Regulation of CD3- lymphocyte function with an antibody against the IL-2 beta chain receptor: modulation of NK and LAK activity and production of IFN gamma

To elucidate the role of interleukin 2 (IL-2) activation in CD3- lymphocytes, we examined the ability of monoclonal antibody (MAb) TU27, developed against the IL-2 receptor (IL-2R) p75 protein (IL-2R beta), to block lymphocyte activation with exogenous IL-2, as well as its innate ability to activate lymphocytes as a result of its surface ligand interaction. The binding of the TU27 MAb and the results of 125I-IL-2 cross-linking experiments suggest that the IL-2R beta chain is expressed primarily on CD3-, CD56+ lymphocytes; although the protein was also detected in a small portion of CD3+ cells, its expression appeared to be donor dependent. In the present study, we found that TU27 totally blocked natural killer (NK) cell activation in a 4-h assay but had no effect on basal levels of NK activity. When treatment was extended to 24 to 72 h, the MAb was able to block the induction of both NK and lymphokine-activated killer (LAK) activity. Of interest was the observation that MAb treatment alone augmented NK activity and subsequent interferon gamma (IFN gamma) production in CD3- lymphocytes but did not activate LAK activity or induce cell growth. Collectively, these results indicate that TU27 not only reacts with p70-75 IL-2R beta but can abrogate IL-2 binding and subsequent activation events. In addition, some CD3- lymphocyte functions (e.g., NK activity and IFN gamma secretion) are directly induced by the binding of MAb to p70-75 through signals that only partially mimic IL-2.     

IL-2R beta agonist P1-30 acts in synergy with IL-2, IL-4, IL-9, and IL-15: biological and molecular effects

From the sequence of human IL-2 we have recently characterized a peptide (p1-30), which is the first IL-2 mimetic described. P1-30 covers the entire alpha helix A of IL-2 and spontaneously folds into a alpha helical homotetramer mimicking the quaternary structure of a hemopoietin. This neocytokine interacts with a previously undescribed dimeric form of the human IL-2 receptor beta-chain likely to form the p1-30 receptor (p1-30R). P1-30 acts as a specific IL-2Rbeta agonist, selectively inducing activation of CD8 and NK lymphocytes. From human PBMC we have also shown that p1-30 induces the activation of lymphokine-activated killer cells and the production of IFN-gamma. Here we demonstrate the ability of p1-30 to act in synergy with IL-2, -4, -9, and -15. These synergistic effects were analyzed at the functional level by using TS1beta, a murine T cell line endogenously expressing the common cytokine gamma gene and transfected with the human IL-2Rbeta gene. At the receptor level, we show that expression of human IL-2Rbeta is absolutely required to obtain synergistic effects, whereas IL-2Ralpha specifically impedes the synergistic effects obtained with IL-2. The results suggest that overexpression of IL-2Ralpha inhibits p1-30R formation in the presence of IL-2. Finally, concerning the molecular effects, although p1-30 alone induces the antiapoptotic molecule bcl-2, we show that it does not influence mRNA expression of c-myc, c-jun, and c-fos oncogenes. In contrast, p1-30 enhances IL-2-driven expression of these oncogenes. Our data suggest that p1-30R (IL-2Rbeta)(2) and intermediate affinity IL-2R (IL-2Rbetagamma), when simultaneously expressed at the cell surface, may induce complementary signal transduction pathways and act in synergy.     

Long-term culture growth of CD4-CD8- lymphocytes exhibiting elevated non-MHC-restricted cytotoxic activity.

We have developed a culture system for "long-term" growth of human lymphokine-activated killer (LAK) cells exhibiting an elevated, wide-spectrum anti-tumor cytotoxicity. The system allows the exponential growth of monocyte- and B-lymphocyte-depleted CD4-CD8- lymphocytes in the presence of human AB serum and recombinant human interleukin-2 (IL-2) (2 x 10(2) U/ml) combined with interleukin (IL-1) beta (50 ng/ml). After 21 days in culture, these cells undergo massive amplification (i.e., the cell yield rises up to 30-120 times the starting values), and exhibit a marked anti-tumor cytotoxic activity against a panel of natural killer (NK)-resistant tumor cell lines. Interestingly, this activity correlates with the high level of perforin RNA. The membrane phenotypes of the final cell population, assessed by a panel of monoclonal antibodies (MoAbs) indicate a mixed population comprising two cell types in variable proportions (i) NKH-1+, T cell receptor (TCR) alpha/beta-, TCR gamma/delta-, CD3-, Leu 23+; (ii) NKH-(+), TCR alpha/beta-, TCR gamma/delta+, CD3+, Leu 23+. This culture system may provide a tool for cellular and molecular studies on the mechanisms of anti-tumor cytotoxicity, as well as the basis for new adoptive immunotherapy protocols in advanced cancers.     

Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

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.     

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)     

Targeted deletion of CD44v7 exon leads to decreased endothelial cell injury but not tumor cell killing mediated by interleukin-2-activated cytolytic lymphocytes

In the current study, we investigated the nature and role of CD44 variant isoforms involved in endothelial cell (EC) injury and tumor cell cytotoxicity mediated by IL-2-activated killer (LAK) cells. Treatment of CD44 wild-type lymphocytes with IL-2 led to increased gene expression of CD44 v6 and v7 variant isoforms and to significant induction of vascular leak syndrome (VLS). CD44v6-v7 knockout (KO) and CD44v7 KO mice showed markedly reduced levels of IL-2-induced VLS. The decreased VLS in CD44v6-v7 KO and CD44v7 KO mice did not result from differential activation and expansion of CD8+ T cells, NK, and NK-T cells or from altered degree of perivascular lymphocytic infiltration in the lungs. LAK cells from CD44v7 KO mice showed a significant decrease in their ability to adhere to and mediate lysis of EC but not lysis of P815 tumor cells in vitro. CD44v7-mediated lysis of EC by LAK cells was dependent on the activity of phosphatidylinositol 3-kinase and tyrosine kinases. Interestingly, IL-2-activated LAK cells expressing CD44hi but not CD44lo were responsible for EC lysis. Furthermore, lysis of EC targets could be blocked by addition of soluble or enzymatic cleavage of CD44v6-v7-binding glycosaminoglycans. Finally, anti-CD44v7 mAbs caused a significant reduction in the adherence to and killing of EC and led to suppression of IL-2-induced VLS. Together, this study suggests that the expression of CD44v7 on LAK cells plays a specific role in EC injury and that it may be possible to reduce EC injury but not tumor cell killing by specifically targeting CD44v7.     

Lysis of human monocytes by lymphokine-activated killer cells

Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.     

Activated endothelial cells resist lymphokine-activated killer cell-mediated injury. Possible role of induced cytokines in limiting capillary leak during IL-2 therapy

We previously demonstrated that IL-2 promotes the adhesion of NK cells to endothelial cells (EC) and that EC are readily lysed by lymphokine-activated killer (LAK) cells in vitro, suggesting that cell mediated endothelial injury may contribute to the capillary leak syndrome observed in patients treated with IL-2. In this investigation, we sought to determine the effects of EC activation on the in vitro susceptibility of EC to LAK cell-mediated cytolysis. Despite increased binding of CD16+ lymphocytes to TNF-activated EC monolayers, prior exposure of EC to any of several IL-2-inducible cytokines including TNF-alpha, IL-1 beta, and IFN-gamma not only failed to render the EC more vulnerable to cytolysis but increased their resistance to LAK cells in 111Indium release cytolysis assays. This decrement in susceptibility to cytolysis resulting from prior exposure to cytokines preceded any detectable increase in HLA class I or II Ag expression. In cold target competition experiments with LAK cell effectors and radiolabeled K562 target cells, TNF-primed EC were no more competitive than unstimulated EC, and in assays with unstimulated PBMC effectors, the addition of unlabeled TNF-activated EC actually increased the cytolysis of the radiolabeled tumor cells. The effects of various cytokines and lymphocyte preparations on EC permeability were also evaluated. In these experiments, saphenous vein EC were cultured on porous filter disks, exposed to cytokines or lymphocytes, and the diffusion of 125I-BSA through the filters was then measured. Exposure to IL-2, IFN-gamma, or TNF-alpha did not increase the diffusion of the BSA through the EC-coated filters, whereas LAK cells markedly increased their permeability. Consistent with the results of the cytolysis assays, pretreatment of the EC with TNF, IL-1, or IFN-gamma diminished the LAK cell-induced increase in BSA diffusion. These results suggest that although circulating IL-2-inducible cytokines such as TNF and IFN-gamma may activate EC in vivo and contribute to lymphocyte margination and lymphopenia, they may not be directly responsible for the IL-2-induced capillary leak syndrome and may actually protect EC from LAK cell-mediated injury.     

IL-7 induces human lymphokine-activated killer cell activity and is regulated by IL-4.

