Induction and clinical utilization of lymphokine-activated killer cells in patients with gastrointestinal tract cancers

In 18 patients with cancers of the gastrointestinal tract, lymphokine-activated killer (LAK) cell activity was studied and compared with that of healthy subjects. After cultivation with 10(3) iu/mL of recombinant interleukin-2, the cytotoxicity of patients' lymphoid cells was increased from 13.6 +/- 6.8% to 76.2 +/- 19.5% against Daudi cells and from 12.8 +/- 8.1% to 76.2 +/- 19.5% against K-562 cells. Based on these results, autologous LAK cells were given to patients. LAK cells injected into subdermal metastatic tumours demonstrated a significant inhibitory effect on tumour growth in comparison with that of control tumour nodules. Of four patients with metastatic tumours in the liver, to whom LAK cells were administered via the hepatic artery, tumour size was reduced by about 25% (minor response) in one patient, with a decrease of computerized tomography attenuation in the tumours occurring in the other three patients.     

Adoptive immunotherapy for unresectable or recurrent pancreatic cancer,using lymphokine-activated killer cells or cytotoxic T cells

Adoptive immunotherapy (AIT) has been reported to be effective for malignancies in some cases. We hypothesized that AIT may be effective for the treatment of pancreatic cancer. Seven patients with unresectable or recurrent pancreatic cancer underwent AIT, carried out with lymphokine-activated killer (LAK) cells or cytotoxic T cells (CTLS) and recombinant interleukin-2 (IL-2). The clinical and immunological effects were evaluated. Of four patients who received CTLs, one had a partial response, one had a minor response, and two showed no change. The three patients who received LAK cells had progressive disease. The CTLs had a significantly higher proportion of CD8 positive T cells and cytotoxic T cells than the LAK cells (P < 0.05), while the LAK cells had a significantly higher proportion of NK cells than did the CTLs (84 ± 12% vs 55 ± 15%,P < 0.05). The LAK activity of the CTLs (61 ± 14%) was significantly higher than that of the LAK cells (42 ± 8%,P < 0.05). Seven days after treatment with LAK cells or CTLs, lymphocyte subsets in the peripheral blood were examined. The proportion of CD8-positive T cells after CTL transfer (46 ± 5%) was greater than that before CTL transfer (28 ± 1%,P < 0.05). The proportion of cytotoxic T cells after CTL transfer increased from 23 ± 1% to 37 ± 2% (P < 0.05). However, these changes were not observed during LAK cell transfer. The proportions of suppressor inducer T cells, helper T cells, and suppressor T cells did not change during therapy. These results suggested that AIT, using CTLs, merits further clinical investigation in patients with pancreatic cancer.     

Evaluation of natural killer and lymphokine-activated killer (LAK) cell activity in vivo in patients treated with high-dose interleukin-2 and adoptive transfer of autologous LAK cells

Summary This study investigated the development of peripheral blood natural killer (NK) and lymphokineactivated killer (LAK) cell activity in vivo in cancer patients treated with high doses of recombinant interleukin-2 and autologous LAK cell infusion. It was found that interleukin-2 administration by bolus injection resulted in an early but transient rise of NK and LAK cell activity in vivo during the first 5–9 days of treatment (postpriming period), whereas continuous infusion of interleukin-2 caused an increase in both cytotoxic activities in the second phase of the treatment, concomitant with infusions of autologous LAK cells. Elevated NK but not LAK cell activity in vivo in the continuous-infusion patients persisted up to 180 days after completion of therapy. In both bolus and continuous interleukin-2 protocols, augmented NK cell activity in vivo appeared to be correlated with a beneficial response to therapy.Supported by the US-Poland Cancer Program (NCI) and by NCI Contract NO1-CM-73705     

Resistance of some leukemic blasts to lysis by lymphokine activated killer (LAK) cells

