations in high-risk MDS was significantly reduced compared with these populations in normal subjects. After cultivation with IL-2, these three cell populations were still reduced in the corresponding MDS groups and the percentage of CD3-CD56+ cells were significantly reduced in high-risk MDS. There was a positive correlation between the percentage of K562 cells lysed by MDS LAK cells and the percentage of CD3-CD56+ lymphocytes in MDS LAK cells. These aberrant lymphocyte subpopulations appeared to explain, at least in part, the reduced LAK cell cytotoxicity in MDS. These results present a possibility that IL-2 and LAK therapies are ineffective for most high-risk MDS patients, whereas they have potential value for low-risk MDS patients whose lymphocyte cytotoxicity is usually preserved.

Assessment of lymphokine-activated killer activity and gamma-interferon production in patients with small hepatocellular carcinomas.

We have previously reported depressed gamma-interferon production and depressed lymphokine-activated killer and natural killer activity in patients with relatively large hepatocellular carcinomas. These parameters were normal in cirrhosis. Some evidence had suggested a gamma-interferon production defect as the main cause of depressed lymphokine-activated killer activity in hepatocellular carcinoma, (i.e., gamma-interferon enhances lymphokine-activated killer and natural killer activity and gamma-interferon antibody inhibits lymphokine-activated killer induction). However, we were unable to clinically define the precise mechanism operating here because gamma-interferon production and lymphokine-activated killer activity were both defective in advanced hepatocellular carcinoma. In recent years, it has become possible to detect even small hepatocellular carcinomas on ultrasonography and to confirm them by fine-needle biopsy. In this study, we assessed those immune parameters in 48 patients with hepatocellular carcinomas less than 2 cm in diameter to confirm depressed immune function and to clarify the mechanism of these defects. Lymphokine-activated killer activity was defective in 31 patients (64.6%), whereas gamma-interferon production was defective in only 1 of these patients (2.1%). This observation argues against the hypothesis that defective gamma-interferon production is the primary defect and provides new insight into the mechanism of progression of defective immune function in hepatocellular carcinoma. Thirty-one of the 48 hepatocellular carcinoma patients were treated surgically, and these immune parameters were followed for 6 mo. The recovery of lymphokine-activated killer activity in all hepatocellular carcinoma patients with low lymphokine-activated killer activity suggests the tumor burden as the cause of defective lymphokine-activated killer activity.

[The preclinical assessment of the activity and pharmacological action of native human interleukin-2]. / Predklinicheskaia otsenka aktivnosti i farmakologicheskogo deistviia nativnogo interleikina-2 cheloveka.

It was demonstrated in in vivo and in vitro experiments that interleukin-2, obtained by cultivation of donor lymphocytes and purified by gel filtration, induces the production of mouse and human killer lymphocytes possessing high cytolytic activity against tumor cells. Interleukin-2 does not cause irreversible changes of the physiological and morphologic indices of vital activity in mice.

Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts.

Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant (specific lysis was 7.8% +/- 1.1, p less than 0.01). In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively (p less than 0.05), as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites. Neither human NK cells (freshly isolated or activated by rIL-2 or rIFN-alpha) nor LAK cells were cytotoxic for purified preparations of cysts of Toxoplasma isolated from the brains of chronically infected mice.