Generation of lymphokine-activated killer cell activity from human thymocytes

We have generated lymphokine-activated killer (LAK) cells from human thymocytes in order to assess the relationship between LAK cells and T cells. Fresh thymocytes lack natural cytotoxic activity, and cytotoxicity cannot be stimulated by short term (1 hr) incubation with interferon or recombinant interleukin 2 (rIL-2). In addition, thymocytes are phenotypically devoid of cells bearing the natural killer (NK)-associated markers cluster designation (CD) 16 and NKH-1. After culture for 5 to 8 days with rIL-2, thymocytes display high levels of cytotoxic activity against both NK-sensitive and NK-resistant targets. Thymocytes require slightly more IL-2 than do peripheral blood lymphocytes to generate LAK activity. We have examined the phenotype of the thymocyte LAK precursor and effector cells. Thymocyte LAK precursors are of low to medium density, CD1-negative, and predominantly CD3-negative. Although CD3-positive cells proliferate in response to rIL-2, they are low in cytolytic capabilities. The effector cells, like the LAK precursors, are low to medium density lymphocytes. The cytotoxic cells are predominantly CD3-negative, and cytotoxic activity cannot be blocked with the use of anti-CD3 monoclonal antibodies. The effector cells also lack most NK-associated markers (HNK-1, and the CD16 markers Leu-11b and B73.1) but possess the NK-associated marker NKH-1 (N901). The responsive cell appears to be at a very early stage of thymic development, and it does not appear to either require or express the CD3-T cell receptor complex.     

Generation of lymphokine-activated killer cell activity from non-NK precursor cells

It is possible to generate high levels of lymphokine-activated killer (LAK) activity in short-term culture from cells enriched for natural killer (NK) activity. To determine whether LAK activity can also be generated from non-NK cells, we have depleted peripheral blood lymphocytes (PBL) of NK cells prior to culture with IL-2. NK activity in PBL is correlated with the intensity of staining with the lysosomotropic vital dye quinacrine. Quinacrine dim PBL, which are devoid of lytic NK cells, are capable of developing LAK activity following culture with IL-2. We have also separated PBL using the NK-associated NKH-1 marker. Depleting NKH-1+ cells eliminates NK activity but the ability to develop LAK activity is retained. NKH-1-depleted cells generate less LAK activity than unseparated or NKH-1-positive cells and do not proliferate as well as unseparated cells to IL-2. When NK-depleted cells are subsequently examined for the expression of the NKH-1 antigen, this marker is absent from most cells at Day 3 of IL-1 culture, but is expressed on an increasing number of cells by Days 6-8. These results suggest that LAK derived from non-NK cells is functionally and phenotypically similar to LAK from PBL-containing NK cells, and may be the result of the activation of an NK precursor population.     

Characterization of interleukin 2-expanded human peripheral blood lymphocytes: not only NKH-1+-NK cells but also NKH-1+ and NKH-1- T cells are LAK effectors

In vitro culture of human peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the expansion of lymphocytes including lymphokine-activated killer (LAK) cells. Using flow cytometry, studies were undertaken to determine the phenotype and LAK activity of each subset of lymphocytes expanded in vitro as a result of incubation for 2 weeks with 2500 U/ml of recombinant IL-2. Such expanded PBMC, when examined by two-color staining with various combinations of anti-CD3, 4, 8, 16, and NKH-1 monoclonal antibodies, consisted of the following six subgroups of cells: (1) CD3+4+8-, (2) CD3+4-8+, (3) CD3+4-8-, (4) CD3-16+NKH-1+, (5) CD3-16-NKH-1+, and (6) CD3-16-NKH-1-. Of the six subgroups, all five subgroups that could be tested, i.e., CD3+ T cells (CD3+4+8-, CD3+4-8+, CD3+4-8-), CD16+ natural killer (NK) cells (CD3-16+NKH-1+), and CD3-16-NKH-1- non-T non-NK cells, possessed LAK activity. Both NKH-1- as well as NKH-1+ T and non-T cells possessed LAK activity.     

