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March 1993, Volume 43, Issue 3

Original Article

ANTI-TUMOR EFFECT OF HUMAN LYMPHOCYTESAND INTERLEUKLN-2

Farzal Anwer  ( Al Hada Armed Forces Hospital, Tail, saudi Arabia. )
Mehdi Mesri  ( Department of Experimental and Clinical Microbiology, Sheffield Medical School, Sheffield, England. )
Robert C. Rees  ( Department of Experimental and Clinical Microbiology, Sheffield Medical School, Sheffield, England. )
Shahana U. Kazmi  ( Department of Microbiology, University of Karachi, Karachi. )

ABSTRACT

We studied the anti-tumor effect of control human lymphocytes and interleukin-2 (lL-2) activated lymphocytes (lymphokine activated killer cells, LAK-cells), on two different cell lines: SW742 human colon adenocarcinoma and K562 human myeloid leukaemia cell line. Our results indicate that lL-2 augment the anti-tumor activityy of human lymphocytes and these LAK-cells lyse the tumor cells very efficiently. Furthermore, we treated the target cells (SW742 and K562) with different cytokines in order to establish whether these cytokines have any effect on susceptibility to lysis by LAK-cells. Anti-tumor activity of human lymphocytes and IL-2 is discussed in this study (JPMA 43: 45, 1993).

INTRODUCTION

Interleukin-2 (IL-2) was originally described as T cell growth factor and is significant to the growth and function of a variety of cell types of the immune system. IL-2 stimulates proliferation of activated cells of the lymphoid lineage and cells responsive to its action include activated Tlymphocytes, natural killer cells (NK), lymphokine activated killer (LAK) cells, B lymphocytes and macrophages1-5. Grimm et al reported that human peripheral lym­phocytes cultured for four days with 1,000 units of interleukin-2 become able to lyse fresh tumor cells with very high efficiency6. in experimental murine system, the adoptive transfer of the lymphokine activated killer cells along-with repeated high doses of IL-2 induces a dramatic reduction in established tumor metastasis7. Clinical trials have shown that impressive tumor regres­sion can be obtained with this protocol in about 20%-of patients with advanced metastatic renal cell carcinoma and melanoma8,9. There are several types of lymphoids cells capable of mediating target cell lysis. These include classic cytotoxic T cells (CTL), natural killer (NK) cells, lym­phokine-activated killer (LAK) cells and the antibody-de­pendent cell-mediated cytotoxicity (ADCC) effector population. Different types of effect or cells can be differentiated from one another because they carry cell surface proteins that act as identifying markers. Extensive studies have now been done on the phenotype of both the lymphocytes which develop LAX activity after culture with IL-2 (i.e., progenitor of cells with LAK activity) and the effect or cells themselves. Although the initial studies on LAK activity suggested a shift in phenotype, from progenitor lacking T-cell as well as NK-cell markers to effect or cells with T-cell marker, most of the subsequent studies have indicated that the progenitors and effect or cells express a very similar pattern of markers10-12. It is now clear that most of the LAX activity from blood lymphocytes is generated from cells with the same characteristic as NK cells. NK cells have been closely associated with large granular lymphocytes (LGL) and the CD16 antigen has been found on almost all NK active cells13. The progenitor of LAX activity in human peripheral blood are mainly LGL with the CD3, CD16+, NKHY phenotype in the present study we have reported the anti-tumor effects of control lymphocytes (without IL-2) and IL-2 activated lymphocytes on two different cell lines: SW742 colon adeno carcinoma cell line and K562 myeloid leukaemia. We also investigated the susceptibility to lysis of LAX cells of cytokine treated (TNF alpha, IL-1alpha and INF alpha), K562 and SW742 cell lines. This was based on the hypothesis that the differences between the cell lines (in their susceptibility to lysis) could result from the expression or accessibility of some membrane structures (by cytokines treatment of a cell line), which play an important role in the formation of conjugates between targets and CD3 effect or lymphocytes followed by lysis.