Induction of lymphokine-activated killer (LAK) activity by IL-2 has been described and characterized as broadly cytolytic activity against both fresh and cultured tumors. rIL-7 in the absence of IL-2 also induces LAK activity in human cells. This activity is unique for IL-7, because it is not shared by other cytokines including IL-1, IL-4, IL-6, and TNF-alpha. IL-7 also induces either de novo or increased expression of the surface markers CD25 (Tac, IL-2R alpha-chain), CD54 (ICAM-1), Mic beta 1 (IL-2R beta-chain) and CD69 (early T cell activation Ag). IL-7-induced LAK activity is independent of IL-2 secretion, because it is not abrogated by IL-2 antisera. The LAK precursor responding to IL-7 stimulation is enriched in the null cell fraction as has been demonstrated for IL-2-induced LAK cells. TGF-beta and IL-4 interfere with generation of LAK activity by IL-7. Anti-IL-4 antiserum enhances IL-7-induced LAK activity and augments induction of surface marker expression by IL-7. This may be indirect evidence that IL-7 stimulation leads to induction of IL-4 activity. Our results describe the activation of mature lymphoid cells by IL-7. This and the previously described role of IL-7 in lymphohemopoiesis makes it a cytokine of potential therapeutic value for treatment of immunodeficiency states and possibly the immunotherapy of cancer.     

Regulation of lymphokine-activated killer cell induction by human recombinant IL-1 receptor antagonist. Obligate paracrine pathway of IL-1 during lymphokine-activated killer cell induction.

IL-2-stimulated human lymphocytes, referred to as lymphokine-activated killer (LAK) cells, can develop a broad range of lytic activity against fresh tumor cells and cultured tumor cell lines. IL-1, a pleiotropic cytokine shown to synergize with IL-2 on LAK induction, is endogenously synthesized and secreted by LAK cells. Immunoblot analysis demonstrated that IL-2-stimulated PBL produced the 31- to 34-kDa pro-molecules of IL-1 within 24 h and maintained their expression for at least 96 h. The role of secreted IL-1 has been examined using rIL-1R antagonist (IL-1ra). The addition of IL-1ra to LAK activation culture resulted in dose-dependent inhibited lytic activity, which was more apparent in LAK cells cultured with higher doses of IL-2. However, IL-1ra had no effect on proliferative responses elicited in LAK cells by IL-2. Moreover, when IL-1 binding was blocked by IL-1ra, the expression of the IL-2R p55 subunit was reduced compared with control LAK cells. The effect of IL-1 binding blockade on expression of other cytokine mRNA was further examined by polymerase chain reaction analysis, and, specifically, inhibition of both TNF-alpha and TNF-beta mRNA expression by IL-1ra was observed in PBL stimulated with IL-2. The reduced biologic activity of TNF in culture supernatants correlated well with the inhibition of mRNA expression. These findings suggest that autocrine/paracrine IL-1 is involved in the initial generation of LAK activity and, in particular, that TNF expression could be induced via an IL-1 autocrine pathway.     

Human recombinant interleukin 2-activated sheep lymphocytes lyse sheep pulmonary microvascular endothelial cells

Administration of lymphokine-activated killer (LAK) cells in combination with interleukin 2 (IL-2) has been effective in reducing tumor mass in humans, but has been accompanied by significant toxicity. We used a chronic awake sheep model to investigate the cause of the vascular leak syndrome associated with IL-2 administration. Sheep repeatedly infused with human recombinant IL-2 (hrIL-2) developed mild pulmonary hypertension, systemic hypotension, acidemia, hypoxemia, and increased flow of protein rich lung lymph. We hypothesized that LAK cells may damage lung endothelium in vivo and cause increased lung vascular permeability. Sheep peripheral blood and lung lymph lymphocytes incubated in vitro with hrIL-2 generated cytotoxic activity for human K-562 cells and sheep pulmonary microvascular endothelial cells. In addition, cytotoxic effector cells were isolated from the peripheral blood of a sheep which had received hrIL-2. These observations suggest that LAK cells possess the ability to damage endothelial cells and may contribute to an increased pulmonary vascular permeability observed following hrIL-2 infusion in sheep.     

The roles of interleukin-15 receptor alpha: trans-presentation, receptor component, or both?

Interleukin-15 receptor alpha (IL-15R alpha) is a high affinity IL-15 binding protein that is crucial for mediating IL-15 functions such as memory CD8 T cell proliferation and NK, NK/T cell, and intestinal intraepithelial lymphocyte development. However, the mechanism by which IL-15R alpha mediates IL-15 functions is unique among cytokines. Originally, IL-15R alpha was thought to be a component of a heterotrimeric receptor complex containing the IL-2/IL-15R beta and common gamma chains (gammaC) that were required for mediating signaling. Although IL-15R alpha may in some cases act as a component of this receptor complex, more recent evidence indicates that IL-15R alpha predominately functions by presenting IL-15 to opposing cells expressing the IL-15R betagamma signaling components. This theory is consistent with the broad, non-lymphoid expression pattern of IL-15R alpha and the evidence that IL-15R alpha expression by lymphocytes is dispensable for IL-15 action in vivo. This new concept of cytokine delivery will allow us to better understand the regulation and function IL-15.     

