Natural killer and lymphokine-activated killer cell activities from human marrow precursors. II. The effects of IL-3 and IL-4

Both IL-3 and IL-4 have multi-CSF activity on early marrow progenitors. We have examined the effect of IL-3 and IL-4 on the differentiation of NK cells from their marrow-derived precursors and have further examined the interactions of these cytokines with IL-2 and IL-1. We tested marrow which had been depleted of mature cells and of E rosette-positive cells (including NK cells) by treatment with soybean lectin and SRBC (SBA-E-BM). The cytolytic activities of the SBA-E-BM samples were tested in 51Cr-release assays after 7 days of liquid culture. K562 targets were used as a measure of NK activity and NK-resistant Daudi targets were used to measure lymphokine-activated killer (LAK) cell activity. Neither NK nor LAK activity was detectable in marrow cultured in medium without cytokines, or in medium containing IL-3, or IL-4 alone. Both of these cytokines were shown to be inhibitory to the IL-2-induced generation of NK and LAK activity from SBA-E-BM at concentrations as low as 1 U/ml. The inhibitory activity of both IL-3 and IL-4 was found to occur early in the marrow cultures, with little or no inhibitory effects seen if added 48 h after IL-2. IL-3 appeared to be specifically inhibitory to NK cell precursors since addition of IL-3 to cultures of PBMC did not inhibit IL-2-induced lytic activities. In contrast, IL-4 was equally inhibitory to the activation of marrow and peripheral blood NK cells by IL-2. Mixing experiments demonstrated that the reduced lytic activity in IL-3 or IL-4 containing marrow cultures were not due to suppression of the NK effectors, nor could marrow cultured in IL-3 or IL-4 serve as targets for IL-2-activated NK cells. Phenotype analysis of the lymphoid cells in marrow cultures containing IL-2 combined with IL-3 or IL-4 revealed fewer cells expressing Leu-11 (CD16), or Leu-19 (CD56) and fewer CD16, CD56 coexpressing cells compared with marrow cultured in medium containing IL-2 alone. The inhibitory activity of IL-4, but not IL-3, could be partially reversed if IL-1 was added to the cultures, suggesting that IL-1 and IL-4 have opposing activities on NK cells responsiveness to IL-2. These interactions between cytokines might be important in the regulation of NK cell differentiation and on the functional activity of mature NK cells.     

Augmentation of cell number and LAK activity in peripheral blood mononuclear cells activated with anti-CD3 and interleukin-2

Summary A wide variety of human cancers currently have no effective treatment and are potential targets for lymphokine-activated killer (LAK) cellular immunotherapy. Relapsed acute lymphocytic leukemia (ALL) and neuroblastoma are two of the major therapeutic challenges in pediatric oncology today. However, one problem which makes LAK immunotherapy in children particularly difficult is obtaining the large numbers of cells required. Present adult therapeutic LAK protocols have utilized short-term (5 day) cultures of interleukin-2 (IL2)-activated cells which are initially obtained from leukophersis. Since routine use of this procedure in small children is not practical, we have investigated a different approach to obtain increased cell numbers by activation of peripheral blood mononuclear cells with OKT3, a mitogenic anti-CD3 monoclonal antibody, and IL2. Cell growth and LAK activity in OKT3+IL2-activated cultures were compared to cultures activated with IL2 alone in 2 children with relapsed ALL and 2 children with stage IV neuroblastoma. OKT3+IL2-activated cultures had marked increases in cell number: after 14 days the OKT3+IL2-activated cultures yielded an approximately 500-fold increase in cell number compared to a 7-fold increase for cultures activated with IL2 alone. In vitro ⁵¹Cr release assays were used to estimate LAK activity of the cultures at 7 and 14 days. When tested against HL60, a natural killer (NK)-resistant tumor cell line, not only were total cytolytic units greatly increased in OKT3+IL2-stimulated cultures but lytic activity on a per cell basis (lytic units/1×10⁶ cells) had also markedly increased on day 14 of culture. Phenotypic analysis demonstrated that 80% to 90% of cells in OKT3+IL2-stimulated cultures were CD3+ T cells. Variable low percentages of CD16+ NK cells were seen in these cultures. In summary, OKT3+IL2 activation resulted in a large increase in cell yield and the development of high level LAK activity using peripheral blood mononuclear cells from children with cancer. This approach may facilitate the utilization of increased cell numbers in future adoptive immunotherapy protocols, especially in pediatric patients.Supported by the Children's Cancer Research Fund, and the USPHS Training Grant T32CA09445Supported by NIH AI17687, AI18326, AI19007, and AI72626     