Peripheral blood mononuclear cells (PBMC) from healthy donors and AML patients in remission were stimulated with phytohemagglutinin (PHA) and recombinant interleukin-2 (IL-2). These stimulated cells (lymphokine activated killer (LAK) cells) showed increased DNA synthesis as measured by 3H-Thymidine uptake. A synergistic effect of PHA and IL-2 was found. LAK cells' ability to kill acute myeloid leukemia (AML) blasts was investigated by the 51Cr release assay. LAK cells showed a cytotoxicity (over 10% specific 51Cr release) against 9/12 leukemic blasts, even at effector/target (E/T) ratios as low as 5:1. However, on average only 22.2% (SD 11.8) and 36.5% (SD 12.5) 51Cr release were obtained in 4- and 18-hour cytotoxicity assays, respectively, at an E/T ratio of 20:1. Leukemic blasts in 3/12 AML cases and normal PBMC were entirely resistant to lysis, even at an E/T ratio of 80:1. Susceptibility to lysis was not correlated to peanut-agglutinin receptor expression. LAK cells were more cytotoxic towards the K-562 cell line (natural killer activity) than unstimulated PBMC.     

Defective immunological functions associated with abnormal lymphokine-activated killer activity in patients with hepatocellular carcinoma

The in vitro lymphokine-activated killer (LAK) activity of peripheral blood mononuclear cells (PBMC) from 36 patients with hepatocellular carcinoma was investigated. The activity was greatly diminished in 13 patients and enhanced in seven patients. A flow cytometric study showed that the percentage of OKM1+, Leu-7+-11b+, and Leu-7-11b+ fractions in PBMC was decreased and the percentage of OKT8+ and Leu7+11- fractions was increased significantly in the 13 patients with lower LAK activity, compared with the values of the seven higher LAK activity patients. Furthermore, the response of PBMC to interleukin-2 (IL-2) was deficient in the lower activity group. However, there was no significant difference in IL-2 production by PBMC, IL-2 receptor (p55) expression of PBMC and mitogen (Con-A, PHA) response of PBMC between the two groups. These findings indicate the possibility that diminished LAK activity in hepatoma patients is due to a decreased number of LAK precursor cells and a defective response of LAK precursor cells to IL-2.     

Induction of allogeneic tumour- and lymphokine-activated lymphocytes against hepatocellular carcinoma

For clinical application of adoptive immunotherapy against hepatocellular carcinoma (HCC), it is not easy to prepare tumour specific effector cells such as cytotoxic T lymphocytes (CTL). To induce potent and broad-spectrum effectors, allogeneic cultured hepatoma cell lines (JHH-4 and HuH-6) were used as stimulators of peripheral blood lymphocytes (PBL) instead of autologous HCC cells. Allogeneic tumour- and lymphokine-activated killer cells (ATLAK) were generated by a mixed culture of lymphocytes and allogeneic cultured tumour cells with recombinant interleukin-2 (rIL-2). The tumour-killing activity of ATLAK induced by HuH-6 was confirmed against HuH-6 and other different HCC cell lines (JHH-2, HuH-7 and PLC). These activated lymphocytes were significantly more potent than lymphokine-activated killer cells (LAK) in [51Cr]-releasing assay. The JHH-4 stimulated ATLAK was reactive not only with JHH-4 but also with JHH-2. The lysis of allogeneic targets could be partially inhibited by anti-CD8 and anti-CD3 but not by anti-CD4. Anti-tumour cytotoxicity in these cultures might be mediated by CD3+CD56- and CD3+CD56+ effectors. These results imply that adoptive immunotherapy for HCC with ATLAK may be more feasible than that with LAK.     

Phenotypical study of human lymphokine-activated killer (LAK) cells

In the present study, T-cell enriched lymphocyte populations were stimulated with recombinant interleukin-2 (IL-2) to obtain lymphokine-activated killer (LAK) cells, and their cytotoxicity was tested against different target cells. The expression of phenotypical markers with a panel of monoclonal antibodies (Moabs) using indirect immunofluorescence techniques and cytofluorometric analysis was studied. The results showed slight variations in T-cell antigen expression and an increase in the expression of immature NK and proliferation associated antigens.     

Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells

Human natural killer (NK) cells constitutively express the beta- and gamma-chains of the interleukin 2 (IL-2)/IL-15 receptor, and both IL-2 and IL-15 are able to activate NK cell proliferation and cytotoxicity. When IL-2-primed human NK cells are exposed to sensitive targets (i.e. K562) they undergo apoptosis mediated by the beta(2)-integrin CD18. Here, we demonstrate that: (i) endothelial cells, similar to K562 tumour target cells, induce apoptosis of IL-2-primed NK cells; (ii) endothelial- and K562 cell-induced apoptosis is significantly lower in IL-15 than in IL-2-stimulated NK cells; (iii) a critical role in the apoptosis of IL-2-primed NK cells is played by the alpha-chain of the IL-2 receptor. Our data show for the first time that IL-2-activated NK cells can die by apoptosis upon contact with the vascular endothelium, which is a necessary step for their extravasation, with a direct pathophysiological relevance on the strategy of adoptive immunotherapy of cancer. On the other hand, IL-15, although generating a similar level of activation of NK cells, largely prevents their apoptotic fate. Therefore, IL-15 produced early in the immune response, when T cells are not yet activated, generates lymphokine-activated killer cells that are efficient killers relatively protected from apoptosis. Once activated, T cells produce IL-2 that overcomes the effect of IL-15 on NK cells, paving the way for their death by apoptosis.     

Lysis of human malignant mesothelioma cells by natural killer (NK) and lymphokine-activated killer (LAK) cells

Malignant mesothelioma is an aggressive tumor of the pleura for which, at present, there is no effective therapy. As interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells lyse many solid tissue malignancies that are unresponsive to conventional forms of therapy, the aim of this study was to evaluate the susceptibility of human malignant mesothelioma cells to lysis by natural killer (NK) and LAK cells. Using a 4-h 51Cr release assay, malignant mesothelioma cell lines grown from six different patients were found to be resistant to NK cell lysis (less than 10% lysis as compared to 50 +/- 3% lysis of the standard NK-sensitive target, K562, p less than 0.001). These malignant mesothelioma cells were, however, susceptible to lysis by LAK cells (58 +/- 4% lysis, p less than 0.001 compared to NK lysis). Similar results were seen using fresh mesothelioma cell targets (4 +/- 2% and 34 +/- 12% lysis for NK and LAK cells, respectively). Optimal LAK cell activation against these targets was achieved by incubating peripheral blood mononuclear cells (2 to 4 x 10(6)/ml) in culture medium containing 1,000 units/ml IL-2 for 3 to 14 days. The degree of LAK cell activation was dependent on the serum source used in culture, with autologous serum being more effective than pooled human AB serum or fetal calf serum at generating LAK cell activity in vitro (p less than 0.05). The results of this study demonstrate that although human malignant mesothelioma cells are resistant to NK cell lysis, IL-2-activated LAK cells effectively kill these targets.(ABSTRACT TRUNCATED AT 250 WORDS)     

The induction of lymphokine-activated killer cells: increased cytotoxicity via long-term cultivation

A long-time culture regime is tested for the production of lymphokine-activated killer cells from human lymphocytes, which combines the advantage of cell expansion with an increased cytotoxicity. The concentrations of IL-2 used are relatively low.     

Therapeutic uses of interleukin-2 and lymphokine-activated killer (LAK) cells

Interleukin-2 (IL-2) is the first of a growing list of lymphokines to be cloned and available for preclinical and clinical evaluation. A product of T-helper lymphocytes, IL-2 augments the cytolytic activity of T-lymphocytes and natural killer (NK) cells, stimulates the proliferation of these cells, and induces the formation of lymphokine-activated killer (LAK) cells. LAK cells exhibit cytolytic activity against a broad range of both freshly isolated and cultured tumor cells, while exhibiting limited cytolytic activity against normal cells. The apparently large therapeutic index suggested by in vitro studies is strongly supported by the antitumor responses seen in preclinical studies. Initial clinical studies reported encouraging response rates, but the actual role of IL-2 and/or LAK cell infusion in cancer therapy has yet to be determined, and may only represent the first step in managing the tumoricidal potential of the immune system.     