Effects of IL-7 and IL-2 on highly enriched CD56+ natural killer cells. A comparative study.

IL-7 has been shown to induce low levels of lymphokine-activated killer cell (LAK) activity in bulk PBMC populations. We report here that immunomagnetically purified CD56+ cells from peripheral blood generated high LAK activity in response to IL-7. The LAK activity induced by IL-7 was comparable to, or slightly lower than, the LAK activity induced by IL-2. When analyzing cells from the same donor, no detectable LAK-generating effect of IL-7 was registered in the PBMC population, in contrast to a substantial effect in the CD56+ population. IL-2 induced 8- to 15-fold higher proliferative activity in CD56+ cells, relative to IL-7. At suboptimal concentrations of IL-2, IL-7 had a synergistic effect on the proliferation. IL-2-neutralizing antibodies did not abrogate the IL-7-induced proliferation or LAK generation. Both IL-7 and IL-2 induced comparable levels of 75-kDa TNFR expression, whereas IL-2R alpha expression was higher in IL-7-stimulated CD56+ cells. Low levels of TNF were produced in response to IL-7 at day 5, as opposed to a 50-fold higher TNF production in response to IL-2. No IL-2 or IL-6 production was detected. Our data indicate that IL-7 has profound and direct effects on CD56+ cells.     

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 mM ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTLp) from C57B1/6 (B6) mice were CD8+, ASGM1-, ORN sensitive. Splenic LAK precursors (LAKp) were CD8-, ASGM1+, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAKp were CD8-, ASGM1+, ORN resistant against YAC-1, whereas LAKp against P815 were CD8+, ASGM1+, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8+ phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8+ cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).     

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.     

Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation.

Guanine ribonucleosides, substituted at the C8 position with either a bromine or a thiol group, have recently been shown to regulate several immunologic responses. We have previously shown that 8-mercaptoguanosine (8MG) can replace the requirement for cytokines in the generation of MHC-restricted CTL. In this paper, we examined the ability of 8MG to induce MHC-nonrestricted killer cells. We found that 8MG did not induce significant lytic activity from normal resting lymphocytes. However, 8MG was able to synergize with minimal amounts of IL-2 in inducing lytic activity similar to lymphokine-activated killers (LAK) in that both NK-sensitive and NK-resistant tumor cells were killed. Both the precursors and effectors of 8MG-LAK activity were similar to NK cells and were CD4- CD8- asialo-GM1+ NK1.1+. Similar to IL-2-induced LAK, 8MG-LAK were B220+. 8MG appeared to "stage" these precursor lymphocytes to become more responsive to IL-2 because optimal induction of 8MG-LAK required preincubation with 8MG before the addition of IL-2. This "staging" appeared to be due to the release of a "second signal" since it was readily inhibited by cyclosporine A. Anti-IFN-alpha beta was as efficient as cyclosporine A in inhibiting 8MG-LAK generation, whereas anti-IFN-gamma and anti-IL-1 did not exhibit significant inhibition. These findings suggest that 8MG can be of possible utility as an IL-2-sparing agent in LAK generation from NK cells.     

IL-4 regulates IL-2 induction of lymphokine-activated killer activity from human lymphocytes

IL-4 is a pluripotent lymphokine acting on various cell types. We investigated the role of human IL-4 on the generation of lymphokine-activated killer (LAK) activity. Human IL-4 alone did not induce LAK activity and inhibited IL-2 induction of LAK activity from unstimulated PBMC, peripheral blood null cells, spleen cells, and lymph node cells in a dose-dependent manner. IL-4 also inhibited several phenomena induced by IL-2 such as cell proliferation, augmentation of NK activity, increase of Leu-19+ cells, and expression of IL-2R(p55) on either CD3+ or Leu-19+ cells. IL-4, however, augmented cell proliferation with other T cell mitogens including PHA, Con A, PMA, or allo-MHC Ag with or without IL-2. In contrast to unstimulated cells, IL-4 alone induced marked cell proliferation and LAK activity as well as Leu-19+ cells from in vitro IL-2 preactivated PBMC or null cells, and did not inhibit IL-2 induced cell proliferation, LAK activity, Leu-19+ cells and IL-2R(p55) expression, but rather augmented them with low doses of IL-2. Although IL-4 alone induced LAK activity from peripheral blood of some patients previously given IL-2, IL-4 inhibited in vitro LAK generation with IL-2 from these cells in most cases. Therefore, IL-4 appears to directly inhibit the IL-2 activation pathway via IL-2R(p70) and prevent resting LAK precursors from proliferating and differentiating into final effector cells. However, once cells were sufficiently preactivated by IL-2, IL-4 induced LAK activity and did not inhibit IL-2 activation of these cells. These data suggest an immunoregulatory role of IL-4 on human null cells and T cells.