Studies on cytotoxicity generated in human mixed lymphocyte culture

Summary High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.This work was supported by NIH grant CA34442     

Generation of lymphokine-activated killer cell activity by low-dose recombinant interleukin-2 and tumor cells

The generation of lymphokine-activated killer (LAK) cells in vitro has been reported to require 100-1000 units of recombinant interleukin-2 (IL2). In this study we investigated the generation of human LAK cells with low-dose IL2 (1-10 U) in combination with human tumor cell lines. A significant LAK activity was generated within 3- to 5-days culture of PBL. Among six human tumor cell lines tested, the K562 cell line had the greatest stimulating activity, and the degree of cytotoxicity was comparative to that of PBL stimulated with higher doses of IL2 alone. The origin of this LAK activity was primarily the E(-) rosetting cell population. Cocultures of E- cells with 1 U/ml IL2 plus K562 had significantly higher cytotoxicity (P less than 0.05) compared to using E+ cells. Phenotypic analysis indicated that 1 U/ml IL2 plus K562 cell stimulation enhanced CD56+ and CD16+ cells. These studies suggest that very low dosages of IL2 with stimulator tumor cells can generate LAK activity comparable to that generated with high dosages of IL2 alone.     

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.     

Evidence that lymphokine-activated killer cells and natural killer cells are distinct based on an analysis of congenitally immunodeficient mice

The in vitro incubation of lymphoid cells in RIL 2 results in the generation of LAK cells that are broadly lytic to autologous, syngeneic, and allogeneic fresh tumor cells, but which do not lyse fresh, normal cells. Strains of mice with congenital immunodeficiencies were tested both for the presence of NK cells and for their capacity to generate LAK cells after in vitro incubation with IL 2. Splenocytes obtained from two immunodeficient mouse strains (NIH-Beige-Nude and NIH-Beige-Nude-XID) failed to generate LAK cells, but displayed significant activity. Splenocytes from another immunodeficient mouse strain (NIH-Beige-XID) generated LAK cells but did not display NK cell activity. This dissociation of activation of LAK cells from NK cells among the immunodeficient strains indicates that the LAK and NK cell lytic systems are distinct.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Similarities between LAK cells derived from human thymocytes and peripheral blood lymphocytes: expression of the NKH-1 and CD3 antigens

Human thymocytes are devoid of NK cells but develop lymphokine-activated killer (LAK) activity after culture with recombinant interleukin-2 (rIL-2). The most active precursor for this activity appears to be a CD3-negative cell. The purpose of these studies was to compare the phenotype and functional activities of thymocyte and peripheral blood lymphocyte (PBL) LAK cells. Following culture, rIL-2-activated thymocytes resemble PBL-generated LAk and PBL NK cells. For each of these populations, lytic activity is highest in NKH-1-positive cells. Two-color fluorescence of each population also indicates that NKH-1+ cells are highly granular, as measured by staining with the lysosomotropic vital dye quinacrine. PBL, PBL-derived LAK cells, and thymus-derived LAK cells have a portion of cells that express both CD3 and NKH-1. However, approximately 60-80% of NKH-1+ cells lack detectable CD3. This suggests that both CD3+ and CD3- cells may be capable of LAK activity. Thymic-derived LAK cells respond to interferon in a manner very similar to NK and PBL-derived LAK cells, but lack the NK-associated CD16 antigen. Thus, despite the absence of NK cells in the thymus, it is possible to generate thymocyte LAK activity which bears a strong resemblance to LAK activity derived from peripheral blood lymphocytes.     