High number of PD‐1 positive intratumoural lymphocytes predicts survival benefit of cytokine‐induced killer cells for hepatocellular carcinoma patients

Background & Aims

Adjuvant cytokine‐induced killer (CIK) cells treatment has shown potential in reducing the recurrence rate and prolonging the survival of patients with hepatocellular carcinoma (HCC). We aimed to identify the best predictive biomarker for adjuvant CIK cells treatment in patients with HCC after curative resection.

Methods

This study retrospectively included 145 pairs of HCC patients by one‐to‐one propensity score matching. One group received CIK cells transfusion after surgery (surgery‐CIK group); the other one group underwent surgery only (surgery‐only group). Immunohistochemistry (IHC) was used to measure PD‐1, PD‐L1, CD4, CD8 and Foxp3 expression in tumour tissues of surgery‐CIK group; IHC of PD‐1 and PD‐L1 was conducted in the surgery‐only group.

Results

The surgery‐CIK group had a significantly higher disease‐free survival (DFS) and overall survival (OS) rates compared to the surgery‐only group. Of all the intratumoural biomarkers, in the surgery‐CIK group, multivariate analysis showed that a high number of PD‐1⁺ tumour infiltrative lymphocytes (TILs) was the only factor that independently predicted favourable OS and DFS. By contrast, in the surgery‐only group, no significant correlations between PD‐1/PD‐L1 expression and survival of patients were identified. Further correlation analysis showed a high number of PD‐1⁺ TILs associated with a high number of both CD4⁺ and CD8⁺ TILs in surgery‐CIK group.

Conclusions

A high number of PD‐1⁺ TILs can serve as a potent biomarker for adopting CIK cells therapy in HCC patients after curative resection.     

Defective function of lymphokine-activated killer cells and natural killer cells in patients with hepatocellular carcinoma

Lymphokine-activated killer activity and natural killer activity in hepatocellular carcinoma patients were assessed. Maximum lymphokine-activated killer activity was induced at 3 to 5 days of incubation, and lymphokine-activated killer activity tended to increase in a manner dose dependent of recombinant interleukin-2. However, the maximum increase of lymphokine-activated killer activity in hepatocellular carcinoma was not as high as that of normal subjects or liver cirrhosis patients. Lymphokine-activated killer activity was impaired in hepatocellular carcinoma as compared to that in normal subjects. Hepatocellular carcinoma seemed to consist of two groups: i.e. a high-lymphokine-activated killer activity group and a low-lymphokine-activated killer activity group. Reduction of natural killer activity was also observed in hepatocellular carcinoma as compared with that in normal subjects and patients with liver cirrhosis. No correlation could be demonstrated between natural killer activity and lymphokine-activated killer activity in normal subjects, liver cirrhosis patients and hepatocellular carcinoma patients. With regard to the presence of HBsAg or alpha-fetoprotein concentration in the sera, there was no significant difference in natural killer and lymphokine-activated killer activity in hepatocellular carcinoma patients. Patients with a small mass lesion showed a low lymphokine-activated killer activity, and depressed lymphokine-activated killer activity was not necessarily related to tumor size. In comparison with the high-lymphokine-activated killer group, the low-lymphokine-activated killer group showed a significant decrease in gamma-interferon production and a preserved function of indocyanine green clearance.     