IL-4 inhibits IL-2-mediated induction of human lymphokine-activated killer cells, but not the generation of antigen-specific cytotoxic T lymphocytes in mixed leukocyte cultures

Human rIL-4 was studied for its capacity to induce lymphokine-activated killer (LAK) cell activity. In contrast to IL-2, IL-4 was not able to induce LAK cell activity in cell cultures derived from peripheral blood. IL-4 added simultaneously with IL-2 to such cultures suppressed IL-2-induced LAK cell activity measured against Daudi and the melanoma cell line MEWO in a dose-dependent way. IL-4 also inhibited the induction of LAK cell activity in CD2+, CD3-, CD4-, CD8- cells, suggesting that IL-4 acts directly on LAK precursor cells. IL-4 added 24 h after the addition of IL-2 failed to inhibit the generation of LAK cell activity. Cytotoxic activity of various types of NK cell clones was not affected after incubation in IL-4 for 3 days, indicating that IL-4 does not affect the activity of already committed killer cells. No significant differences were observed in the percentages of Tac+, NKH-1+ and CD16+ cells after culturing PBL in IL-2, IL-4 or combinations of IL-2 and IL-4 for 3 days. IL-4 also inhibited the activation of non-specific cytotoxic activity in MLC, as measured against K-562 and MEWO cells. In contrast, the Ag-specific CTL activity against the stimulator cells was augmented by IL-4. Collectively, these data indicate that IL-4 prevents the activation of LAK cell precursors by IL-2, but does not inhibit the generation of Ag-specific CTL.     

Evaluation of basic procedures for adoptive immunotherapy for gastric cancer

Peripheral blood lymphocytes (PBL) of gastric cancer patients in advanced stages showed lymphokine activated killer (LAK) activities comparable to those of healthy donors, suggesting potential applicability of LAK cells induced from PBL stimulated with recombinant interleukin-2 (rIL-2) in adoptive immunotherapy (AIT) for gastric cancer. In order to generate a large number of LAK cells from PBL, lymphocytes were cultured with both rIL-2 and phytohemagglutinin (PHA). In this culture, the numbers of cells increased to a greater extent than those in culture with rIL-2 alone but cytotoxic activity did not augment, thus suggesting that this procedure would not afford sufficient clinical effects. On the other hand, a large number of LAK cells with high anti-tumor activities were efficiently induced from spleen cells of the patients by culture of rIL-2; hence clinical usefulness of these LAK cells is anticipated. In regional lymph node lymphocytes (RLNL) cultured with rIL-2, the cytotoxic activities were lower than in those induced in PBL, and a characteristic increase of CD8 + CD11 + suppressor T cells was observed after incubation with rIL-2. Nevertheless, an increase of CD4 + 4B4⁺ helper inducer T cells was also observed in RLNL after the culture with rIL-2. Furthermore, high cytotoxic activities were induced in RLNL in some cases in which metastasis to the regional lymph nodes was not detected. When gastric cancer patients were pretreated with biological response modifiers (BRM), especially with Lentinan, LAK cells from PBL showed higher NK and LAK activities as compared with those of patients without BRM pretreatment.This work was partly supported by a grant from Hokkoku Cancer Research Foundation.     

Induction of natural killer effectors from human thymus with recombinant IL-2

The NKH1 Ag is expressed on all cells in human peripheral blood capable of mediating spontaneous non-MHC restricted cytolytic function (i.e., natural killing). The majority of NK cells do not express CD3 Ag and do not express TCR gene products. However, approximately 20 to 25% of NKH1+ cells coexpress CD3 and TCR proteins. Both NKH1+CD3+ and NKH1+CD3- effectors can proliferate in response to IL-2 which also results in enhancement of cytolytic function. In the present studies, we examined thymocytes after incubation with rIL-2 for the presence of NKH1+ cells and for the development of non-MHC restricted cytolytic function. NKH1+ cells and NK activity could not be detected in fresh thymus. After culture with rIL-2 only, NK activity appeared in 3 days, reached a maximum after 7 days, and was effective against a panel of NK-sensitive targets. NK activity was correlated with the expression of NKH1 on the surface of in vitro proliferating thymocytes and immunofluorescent cell sorting demonstrated that almost all cytolytic activity was mediated by NKH1+ cells. As expected given the thymic origin of these cells, the majority of NKH1+ cells in culture expressed CD3. However, all cultures contained NKH1+CD3- effector cells which represent 15 to 40% of the NKH1+ population. As in peripheral blood, both NKH1+CD3- and NKH1+CD3+ exhibited non-MHC-restricted cytotoxicity, but only CD3+ effectors could be inhibited by anti-T3 mAb. These findings demonstrate that rIL-2 alone can induce subpopulations of thymocytes to proliferate, to express the NKH1 marker and become NK active in vitro. Furthermore, they suggest that the thymus which plays a role in the differentiation of NKH1+CD3+ NK effectors may also play a role in the differentiation or maturation of NKH1+CD3- NK effectors.     