MATERIAL AND METHODS

K562 cell line
­K562 myeloid leukaemia cell line was a generous gift from Imperial Cancer Research Fund Laboratories, London and maintained as anon-adherent cell line in RPM I-I640 plus 10% new born calf serum.
SW742 cell line
The SW742 colon adenocarcinoma cell line was a generous gift from Dr. Russel Greig, Smith Mine and Beecham\\\'s Laboratories and maintained as adherent cell line in RPMI-1640 medium plus 10% foetal calf serum.
Preparation of peripheral blood lymphocytes
Peripheral blood was collected from normal heal­thy volunteer donors by venepuncture into sterile glass bottles containing the anticoagulant heparin (iO u/ml blood). The blood was diluted 1:1 with sterile PBS and carefully layered over LSM (lymphocytes separation medium) in a plastic universal at a ratio of 1:2, LSM:diluted blood with asterile plastic 10 ml pipette. The density gradients were centrifuged at 400g for 35 mins. at room temperature. The rich buffy coat interface, be­tween the LSM and plasma, was harvested carefully with a sterile glass pasteur pipette. The cells were washed once with PBS-FCS at 4°C550g for 15 mins. and twice at 4°C, 400g for 6 mins. The lymphocytes were resuspended in RPMI and counted using the trypan blue exclusion method.
Generation of cytotoxic effector cells
Lymphokine-activated killer (LAX) cells. PBMCs (peripheral blood mononuclear cells) obtained from peripheral blood of healthy donors were resuspended at the concentration of 4X106/ml in RPMI containing 10% AB serum and IL-2 (500 u/mI). Following a 4 day incubation in a 24 well tissue culture plate, at 37°C in a humidified CO2 atmosphere, cells were harvested, washed twice in PBS, resuspended at the concentration of 3X106/ml and used as LAK effectors in the cytotoxicity assay.
4hours51 Cr-release test (cytotoxicity assay)
Target cells (K562, SW742) were grown and treated with cytokines (tumor necrosing factor 1000U/ml, inter­leukin-1 1000lJ/ml, interferon alpha 500U/ml) for 24 hrs. Target cells (in a 0.2 ml volume) were labelled for 1 h at 37°C with 60 mCI of51Cr as sodium chromate (Na251Cr04), washed twice with RPMI containing 10% NBCS, resuspended in 10 ml of medium and incubated for a further 1 h at 3 7°C. Target cells were washed twice again and resuspended at the concentration of 1X105/ml. Cytotoxicity tests were performed in triplicate in round bottomed 96 well flexible assay plates. Effector cells (0.1 mI/well) were incubated with target cells (0.1 mI/well) at ratios of 30 to 1, 15 to 1 and 7.5 to 1 and the plates incubated at 37°C for 4 h in humidified 5% CO2 atmosphere. Supernatant (0.1 ml) was harvested from each well and counted in a gamma spectrophotometer, Canberra Packard Ltd. The percent 51Cr release was determined for each group following subtraction of the spontaneous release and the percentage cytotoxicity calculated by the formula:
(test release) — (spontaneous release) % cytotoxicity =   x 100
100 — (spontaneous release)

RESULTS

Effect of cytokines on K562 susceptibility to lysis by human lymphocytes
Human lymphocytes and K562 cell line (with IL-1a, TNFa and IFNa and without cytokines) were prepared and used as effectors and targets in a 4 h 31Cr-rel ease assay at a maximum effector to target ratio of 30:1.

As shown in Figure 1 there was no significant difference in lysis of cytokine treated and untreated K562 cells by lym­phocytes, lysis was less than 10% in all cases.
Effect of cytokines on K562 susceptibility to lysis by IL-2 activated lymphocytes (LAK cells)
The LAIC cells were produced by IL..2 treatment of PBMC and were used as effectors in a 4 h 51Cr-release assay at a maximum effector to target ratio of 30:1 as before. There was about seven fold increase in lysis of K562 cells by IL-2 activated lymphocytes as compared to lysis by control lymphocytes (Figure 2, Table 1).