Precursor phenotype of lymphokine-activated killer cells in the mouse

Lymphokine-activated killer (LAK) activity has been proposed to functionally differ from natural killer (NK) activity largely on the basis of a broader target cell spectrum and different kinetics of response to interleukin 2 (IL 2). Similarly, it has been proposed that the precursor cells for LAK activity are phenotypically distinct from NK cells. In most precursor studies, phenotype comparisons have been made between fresh NK cells and LAK cells which have been generated by 3 to 5 days of culture in IL 2. In the present study, we utilized positive selection with monoclonal antibodies to characterize the surface phenotype of precursor cells which give rise to rIL 2-augmented NK activity within 24 hr and to classically generated LAK activity which appears after 3 to 5 days of culture in rIL 2. The results demonstrated that highly purified (93 to 95%) Lyt-2+ or L3T4+ T lymphocytes were unable to generate appreciable amounts of either augmented NK activity or LAK activity when cultured with rIL 2, whereas the highly purified (98%) Lyt-2-, L3T4-, asialo GM1+ lymphocyte subset gave rise to both augmented NK and LAK activities. These findings demonstrate that both augmented NK and LAK activities can arise from precursors expressing the same phenotype. Overall, the results suggest that NK cells in mouse spleen constitute a major precursor component for the generation of LAK activity from that organ.     

Human natural killer cell adhesion molecules. Differential expression after activation and participation in cytolysis.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.     

The role of IL-4 in proliferation and differentiation of human natural killer cells. Study of an IL-4-dependent versus an IL-2-dependent natural killer cell clone

The role of IL-4 in proliferation and differentiation of human NK cells was studied using newly established sublines of an IL-4-dependent NK cell clone (IL4d-NK cells) and an IL-2-dependent NK cell clone (IL2d-NK cells) derived from a parental conditioned medium-dependent NK cell clone (CM-NK cells). IL-4 induced the higher proliferation of CM-NK cells, but abolished their NK activity and decreased CD16 and CD56 Ag expression. In contrast, IL-2 induced the higher NK activity and increased CD16 and CD56 Ag expression. Addition of anti-IL-4 antibody to the culture of CM-NK cells with CM inhibited the proliferation, but slightly increased NK activity, and largely increased CD56 Ag expression. Addition of anti-IL-2 antibody to the culture of CM-NK cells with CM inhibited both proliferation and cytotoxicity. Proliferation of IL4d-NK cells, which is totally dependent on rIL-4, is greater than that of IL2d-NK cells, which was greater than parental CM-NK cells. Morphologically, IL4d-NK cells are small and round, whereas IL2d-NK cells are large and elongated. Anti-IL-4 antibody inhibited proliferation of IL4d-NK but not IL2d-NK cells, whereas anti-IL-2 antibody inhibited that of IL2d-NK but not IL4d-NK cells. IL-2 was not detected in the supernatant from IL4d-NK cells, nor was IL-2-mRNA expressed in IL4d-NK cells. In contrast, IFN-gamma production and protein expression in IL4d- and IL2d-NK cells were detected. NK cell activation markers (CD16 and CD56) were expressed on IL2d-NK cells but not IL4d-NK cells. IL4d-NK cells were not cytotoxic to any tumor cells tested, whereas IL2d-NK cells displayed potent NK activity and lymphokine-activated killer activity. IL4d-NK cells failed to bind K562 tumor cells, whereas one-third of the IL2d-NK cells did. IL4d-NK cells responded to rIL-2, proliferated, and differentiated into cytotoxic NK cells, whereas IL2d-NK cells failed to respond to rIL-4 and died. These results raise a possibility that IL4d-NK cells or IL2d-NK cells primarily represent the immunologic properties of immature or activated types of human NK cells, respectively. Our results provide the first evidence of the capability of IL-4 to support continuous proliferation of a lymphocyte clone with immature NK cell characteristics and to stimulate IFN-gamma production in the clone. IL-4 is suggested as a potential growth factor for certain types of human NK cell progenitors.     