Resting human peripheral blood lymphocytes can be activated to cytolytic function by antibodies to CD3 in the absence of exogenous interleukin-2

We investigated the ability of anti-CD3 antibodies to activate resting human peripheral blood lymphocytes (PBL) to a cytolytic function. We found that two anti-CD3 antibodies, but not an anti-CD4, anti-CD8, or anti-CD2 antibody, could activate resting unseparated PBL to become killer cells in the absence of exogenous interleukin-2 (IL-2), although exogenous recombinant IL-2 (rIL-2) synergized with anti-CD3. We also found that these anti-CD3 antibodies were active in the absence of rIL-2 only when linked to a solid surface such as a Sepharose bead or a plastic tissue culture plate. Cytolytic activity was measured in several ways: (i) by the ability of activated PBL to lyse the NK-sensitive line K562, and (ii) by the ability of these cells to lyse a CD10+ (CALLA+), NK-resistant target in the presence of either concanavalin A (lectin-dependent lysis) or an anti-CD10-anti-CD3 heterodimer. At least two different types of cytolytic cells were activated by anti-CD3 antibodies, an NK-like cell, which was CD2+CD3-CD4-CD8-CD16+-NKH1a+, and a CTL-like cell, which was CD2+CD3+CD4-CD8+CD16-NKH1a-. The former cell lysed the K562 line and the latter cell lysed Namalwa in the presence of the anti-CD10-anti-CD3 heterodimer or concanavalin A. The NK-like cell was probably activated by endogenous IL-2 produced by the anti-CD3-activated CD3+ cells and both the NK and CTL-like cells required the presence of adherent cells for maximal activity. The dose response and the kinetics of anti-CD3 activation of PBL to cytolytic activity were also studied. The use of the anti-CD3-activated cytolytic cells as effectors in anti-CD3 heterodimer-mediated lysis of tumor cells may be a novel approach to the therapy of cancer, and a comparison with the well-studied rIL-2/lymphokine-activated killer (LAK) system is discussed.     

Heterogeneous lymphokine-activated killer cell precursor populations

Summary We developed a monoclonal antibody (mAb) 211, which recognizes the precursors in peripheral blood of lymphokine-activated killer cells (LAK) induced by recombinant interleukin-2 (rIL-2). In conjunction with complement mAb 211 also eliminates natural killer cells (NK) and a majority of the cytotoxic T lymphocytes. B cells and monocytes do not express the 211 antigen. Since mAb 211 recognized such a large percentage of peripheral blood lymphocytes we examined which 211⁺ subpopulation was the predominant precursor of rIL-2-induced LAK cells using two-color fluoresence-activated cell sorting (fluorescein-conjugated 211 mAb plus phycoerythrin-CD11b). This method identified the 211⁺/ CD11b⁺ population as the predominant phenotype of the rIL-2-induced LAK precursor. In addition, we directly compared the phenotype of the LAK precursor induced by delectinated T-cell growth factor (TCGF) to that induced by rIL-2. The 211-depleted population, which was devoid of NK cells and LAK precursors (inducible by rIL-2), was capable of generating LAK activity when TCGF was used as the source of lymphokine. LAK cells induced by TCGF from the 211-depleted population lysed a fresh sarcoma and an NK-resistant cultured melanoma tumor target but not the Daudi cell line, which was lysed by rIL-2-induced LAK cells. Lymphoid subpopulations, depleted using NKH1a mAb, behaved similarly, generating high levels of lysis against the two solid tumor targets when cultured with TCGF but not with rIL-2. CD 3-depleted populations showed enrichment for LAK precursors using either rIL-2 or TCGF. These results indicate that while rIL-2-induced LAK precursors cannot be separated from cells with NK activity, TCGF-induced LAK cells can be generated from populations of peripheral blood mononuclear cells without NK activity.     

