Scand. J. Immunol. 36, Suppl. 11, 185-186, 1992

Generation of Lymphokine-Activated Killer Cells by Adherent LGL Phenotype Cells and Non-Adherent T Lymphocytes R. OUESLATI, M. MAALEJ* & M. CHOUIKHA Laboratoire d'Immunologie, Hopital Militaire de Tunis, and *Institut Salah Azaiez, Tunis, Tunisia

Oueslati R, Maalej M, Chouikha M. Generation of Lymphokine-Activated Killer Cells by Adherent LGL Phenotype Cells and Non-Adherent T Lymphocytes. Scand J Immunol 1992;36(Suppl.ll):l85-6 In this work we separated human blood lymphocytes (PBL) in two populations (A-LAK and NALAK cells) by the adherence plastic method. A maximum adherence of cells was obtained after 2 days of PBL incubation in LAK medium containing 500 U/ml rIL-2. The A-LAK cells had LGL phenotype but 40% of them had a macrophage phenotype marker and less than 20% weakly expressed a T-cell marker. This population, when reincubated in culture, produced an increasing titre of interferon. At the same time, a significant NK activity against K562 target cells was measured just after enrichment; these enriched adherent cells also developed an increased LAK activity against DAUDI cell lines, ninefold more at 6 days than when assayed just after enrichment. In contrast, 75% of the NA-LAK enriched cells expressed T-cell marker; these produced two- to threefold less interferon than A-LAK cells at all time-points. The NA-LAK lymphocytes enhanced principally LAK activity measured by 70% lysis against DAUDI target cells tested at 6 days of culture. Further studies are in progress to determine the nature of the effector cells that mediated LAK activity. R. Oueslati, Laboratoire d'Immunologie, Hopitai Mititaire de Tunis, 1008 Montfleury-Tunis, Tunisia

Recent advances in adoptive immunotherapy have used cytotoxic lymphocytes with broad antitumour cytotoxicity in combination with high dose recombinant interleukin 2 (rIL-2) [1, 2]. The efficiency of this therapy in experimental systems as well as optimum modulation of a number of immune parameters in patients has been limited because the number of cultured lymphocytes and level of rIL-2 administered have often been toxic [3]. These limitations have compromised the general use of this form of immunotherapy in a clinical setting. In order to optimize this therapy it is important to identify the subsets of lymphocytes that mediate this activity. In the rat system, Vujanovic showed that large granular lymphocytes (LGL), enriched by an adherence technique method, mediated NK and LAK activity [4]. In this study, exploiting the same method for isolation of LGL cells, we demonstrate in a human system that adherent LAK (A-LAK) cells largely of LGL phenotype generate NK and LAK activity. At the same time, non-adherent LAK (NA-

LAK) cells, dominated by phenotypic T cells, also developed LAK activity derived from heterogeneous precursors cells.

MATERIALS AND

METHODS

To generate LGL cells, mononuclear cell suspensions were passed over a nylon wool column to remove B cells and macrophages. The depleted cells (PBL) were adjusted to a concentration of 2 x lO^ml in the culture medium containing 500 U/ml of rlL-2 (LAK medium), and 18-20 X 10* cells were cultured in a plastic flask (T25, Falcon Flask) positioned on its flat side for various culture periods (1 to 4 days). After this the NALAK cells were decanted and the A-LAK washed with PBS and removed by addition of 0.005/0.02% Trypsin/ EDTA in PBS solution for 2 min at 37°C. The NA-LAK and A-LAK cells collected were counted by trypan blue exclusion and resuspended at 2 x 10*/ml in fresh LAK medium. These cells were also reincubated for various times. The surface phenotype of cells was identified by monoclonal antibodies in conjunction with an indirect 185

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immunofluorescence test, and the cytotoxic function of NK or LAK cells was examined by a short-term specific "Cr-release assay (4 h) [5].

immtjne-specific T cells capable of mediating destruction of tumour trough specific recognition of tumour-associated antigen.