Interferon is able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells

It is known that IL-2 induces lymphocytes to produce interferon-gamma (IFN-gamma) and this IFN type is particularly efficient in inducing tumor cell resistance to natural killer (NK) cell-mediated lysis. We have investigated the effect of IFN on tumor cell sensitivity to LAK cell-mediated cytotoxicity. Pretreatment of the human K562 leukemia and HHMS melanoma with IFN-gamma and the Daudi lymphoma with IFN-alpha caused a significant reduction in sensitivity to lysis by human LAK cells generated in vitro in the presence of human recombinant IL-2 (100 U/ml). The LAK activity was mediated by cells expressing NK cell markers (CD16,NKH1) as well as by cells with T cell markers (CD3, CD5). IFN-treated K562 cells were protected from lysis mediated by all these populations. Supernatants from LAK cultures containing IFN-gamma were able to induce NK and LAK resistance when used to pretreat K562 overnight. Antibodies to IFN-gamma but not to IFN-alpha were able to neutralize this activity. Taken together, these results indicate that the production of IFN-gamma by LAK cells may be of importance in induction of tumor cell resistance to LAK cell-mediated lysis.     

Regulation of human cytolytic lymphocyte responses by interleukin-12.

IL-12 is a heterodimeric cytokine which has been shown to cause the proliferation of activated T and NK cells, to enhance the lytic activity of NK cells, and to induce IFN-gamma production by resting and activated T and NK cells. We previously reported that IL-12 could synergize with IL-2 to activate human LAK cells in the presence of hydrocortisone but that IL-12 alone was inactive. We herein show that in the absence of hydrocortisone, IL-12 by itself can activate human LAK cells. IL-12-induced LAK cell activity was mediated predominantly by CD56+ lymphocytes. Activation of LAK cells by IL-12 appeared to be independent of IL-2 since it was not inhibited by neutralizing anti-human IL-2. However, IL-12- and IL-2-induced LAK cell activation could be partially inhibited by anti-human TNF-alpha. Moreover, IL-12 produced in situ appeared to play a role in IL-2-induced LAK cell activation since rat monoclonal antibodies to human IL-12 could partially inhibit the generation of LAK cells in response to IL-2. In addition to its effects on LAK cell responses, IL-12 could facilitate specific allogeneic human CTL responses. However, IL-12-facilitated CTL responses were blocked by neutralizing anti-human IL-2 indicating a requirement for IL-2 produced in situ. The ability of IL-12 to facilitate both nonspecific LAK and specific CTL responses suggests that it may be useful as a therapeutic agent against some tumors and infectious diseases.     

Functional and phenotypic modifications induced by IL-4, as single agent or in combination with IL-2, on PBMC preactivated in vivo by alpha-interferon + interleukin-2 therapy

The effect of human IL-4, used as a single agent or in combination with low or high dose IL-2, upon LAK-cell proliferation and activation has been tested on PBMC from patients treated with alpha 2-IFN and IL-2. Four days in vitro culture with IL-4 did not induce any LAK-cell activation; IL-4 induced the proliferation of CD3+ CD4+ T-cells, but decreased the percentage of NK cells in culture samples. When combined with high dose IL-2, IL-4 improved the recovery of MN cell without modification of T-cell subsets; however, IL-4 had no major effect on IL-2-induced NK or LAK cell activity. The combination of IL-4 and low dose IL-2 still significantly improved the total MN cell recovery but did not modify the distribution of T and NK lymphocytes; IL-4 inhibited low dose IL-2-induced NK and LAK cell activity, and increased the BL-esterase activity induced by high or low dose IL-2. The combination of IL-4 and IL-2 did not induce any large variation in the percentage of IL-2R (p55) expressing cells. In all tested conditions, IL-2R (p55) was mainly expressed on CD4+ T cells; less than 2% of the cells coexpressed the NK cell marker CD56 and IL-2R (p55). The effect of IL-4 upon IL-2-induced LAK cell expansion is thus very different on PBMC pre-activated in vivo by alpha IFN + IL-2 therapy than on PBMC pre-treated in vitro with IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and differentiation of NK lineage cells in long-term bone marrow cultures in the absence of exogenous growth factors.