In vivo effects of recombinant IL-2. I. Isolation of circulating Leu-19+ lymphokine-activated killer effector cells from cancer patients receiving recombinant IL-2

This study was designed to isolate and phenotypically characterize lymphokine-activated killer (LAK) cells generated in vivo during administration of high dose rIL-2 to cancer patients. The development of circulating LAK effector cells in these patients was demonstrated by the ability of fresh PBL to exhibit lytic activity against the NK-resistant Daudi cell line and fresh tumor cells without prior in vitro culture with rIL-2. Kinetic studies demonstrated that circulating LAK effector cells are detectable 4 to 6 wk after the initiation of rIL-2 therapy. Cells isolated by FACS revealed that circulating LAK cells are Leu-19+, Leu-17+ but CD5-. We have previously reported that circulating Leu-19+ cells are heterogeneous with regard to the expression of CD16 and CD8. Since sorting of cells expressing Leu-19 and either low quantities of CD8 or CD16 resulted in cytolytic activity in both the positive and negative fractions, these latter two markers do not identify subpopulations of Leu-19+ cells with or without LAK cytolytic activity. Although all LAK cells generated in vivo were Leu-19+, we generated LAK cells from the Leu-19- subpopulation after in vitro culture with rIL-2, suggesting that at least some of in vitro generated LAK cells are derived from Leu-19- precursor cells. These LAK cells did not, however, express the Leu-19 surface marker. Based on the functional data reported in this paper, we conclude that circulating LAK effector cells are a phenotypically heterogeneous population that express surface Ag in association with NK cells and not T lymphocytes.     

Suppression of lymphokine-activated killer induction by neutrophils

Peripheral blood polymorphonuclear neutrophils (PMN) suppressed the induction of PBL lymphokine-activated killer (LAK) function by rIL-2 in vitro. The suppression depended on the concentration of PMN in the IL-2 culture, and required intact PMN. However, PMN did not require treatment with immunoregulators such as IL-2, LPS, or TNF to express the suppressive activity, and no direct contact with PBL was needed for the suppression. Addition of anti-TNF antibodies had no effect on the suppression, suggesting that no endogenous TNF in the culture was involved in the suppression. PMN did not inhibit LAK function by preventing utilization of IL-2 by PBL or by selective depletion of NKH-1+ cells which constitute the majority of LAK precursors in PBL. The suppression was reversed by superoxide dismutase but not by catalase, suggesting that superoxide anion, not hydrogen peroxide, was involved in the suppression. No other suppressive factor was detectable in PMN culture supernates. Our results of PMN regulating LAK induction in vitro suggest that PMN may have a role in determining the outcome of immunotherapy with IL-2 in vivo.     

Characteristics of interleukin-6-enhanced lymphokine-activated killer cell function

To study the effect of IL-6 on the development of cytotoxic cells, we examined lymphokine-activated killer (LAK) activity generated from human nonadherent PBL. Addition of rIL-6 at the initiation of 5-day PBL cultures significantly increases LAK activity in the presence of low concentrations (between 5 and 25 u/ml) of rIL-2. RIL-6 alone induces no PBL LAK activity but at doses as low as 0.8 u/ml rIL-6 enhances LAK activity with optimal enhancement of LAK at 5.0 u/ml of rIL-6. This enhancement is independent of effects on cells growth as rIL-6 did not affect the cell recovery of PBL cultured in rIL-2. RIL-6-enhanced LAK is mediated by the same type of effector cells as those of LAK from rIL-2 alone with effector cells primarily generated from large granular CD3-negative E rosetting lymphocytes. RIL-6 does not change the time course of LAK development and pretreatment of PBL with rIL-6 has no effect on the PBL response to subsequent rIL-2 induction of LAK. Addition of rIL-6 to LAK cultures 2 hr before the cytotoxicity assay shows equal enhancement as addition at the initiation of the culture. However, rIL-6 requires the presence of both rIL-2 and another factor in the supernatant from LAK cultures in order to enhance LAK. Our results indicate that IL-6 can modulate LAK activity at a very late stage of LAK development, and that the enhancement by IL-6 is dependent on the presence of IL-2 and another soluble factor generated during rIL-2 culture.     

Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: I. phenotypic and functional analysis of the peripheral blood mononuclear cells

Summary The efficacy of recombinant interleukin-2 (rIL-2) or rIL-2 plus lymphokine-activated killer (LAK) cells in cancer therapy has been demonstrated by several groups both in experimental models in animals and clinical trials in humans, but their effects in vivo have yet to be clarified.Starting February 1988, we have treated 12 patients affected by advanced renal cancer with rIL-2 + LAK cells according to an open, non-randomized, phase II trial. Immediately before each rIL-2 infusion and during the last day of infusion, immunological tests were performed on the patients' peripheral blood mononuclear cells. During rIL-2 infusion we have observed a slight increase of the spontaneous cell proliferation and of natural killer (NK) and LAK activity; phenotypic analysis showed a significant decrease in the CD4⁺ T-lymphocyte subset, both in percentage and in absolute number. Conversely, before each cycle CD4⁺ cells increased when compared to basal values. No significant variations were observed in the CD8⁺ T-lymphocyte subset. Furthermore, a significant increase of the NK cells (CD3^– CD56⁺ CD16⁺) was evident during rIL-2 infusion.     