RESULTS A N D DISCUSSION Our results showed that the maximal A-LAK cells were generated in our system after 2 days of culture. These cells expressed largely the LGL phenotype marker (60% were Leu7^ and Leullb+). The A-LAK cells also expressed Leu 15 marker macrophage phenotype (40%). These cells were characterized by their production of interferon-y; this cytokine probably limited the proliferation of the A-LAK cells by inhibiting many stimulatory factors [6]. In contrast, 75% ofthe NA-LAK cells expressed Leu4+ T phenotype marker with less than 5% expressing macrophage marker; the LGL phenotype marker was expressed in less than 30% of these cells. Our results demonstrate that the adherence technique is not only a method for enrichment of LGL phenotype cells but is also suitable for achieving enrichment of non-major histocompatibility complex-restricted effector cells [4]. On the other hand, measurements of the cytotoxic activity, as defined by the ability of A-LAK or NALAK cells to lyse the NK-insensitive tumour DAUDI line and NK-sensitive tumour K562 line, showed that just after enrichment the NK cell activity of A-LAK cells was more significant than that in unseparated (PBL) and NA-LAK cells. The kinetics of cytotoxic cells in long-term culture showed a significant increase in LAK activity in all subset cells. Our results are partially in discord with some other studies, which indicate that LAK precursors in the PBL were the mature NK cells [7, 8]. They are, however, in agreement with other reports which have clearly demonstrated heterogeneous cell precursors including NK, T and even B cells mediated LAK activity [6, 9, 10]. In contrast to the A-LAK cells effectors, the NK and LAK activity were mediated by heterogeneous NA-LAK cell effectors. This heterogeneity is important because NA-LAK cells, dominated by T cells, may contain the

REFERENCES 1 Mule JJ, Shu S, Schwartz SL, Rosemberg SA. Successful adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant IL-2. Science 1984;225:I487. 2 Salup RR, Wiltrout RH. Adjuvant immunotherapy of established murine renal cancer by InterIeukin-2 stimulated cytotoxic lymphocytes. Cancer Res 1986;46:3358. 3 Boldt DH, Mills BJ, Gemlo BT et al. Laboratory correlates of adoptive immunotherapy with recombinant interleukin-2 and lymphokine-activated killer cells in humans. Cancer Res I988;48:4409. 4 Vujanovic NL, Herberman RB, Olszowy MW, Cramer DV, Salup RR, Reynolds CW, Hiserodt JC. Lymphokine-activated killer cells in rats: analysis of progenitor and effector cell phenotype and relationship to natural killer cells. Cancer Res 1988;48:884. 5 Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH. The relationship of CDI6 (Leull) and Leul9 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol 1986; 136:4480. 6 Hao LI, Kniep E, Emmendorffer A, Lohmann Mathes ML. Differentiation of macrophage precursors to cells with LAK activity under the influence of CSF-1 and high dose IL-2. Scand J Immunol I991;33:511. 7 Grim E, Ramsey A, Mazumder A, Wilson DJ, Djeu JY, Rosenberg SA. Lymphokine-activated killer cell phenomenon II. Precursor phenotype is serologically distinct from peripheral T lymphocytes, memory cytotoxic thymus-derived lymphocytes, and natural killer cells. J Exp Med 1983;157:884. 8 Ramsdell FJ, Lindemann RA, Shau AH, Gray JD, Golub H. Generation of lymphokine-activated killer cell activity from non-HK precursor cells. Cell Immunol 1982;116:287. 9 Ochoa AC, Hasz DE, Rezonzew R, Anderson PM, Bach FH. Lymphokine-activated killer activity in long-term cultures with anti-CD3 plus interleukin2: identification and isolation of effector subsets. Cancer Res I989;49:963. 10 Tilden FB, Itoh K, Balch CM. Human lymphokineactivated killer (LAK) cells: Identification of two types of effector cells. J Immunol 1987; 138:1068.

Generation of lymphokine-activated killer cells by adherent LGL phenotype cells and non-adherent T lymphocytes.

In this work we separated human blood lymphocytes (PBL) in two populations (A-LAK and NA-LAK cells) by the adherence plastic method. A maximum adheren...
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