Neither lytic NK cells nor IL-2-responsive NK precursors were produced in myeloid (Dexter) long-term bone marrow cultures (LTBMC). However, when myeloid LTBMC were switched to lymphoid (Whitlock-Witte) conditions and reseeded ("recharged") with fresh bone marrow cells (BMC), nonadherent cells with NK lytic activity and NK 1.1+ phenotype were produced within 1-2 weeks without the addition of exogenous IL-2 to the cultures. NK- and T cell-depleted BMC proliferated extensively in switched cultures and in 2 weeks generated cells that lysed the NK target YAC-1 but not the LAK target P815. The presence of NK precursors in the cultures was confirmed by reculturing nonadherent cells harvested from recharged LTBMC in fresh medium containing 50 U rIL-2/ml. High levels of NK lytic activity were generated. Sequential expression of NK 1.1 and IL-2 responsiveness followed by lytic activity was demonstrated by harvesting cells early after recharge, prior to the appearance of lytic cells. Elimination of NK 1.1+ cells depleted the ability to respond to IL-2 in secondary culture. Our studies demonstrate that myeloid-to-lymphoid switched LTBMC support the proliferation and differentiation of NK lineage cells from their NK 1.1-, nonlytic progenitors in the absence of an exogenous source of growth factors.     

Lymphokine-activated killer (LAK) cells. VI. NK1.1+, CD3+ LAK effectors are derived from CD4-, CD8-, NK1.1- precursors.

Normal murine splenocytes cultured with IL2 for 6, but not 3, days contained an NK1.1+, CD3+ lytically active subset. These lymphocytes were not derived from NK1.1+ precursors since NK1.1+ cells, purified by flow cytometry, failed to express CD3, as determined by the 145-2C11 mAb, on their surface even after culture with IL2 for 6 days. Instead, the precursors of the NK1.1+, CD3+ effectors were contained in a B cell-depleted CD4-, CD8-, NK1.1- splenic subset. Freshly obtained CD4-, CD8-, NK1.1- splenocytes were mostly CD3+, CD5+, B220-, had no spontaneous lytic activity against YAC-1, and were unable to mediate anti-CD3 directed lysis against FcR-bearing target cells. Culture of the CD4-, CD8-, NK1.1- splenocytes with IL2, for 6 days, resulted in the development of NK1.1+, CD3+, B220+ effectors 40% of which were CD5dim and 20-25% of which expressed TCR-V beta 8 as determined by the F23.1 mAb. The acquisition of NK1.1, B220, and lytic activity by this triple-negative subset was readily inhibited by cyclosporine A (CSA). On the other hand, CSA had no effect on the acquisition of B220 or lytic activity by NK1.1+ precursors obtained by flow cytometry sorting. Moreover, all of the NK1.1+ cells generated by IL2 culture of splenocytes obtained from mice depleted of NK1.1+ lymphocytes (by in vivo injection of anti-NK1.1 mAb) coexpressed CD3 on their surface and were thus distinct from classical NK cells. These findings demonstrate that splenic NK cells do not express or acquire CD3; that the NK1.1+, CD3+ LAK effectors are derived from an NK1.1- precursor; and that CSA is exquisitely selective in its inhibitory effect on LAK generation.     

Feeder cells enhance oncolytic and proliferative activity of long-term human bone marrow interleukin-2 cultures and induce different lymphocyte subsets