As shown in Figure 2, TNF a and IL-la treated K562 cells did not show -any considerable change in lysis by LAK cells, although some decrease was noticed in IFNa. Enhanced expression of MHC class I antigens with TNFa and IFNa did not correlate with a decrease in K562 susceptibility to lysis by LAK cells.
Effect of cytokines on SW742 cell line susceptibility to lysis by human lymphocytes
Cytokine treated SW742 cell line (TNF a 1000 u/mi and IFN a 500 u/ml for 24 hours) and without cytokines was used as target for lymphocytes in this experiment. A 4 h “Cr-release assay was set up as described before.

As it is shown in Figure 3 that there was no significant killing of SW742 cell line by lymphocytes nor there was any effect on killing by cytokine treated SW742 cells. Lysis was less than 5% in all cases.
Effect of cytokines on SW742 susceptibility to lysis by IL-2 activated lymphocytes (LAX cells)
The LAK cells were used as effectors in a 4 h 51Cr release assay at a maximum effector to target ratio of 30:1 as before. There was extensive killing of SW742 cells by IL-2 activated lymphocytes (compare Figure 4 with Figure 3, also see Table II).

As it is shown in Figure 4.

TNF alpha did not appear to alter susceptibility to lysis by LAK cells. JFN alpha, however, considerably decreased SW742 susceptibility. Although both TNF alpha and IFN alpha increased MCH class I expression, decrease in cytolytic susceptibility was only noticed with IFN a treatment of the target cells. This may further indicate that LAK cells have different specificities compared to NK or IFN alpha activated NK cells.