Generation of lymphokine-activated killer activity in T cells. Possible regulatory circuits.

CD4+ and CD8+ T cells do not develop significant lymphokine-activated killer (LAK) activity when PBL are cultured with IL-2 or even when they are activated with a T cell stimulus such as OKT3 mAb. The possibility that a T cell regulatory mechanism prevents the development of LAK activity by CD4+ or CD8+ cells in OKT3 mAb and IL-2 cultures was tested by depleting CD8+ or CD4+ cells from PBL before stimulation with OKT3 and IL-2. Under these conditions, the remaining CD4+ and CD8+ cells were able to generate non-MHC-restricted lysis of NK-resistant tumor targets. Our data suggested that a regulatory signal was present in the culture to prevent the development of lytic function by T cells. T cells removed from the PBL cultures were, upon culture with IL-2, able to generate high LAK activity, suggesting that inhibition of the CD4+ or CD8+ T cell-mediated LAK activity was an active ongoing process, which blocked the lysis at the level of the activated cell and not the precursor cell. Mixing experiments demonstrated that the CD4+ or the CD8+ cells isolated from the PBL cultures were able to inhibit the development of lytic function in the CD4-depleted and CD8-depleted cultures. Transforming growth factor-beta (TGF-beta) has been shown to block LAK activity of NK cells in IL-2-stimulated cultures. When TGF-beta was added to CD4(+)- or CD8(+)-depleted cultures, it also inhibited LAK activity of T cells in a dose-dependent fashion, without interfering with T cell growth. Lytic activity returned to activated levels when TGF-beta was removed from the culture medium, thereby demonstrating the reversibility of TGF-beta inhibition.     

Studies on the relationship of human natural killer and lymphokine-activated killer cells with lysosomal staining and analysis of surface marker phenotypes

We have previously demonstrated that natural killer (NK) cells are lysosome-rich and stain more intensely with lysosomotropic agents such as neutral red and quinacrine (Qu) than do non-NK cells. In this study we combined the quantitation of Qu staining with surface marker staining to define subpopulations of NK cells. While all NK activity was contained within the Qu+ population, most but not all NK cells expressed the surface marker CD16. A subpopulation of NK cells was found to be Qu+CD16- composed of medium- to large-sized cells with a granular appearance on Giemsa staining. Culture with interleukin-2 (IL-2) induced enhanced cytotoxicity in peripheral blood lymphocytes (PBL) against NK-sensitive and NK-resistant tumor cells. Like NK cells, these lymphokine-activated killer (LAK) cells were predominantly Qu+CD16+. However, some LAK cells were Qu+CD16-. The Qu+CD16+ cells were typical large granular lymphocytes (LGL). The Qu+CD16- cells were also large lymphocytes, more than 50% of which were proliferating. However, the granulation in some Qu+CD16- cells, as detected by Giemsa staining, was more prominent and numerous than others in the same population. No LAK activity was ever detected in Qu- cells, which were uniformly small lymphocytes. Quantitation of Qu staining in effector cells was therefore demonstrated to have a good correlation with NK and LAK functions, and with surface markers can help to characterize both types of cells. Moreover, these results indicate that both NK and LAK populations include a small subset of CD16- cells in each.     

Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.     

Potentiated lymphokine-activated killer cell activity generated by low-dose interleukin-2 and mismatched double-stranded RNA

Summary Lymphokine-activated killer (LAK) cell activity was measured in human peripheral blood mononuclear cells (PBMC) treated in vitro for 3 days with recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA). Lytic activity was measured utilizing K562 (NK-sensitive) and 786-0 (NK-resistant) target cells. PBMC cultured with rIL-2 (10–1000 BRMP U/ml) alone showed concentration-dependent lytic activity against the 786-0 target cells, while cells cultured in unsupplemented medium or medium supplemented with mismatched dsRNA (200 µg/ml) alone could not lyse the 786-0 targets. The combination of mismatched dsRNA with suboptimal concentrations of rIL-2 (10–30 U/ml) showed enhancement of both natural killer (NK) and LAK cell activities. The uptake of [³H]thymidine by treated effector cells was dependent on time and rIL-2 concentration and was not increased in the cells treated with low-dose rIL-2/mismatched dsRNA, compared to those treated with low-dose rIL-2 or mismatched dsRNA alone. Similarly, changes in the expression of CD3, CD4, CD8, CD57, CD16 and CD25 cell surface antigens were dependent on rIL-2 concentration and not altered by the presence of mismatched dsRNA. These results indicate that mismatched dsRNA can potentiate rIL-2-induced LAK cell activity by increasing the functional activity per cell, rather than by increasing the number of activated cells.     