Summary The effect of feeder cells on oncolytic activity of lymphocyte subsets and their growth was evaluated in long-term human bone marrow interleukin-2 (IL-2) cultures. Two B-lymphoblastoid cell lines (Daudi and Epstein-Barr-virus-transformed BSM) and two human leukemias, AML-M5, were used as feeder cells. The most prominent effects were seen in cultures stimulated with Daudi cells. In these cultures, cytotoxic activity was 100–1000 times increased against a broad range of target cells and the total cellular expansion was more than 40 times higher than in control cultures. This Daudi-related effect appeared to be mediated by natural killer (NK) cells, since cellular expansion occurred mostly in the CD16⁺ and CD56⁺ CD3^– NK cell subset. In cultures stimulated with BSM and acute myelogenous leukemia (AML) feeder cells, the increase in proliferation was similar, but the enhancement of cytotoxicity, even though significant, was less prominent. Although all feeder cells were effective in stimulation of bone marrow reactivity, the highest cytotoxicity was always observed with feeder cells autologous to the targets, indicating some degree of specificity. This was especially evident in cultures stimulated with autologous versus allogeneic AML feeder cells. In contrast to Daudistimulated IL-2 cultures, in which the highest expansion of CD3^– CD56⁺ NK cells was observed, in BSM and AML cultures, the CD3⁺ CD56^+/- T cell subsets were more prolific. This indicates that the response and phenotypic heterogeneity of bone marrow cultures depends on the type of feeder cells used. This observation indicates that the preferential stimulation of a pertinent lymphocyte subset for therapeutic purposes may be possible.Recipient of Florence Maude Thomas Cancer Research Professorship     

Interleukin 3 is a major negative regulator of the generation of natural killer cells from bone marrow precursors

We have established a bone marrow culture system in which mature natural killer (NK) cells can be generated from inactive precursors by interleukin 2. Recombinant interleukin 3 (IL 3) almost completely blocked the induction of NK cells in this culture system as judged by cytotoxic activity, as well as appearance of cells with NK phenotype. The dose-response curve for inhibition of the generation of NK activity with IL 3 parallelled the growth promoting activity on the strictly IL 3-dependent cell line L/B. The effect of IL 3 was selective for the precursor stage of the NK cell, because mature NK cells were not affected by culture with IL 3 for the same period of time. Moreover, the effect of IL 3 was confined to the first 24 hr of culture, indicating an effect on an early stage of NK cell differentiation. IL 3 did not increase the small normally occurring NK-sensitive population in bone marrow, and did not affect the activity of a variant cytotoxic cell with specificity for adherent target cells, the natural cytotoxic cell. Concomitantly with downregulation of NK cell generation, IL 3 induced strong proliferation in the bone marrow cultures and an increase in the percentage of cells expressing the T cell marker Thy-1. A model for regulation of NK cells based on competition of growth factors for target cells with a common progenitor is discussed.     

Generation of NK cell activity from human bone marrow

This study was designed to examine the effect of interleukin 2 (IL 2) on cytotoxic activity of human bone marrow cells and to characterize the IL 2-dependent killer cells and the cell population required for their induction. We have demonstrated that the most aggressive IL 2-dependent killer cells (directed against leukemic and solid cancer targets) exhibited LGL morphology and expressed NK cell-associated antigens NKH1 and CD16, but not T cell-associated antigens CD3, CD4, CD5, or CD8. Similarly, the bone marrow cell population necessary for induction of killer cells with highest cytotoxic activity displayed NK cell surface characteristics, as exemplified by CD16 and Leu-7 antigens. On the contrary, very low or no lytic activity was generated from the bone marrow cell population expressing T cell markers CD3 and CD5. These data indicate that the IL 2-dependent bone marrow-derived killer cells with antitumor activity were activated NK cells. If T cells are involved at all in IL 2-dependent bone marrow killing, their potency is inferior to that of activated NK cells. The clinical applications of these studies are discussed.     

Immunologic and clinical aspects of natural killer cells in human leukemia

We have studied peripheral-blood, splenic and bone marrow natural killer (NK) activity in patients with leukemia. These studies demonstrated that leukemic patients displayed defective NK activity in all of these tissues. However, NK defect could be corrected by culture of effector cells with interleukin-2 (IL-2). The phenotypic analysis of IL-2 cultures showed clearly the heterogeneity of lymphocyte subsets. The characterization studies demonstrated that CD56+, CD3- NK cells manifested most potent lysis of leukemia, CD56+, CD3+ T cells mediated some, but low, antileukemia activity and CD56-, CD3+ T lymphocytes were devoid of cytotoxicity.