DISCUSSION

For the past two decades attempts have been made to develop immune therapies for cancer treatments on the basis of stimulating the host immune response to the tumor. These approaches have based on attempting to immunize against specific tumor cells or to immunize with non-specific stimulants (like BCG) in the hope that general immune stimulation would concomitantly in­crease the host anti-tumor response. The inability to stimulate sufficiently strong response to putative tumor antigens and the general immune incompetence of the tumor bearing host were the factors that argued against the success of this approach. In fact these clinical attempts were unsuccessful and have largely been abandoned15. An alternative therapeutic approach to the im­munologic treatment of cancer is that of adoptive transfer of immune cells7. Adoptive immunotherapy is defined as the transfer of active immunologic agents, such as cells with anti-tumor reactivity that can mediate, either direct­ly or indirectly anti-tumor effects in the tumor-bearing host. The major obstacle to the development of success­ful adoptive immune therapies has been the availability of appropriate cells for use in adoptive transfer. The cells of immune system are best to be used as an important tool for their possible advantages in the adoptive im­munotherapy. It is not technically precise to use the term ‘LAK cell”, since a variety of cells can mediate to LAK activity although CD3 lymphocytes constitute the major population of IL-2- induced cytotoxic effectors16. Flow cytometric analysis has shown that the expression of both ICAM-1 and CD2 increases on IL-2 activated LAX cells, resulting in increased binding of these cells to tumor cells such as lymphomas17. These results are in agreement with the report by Wiekbe et al16. who showed that LAK susceptibility is usually unaltered after K562 TNF treat­ment. These resultS may emphasise the hypothesis that LAX cells are “ultra activated” entities with different specificities. The nature of the molecules involved in recognition of targets by LAX cells is not known, although the existence of two new cell surface molecules have been reported18. The possibility exists that the CD3 cytotoxic effectors recognise their targets through non-specific adhesion mechanisms possibly LFA-1/ICAM-1 and/or CD2/LFA-3. Since the natural or IL-2 activated CD3 cytotoxic cells are armed lymphocytes which contain granules or cytotoxic proteins, the stable adhesion of effector cells to the targets might be the triggering signal inducing the destruction of the targets19. Lymphotoxicity is mediated by various subsets of effector lymphocytes. One type of cytotoxicity, for instance, is mediated by well characterised immune killer cells which bind to their target via an antigen-specific heterodimeric T-cell receptor and recognise an antigen associated with histocompatibility molecules. Another type of cellular cytotoxicity is mediated by the CD2+ CD3 lymphocyte subset which does not express any known specific recognition structure and represents the majority of natural killer (NK) lym phocytes and lym­phokine activated killer cells (LAX)”. Most of the cells of this subset express surface markers such as CD16 and CD56. However, NK/LAK cytotoxicity is often designated as non-immune and non-MHC-restricted cytotoxicity. A population of CTL (CD3 ~) is also involved in non- MHC restricted killing, termed non-MIIC restricted cytotoxic T cells. The CDY lymphocytes appear to be extremely important for neoplastic surveillance, since LAK effectors can efficiently destroy certain tumour cells in vitro and in vivo. Cytotoxic effector cells mediate target cell lysis through a series of stages: (1) target cell recognition, (2) cell adhesion, (3) effector cell triggering, (4) effector cell activation, (5) secretion of cytotoxic factors, (6) factor binding to the target cell, (7) target cell changes, (8) cell death and (9) effector cell recycling. T-lymphocytes recognise peptide sequences associated with MHC class I antigen via their CD3/TCR complex, whereas other surface molecules (yet unknown) are associated with target recognition by NK cells4. Adhesion of lymphocytes to a target cell occurs through a series of adhesion proteins present on the effector cell surface. Two major pathways of adhesion have been identified in T-lym­phocyte adhesion to target cells. Firstly, LFA- 1 on CTh interacts with ICAM-1 on the target cell20. Secondly, the effector cell CD2 molecule interacts with LFA-3 on the target cell21. CD56 molecule (NKH-l/Leu 19 antigen2 appears to be involved in target cell binding on NK cells. Following the binding of effector cells to targets, trans­membrane signalling activates certain intracellular processes, leading to the secretion of granules from cytotoxic T-cells or NK-cells which are responsible for delivering the ‘lethal hit’ to the target. Although the process of killing by NK-cells and cytotoxic T- lym­phocytes is in many respects similar, granules only appear in CTL upon activation, whereas lytic LGL (NK cells) already contain these granules4. It has been shown that treatment of target cells with IFN results in target cell resistance to cytotoxic attack (Figure 4), due to the failure of the effector granules to become reoriented after target cell binding and hence preventing the triggering of already bound LAX cells. The data presented in this study shows that the inhibitory effect of TNFa on K562 and SW742 suscep­tibility to lysis by LAK cells is negligible as compared to the effect of IFNA. Therefore, it may be possible to speculate that although LAX activities are similar in their lytic properties (e.g., target selection), their mechanism of recognition and lytie attack may be divergent. Further­more, cytokine treatment of targets may greatly alter the recognition structures on targets. Preliminary investiga­tion of recognition events by Ortaldo and Longo indi­cates that IL-2 activated effector cells do not recognise the same structure that is recognised by freshly isolated NK cells22. Extensive amount of research is being done to understand the exact mechanisms of immunotherapy of cancer by using immune cells, because the normally functioning immune system protects against disease, it is intriguing to think’ that therapeutic manipulation of the immune response can combat malignancy. Our under­standing of the immune system has expanded dramatical­ly during the past decade, revealing an immensely complicated network of effector cell-target cell interac­tions. We are still far from precise understanding of the relationships between tumor cells and immune effector cells, perhaps exogenously administered agents, such as lymphokines and tumor-specific antibodies, can lead to effective anti-tumor response from a patient’s own immune effector cells. On the other hand, successful immunotherapy may involve this present approach, with adoptive transfer of in vitro-activated immune competent cells. En this model the task for the researchers is to define relevant T-cell subpopulations that will participate in tumor regression in vivo. Optimal immunotherapy will require significant understanding of tumor cell biology, as well as the long list of lymphokine signals capable of modulating NK cells, LAX cells and CTL function. The problem of homing of effector populations to tumor sites may also be addressed through knowledge of the role of adhesion molecules. It is important to note that none of the immunotherapeutic trails to date addresses the issue of immune system maintenance after therapy. Immune system management of survivors of treatment will be an increasingly important clinical research problem. Until the immunologic deficits characteristics of patients with cancer are reversed, it is expected that complete responders will represent the minority of clinical trials. As our knowledge of the immune system increases, so must clinical research expand to explore new possibilities for therapy. It is anticipated that through the use of new approaches to immunotherapy, including autologous transfer of anti-tumor effector cells, IL-2 therapy, monoclonal antibodies, retrovirus­mediated gene transfer and chemotherapeutic agents, future patients with cancer will realise significant in­creases in survival.

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