Phenotypic characterization of lymphokine-activated killer cells from human lymph node lymphocytes

We previously reported that lymphokine-activated killer (LAK) activity can be generated in human lymph node lymphocytes (LNL) at the same level as that in peripheral blood lymphocytes (PBL), despite the absence of active natural killer (NK) cells. In the present study, we investigated the surface phenotype of LNL-LAK cells by fractionation of lymphocytes, using a panning method. LNL isolated from lung cancer patients were cultured in the presence of recombinant interleukin 2 for 8 days and separated into T cells and non-T cells according to the expression of CD3 antigen. LAK effectors were enriched in the CD3- non-T cells. However, the CD3+ cells also mediated a low but substantial level of LAK activity, which was attributed to a CD8+ T-cell subset. Further investigation of the CD3- cells revealed that most of the CD3- effector cells expressed neither B-cell (CD20) nor NK-cell (CD16) markers. Precursors of this CD3-CD20-CD16- (null) population appeared to be also CD3-, CD20-, and CD16-. From these results, we would stress the significant contribution of CD3-CD20-CD16- null cells to the LAK phenomenon, which has not been focused on in PBL.     

Increased cytotoxicity and CD16 (Leu 11) expression in long-term, IL-2-activated human peripheral blood mononuclear cells

CD16 (Leu 11) positive cells are believed to be the effector cells for the so-called LAK phenomenon. Current evidence suggests that this cell population is comprised predominantly of IL-2-activated CD3 negative Leu 11+ NK cells and a minor proportion of Leu 11+ CD3+ MHC unrestricted type II cytotoxic T cells. The current study demonstrates a continuous increase in the frequency of Leu 11+ (and CD8+) cells and a decline of CD3 and CD4 positive cells during prolonged culture of human PMBL with high levels of rIL-2. Cytotoxicity also increases in this time period parallel with Leu 11 to a maximum of activity on the twelfth day of culture. This correlation suggests that the long-term activated killer cells generated in this period are Leu 11+, CD8+, CD3-, CD4- activated NK cells. With regard to tumor therapy, the long-term culture of PMBL in rIL-2 may be of advantage over short-term activation protocols. If the Leu 11+ cells are in fact the mediators of the therapeutic response, the long-term culture generates up to six times more effector cells. In addition, this method allows significant savings in the expense for leukophoresis, cell culture, and laboratory personnel. The efficacy of long-term, cultured rIL-2-activated Leu 11+ cells for tumor therapy is currently being investigated in clinical trails.     

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.     

Human postnatal CD4- CD8- CD3- thymic T cell precursors differentiate in vitro into T cell receptor delta-bearing cells

The signals required for activation and the differentiation of human triple negative postnatal thymocytes were studied in vitro. Highly purified populations of CD4-, CD8-, CD3- (triple negative) thymocytes were isolated by combined panning and preparative cell sorting and the ability of triple negative thymocytes to proliferate in response to various cytokines determined. Maximal triple negative proliferation was obtained using a mitogenic combination of CD2 antibodies and either rIL-2 or the phorbol ester, PMA. Long term growth (2 to 6 wk) of postnatal triple negative thymocytes was best achieved using CD2 antibodies and rIL-2. After in vitro culture with CD2 antibodies and rIL-2, triple negative thymocytes gave rise to TCR-delta+ cells beginning on day 2 of culture (approximately 15% CD3/TCR-delta+) reaching maximum (approximately 60% CD3/TCR-delta+) on day 7 with stable number of TCR-delta+ cells observed in vitro for up to 6 wk. Analysis of 30 clones of human postnatal triple negative thymocytes demonstrated 9 of 30 (30%) were TCR-delta+, beta F1-, essentially ruling out overgrowth of the triple negative population over time by a minor pool of contaminating TCR-delta+ cells. Thus, these studies have defined an in vitro culture system for human postnatal T cell precursors and demonstrated that precursors of human TCR-gamma delta+ T cells reside in the triple negative thymocyte pool.