Severe intrahepatic cholestasis in patients treated with recombinant interleukin-2 and lymphokine-activated killer cells

Summary Immunotherapy with recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells (LAK) has become a new form of therapy that has been shown to induce remissions in patients with renal cell carcinoma and melanoma. Despite encouraging results, this form of therapy has been associated with increasing toxicity, often requiring therapy in an intensive-care unit. In this report severe intrahepatic cholestasis occurred in two patients receiving rIL-2 and LAK cells. This form of cholestasis appeared to be directly related to rIL-2 administration at a doses of 2×10⁶ U/m² and 3x10⁶ U/m² t.i.d. A liver biopsy showed moderate hepatocellular bile stasis, with lobular and portal inflamation. All other studies for potential cause of this cholestasis were negative, including studies for metastatic disease. When therapy was discontinued, evidence for cholestasis and bile stasis resolved. We conclude that rIL-2 is a drug with a potential to induce severe hepatic injury that is reversible upon cessation of therapy with rIL-2. Further care should be exercised when rIL-2 is administered to patients with abnormal liver function.Supported in part by Dr. I. Fund Foundation, Hoffmann-La Roche Inc., Nutley, New Jersey, and the Sarah C. Upham Trust     

The phenotypic and functional characteristics of umbilical cord blood and peripheral blood natural killer cells

Allogeneic hematopoietic cell transplantation can be curative for patients with high-risk acute leukaemia. Umbilical cord blood (UCB) is an increasingly used source of allogeneic stem cells for patients who are in need of a transplant, but do not have a sibling donor. This review highlights the similarities and differences between the natural killer (NK) cells obtained from adult peripheral blood (PB) and UCB. These two cell sources show similar percentages of NK cells, including the major CD56^dim and CD56^bright subpopulations. UCB also contains an additional CD56⁻CD16⁺ subset, not typically found in PB. In addition, there are a number of progenitor cell populations in UCB that can give rise to NK cells. Some studies showed that UCB NK cells express a relatively higher percentage of inhibitory receptors (CD94/NKG2A and killer-cell immunoglobulin-like receptors) and less adhesion molecules. Resting UCB NK cells also show significantly less cytotoxicity compared to PB NK cells. However, following cytokine stimulation, the cytotoxicity of UCB NK cells can be rapidly increased to levels that are comparable to PB NK cells. Activation and expansion protocols for UCB NK cells are briefly reviewed. Lastly, we outline the early use of UCB NK cells in clinical trials.     

Natural killer cell and islet killer cell activities in Type 1 (insulin-dependent) diabetes

Summary Peripheral blood mononuclear cells from 20 Type 1 (insulin-dependent) diabetic patients were examined for natural killer cell activity using the K562 cell line as⁵¹Cr labeled targets. Mean natural killer cell cytotoxicity mediated by enriched non-T cells from patients (37 ± 4.0%) was lower (p < 0.03) than in controls (56 ± 3.7%). Specificity was evaluated by examining other patient subgroups. Mean non-T cell mediated natural killer cell activity in Type 2 (non-insulin-dependent) diabetic patients and Type 1 patients with long term disease was 65±54% and 62±4.8% respectively (p<0.003 vs new onset Type I patients). Longitudinal studies of new onset Type 1 patients during the remission (honeymoon) phase revealed persistently impaired natural killer cell activity in 3 of 4 patients. In 30 new onset and 11 remission Type 1 diabetic patients, mean non-T cell-mediated cytotoxicity was also measured using dispersed⁵¹Cr labeled islet target cells. Mean islet cytotoxicity mediated by cells from new onset patients was 34±2.4%, whereas in non-diabetic control subjects mean cytotoxicity was 25 ± 1.8% (p < 0.005). During remission, islet cytotoxicity remained at similar or elevated levels in most patients. In patients evaluated simultaneously for K562 and islet cell cytotoxicity, natural killer cell activity was decreased, whereas islet killing was increased. These results suggest a dichotomy in natural killer cell and islet killer cell activities in new onset Type 1 diabetes that could have an important role in the pathogenesis of Type diabetes.     

Immunotherapy of hepatocellular carcinoma with autologous lymphokine-activated killer cells and/or recombinant interleukin-2

Summary Five patients with hepatocellular carcinoma were subjected to immunotherapy: three patients were treated by adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2), and two patients by systemic administration of rIL-2 alone. In one patient with diffuse-type hepatocellular carcinoma and portal vein thrombosis who was treated by infusion of LAK cells (a total number of 1.5x10¹⁰ cells/13 doses) and continuous rIL-2 administration (a total dose of 1.25x10⁸ units) via a percultaneously placed hepatic arterial catheter, the size of the tumor reduced dramatically and the portal vein thrombosis retracted. In two patients who had LAK cells infused (totals of 6.6x10⁹ cells/4 doses and 3.1x10⁹ cells/2 doses, respectively) during hepatic angiogram followed by systemic administration of rIL-2 twice a day, no clinical improvement was noticed. In two patients who received rIL-2 alone systemically (total doses of 8.9x10⁷ and 5.5x10⁷ units, respectively), neither clinical improvement nor severe side effects were observed. The results suggest that adoptive immunotherapy combined with continuous local administration of rIL-2 via a percutaneously placed hepatic arterial catheter may be an effective therapy without apparent side effects for patients with hepatocellular carcinoma who cannot be treated by conventional cancer therapy.     

Effects of interleukin-12 on natural killer cell cytotoxicity and the production of interferon-7 and tumour necrosis factor-a in patients with myelodysplastic syndromes

The effects of Interleukin 12 (IL-12) on natural killer (NK) cell cytotoxicity and on the production of interferon-7 (IFN-7) and tumour necrosis factor-a (TNF-a) were examined in 15 patients with myelodysplastic syndromes (MDS), which are well known to have immunologic defects, and in 11 normal subjects. The NK cell cytotoxicity of all of the normal subjects was augmented by incubation with IL-12 alone, and co-incubation with interleukin 2 (IL-2) further augmented it (type A response). The MDS patients showed varied responses to IL-12/IL-2. Seven patients showed the type A response, resulting in augmented NK cell cytotoxicity which was similar to that in the normal subjects. In five other patients the cytotoxicity was not increased by IL-12 alone, but the combination of IL-12 and IL-2 did augment the cytotoxicity (type B response). The augmented cytotoxicity in these type B patients was lower than that in the normal subjects. In the final three MDS patients the cytotoxicity was low and not affected by IL- 12 and/or IL-2 (type C response). AH patients with refractory anaemia with excess blasts (RAEB) and patients with RAEB in transformation showed a type B or C response. Conversely, six of eight refractory anaemia patients showed a type A response. In MDS patients there was a positive correlation between the percentage of CD3CD56⁺ cells in pre-incubated cells and the cytotoxicity of cells incubated with IL-12/IL-2. The combination of IL-12 and IL-2 augmented IFN-7 and TNF-Q production by nonadherent mononuclear cells in a synergistic or cumulative manner, respectively, in most patients. These results suggest that IL-12, alone or with IL-2, may modulate these important immunologic functions in most MDS patients.     

Evidence that continued remission in patients treated for acute leukaemia is dependent upon autologous natural killer cells

Although it has been known for more than 40 years that allogeneic immune responses cure leukaemias after bone marrow transplantation, autologous leukaemia-specific immunity remains controversial and its impact upon survival has not been established. Here we have tested 25 patients with de novo acute leukaemias, while in remission at completion of their anti-leukaemia therapy, for evidence of autologous cytolytic immunity to their leukaemic cells taken and cryopreserved at disease presentation. We have measured this degree of cell-mediated cytotoxicity in vitro and termed it "leukaemia cytolytic activity" (LCA). Patients whose disease ultimately relapsed had significantly lower LCA than those who remained in remission beyond 2 years (P < 0.001); the absence of LCA when in remission predicted subsequent relapse within 2 years with a sensitivity of 100% and specificity of 77%. LCA was mediated in vitro by CD56+/CD8alpha+/CD3- natural killer cells. We propose that it is this immune response, rather than the chemotherapy per se, which is responsible for continued remission and that measurement of LCA in patients at completion of therapy may be used as an indicator of risk of subsequent relapse. Patients lacking this response will require further treatment, either with an allogeneic donor transplant or an alternative immunotherapeutic strategy.