Interleukin 2 induces proliferation of normal "resting" human T cells in the absence of other known external stimulation

Interleukin 2(IL-2) is known to stimulate the progression of activated T cells from G1 through the rest of the cell cycle. We have demonstrated that addition of purified recombinant human IL-2 (rIL-2) to fresh normal human peripheral blood mononuclear cells (PBM), which were IL-2 receptor (Tac) negative by FACS analysis, stimulated marked proliferation of the PBM. IL-2-induced proliferation was also observed with umbilical cord blood mononuclear cells. Monocyte depletion of PBM resulted in a marked reduction of rIL-2-induced proliferative response which could be restored by adding back autologous irradiated monocytes but not by interleukin 1. The T cells preincubated with rIL-2 showed a five to six times enhanced autologous mixed-lymphocyte reaction (AMLR) compared to controls. The rIL-2-induced proliferative response of PBM was inhibited in a concentration-dependent fashion by preincubation of PBM with an anti-HLA-DR framework monoclonal antibody. The proliferating cells were shown by two-color flow cytometric analysis to be primarily Leu-1+ and Leu-4+ T cells (both leu-3+ and Leu-2+ subsets); however, 6 to 19% of responding cells had surface markers for B cells or NK cells. The data demonstrate that rIL-2 can induce proliferation of "resting" human T cells. The phenomenon may be related to a monocyte-dependent AMLR which induces IL-2 receptors and IL-2 responsiveness in a subset of T cells.     

Effective Induction of Human NK Cells with OK-432 and Further Augmentation of Their Cytolytic Function by rIL-2

Low concentrations of exogenously added recombinant interleukin 2 (rIL-2) were able to augment OK-432-induced natural killer (NK) cell activity. This kind of augmenting effect depended on the dose of rIL-2 and manifested itself only in PBMC stimulated with OK-432 (OK-MC) followed by rIL-2; augmentation did not happen in the reverse order. The existence of CD16⁺/CD25⁺ (IL-2 receptor positive; IL-2R⁺) and CD57⁺/CD25⁺ double positive cells which possess NK cell surface markers in OK-MC markedly increased in a long-term culture (12 days). A strong positive correlation was observed between the IL-2-dependent augmentation of NK activity and the quantitative changes in cell populations that possessed NK cell phenotypes. Treatment of the day-12-OK-MC with monoclonal anti-CD56 antibody plus complement could almost completely abrogate the augmented NK cytotoxicity. Furthermore, this augmenting effect was detectable within 4 hr after addition of rIL-2 at single cell level, suggesting that the effect did not require NK cell's DNA synthesis. Thus it was suggested that OK-432 could promote and upregulate the expression of IL-2 receptor (CD25) on CD56⁺ NK cell populations. Moreover, it was considered that the interaction of low concentration rIL-2 with IL-2 receptors on OK-432-activated NK cells could augment their lytic function.     

IL-12 receptor. II. Distribution and regulation of receptor expression.

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.     

Different sensitivity to interleukin 4 of interleukin 2- and interferon alpha-induced CD69 antigen expression in human resting NK cells and CD3+, CD4-, CD8- lymphocytes.

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.     

IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury

NK cells promptly disappear from the circulation of patients treated with high dose i.v. rIL-2. To further study this process, we evaluated the effects of IL-2 (1000 U/ml) on normal donor PBMC incubated for 1 h on cultured human saphenous vein endothelial cells (EC). Although the NK activity of non-adherent PBMC recovered from flasks coated only with fibronectin increased in the presence of supplemental IL-2, the activity of cells recovered from flasks coated with EC decreased when IL-2 was added to the medium. The percentage of NK (CD16+) cells among the EC-non-adherent PBMC was reduced relative to that of the input cells when IL-2 was added. The percentage of CD16+ cells in the EC-adherent PBMC, as well as their NK activity, increased in the presence of added IL-2. Although EC had no effect on the lysis of labeled K-562 cells by unstimulated PBMC in cold target competition experiments, they were able to compete in cytolytic assays using PBMC previously activated by exposure to IL-2 for 1 h. EC were not lysed by these briefly activated PBMC in 3-h cytotoxicity assays but were lysed by these effectors in 18-h assays and in 3-h assays using PBMC pre-activated by more prolonged culture with IL-2. The ability of IL-2 to induce NK cell adhesion to EC was not blocked by a mixture of neutralizing antisera raised against rTNF-alpha, rIL-1 alpha, and rIL-1 beta, factors known to promote leukocyte adhesion to EC. We conclude that IL-2 rapidly induces NK cell adhesion to EC and propose that this effect accounts for the disappearance of circulating NK cells after the infusion of high doses of IL-2. In addition, these results suggest that NK cells activated by IL-2 in vivo may injure the endothelium and contribute to the extravasation of plasma and the retention of fluid characteristic of IL-2 treatment.     

Functional effects of a monoclonal antibody directed against a distinct epitope on 4F2 molecular complex in human peripheral blood mononuclear cell activation.

The 4F2 antigenic complex is expressed on most human cell lines in culture, on monocytes and activated lymphocytes, but not on resting T and B lymphocytes. Monoclonal antibody (mAb) CB43 recognizes an epitope of the 4F2 heterodimer either located on the light chain or dependent on the conformation of the molecule. The binding of CB43 mAb to peripheral blood mononuclear cells (PBMC) induced a dose-dependent comitogenic effect in the presence of submitogenic concentrations of anti-CD3 mAb. Significant amounts of interleukin (IL)-1 beta but not IL-2 or interferon-gamma were released in the supernatant. Pretreatment of monocytes with CB43 mAb increased the phytohemagglutinin-induced T lymphocyte proliferation. However, CB43 mAb did not exert agonistic effects on activated T lymphocytes. Depletion of CB43+ cells from PBMC decreased the proliferation and generation of cytotoxic effector cells induced by a mannoprotein (MP) derived from Candida albicans cell wall but not by recombinant IL-2. Furthermore, depletion of CB43+ cells from PBMC preactivated with MP or rIL-2 led to a significant decrease in their cytotoxic activity. CB43 mAb did not inhibit the growth of cell lines nor the proliferation of T cells. Thus CB43 mAb identifies a distinct functional epitope on the 4F2 molecular complex and might be useful in further studying the role of this molecule in cellular activation.     

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.     

Gamma-irradiated peripheral blood mononuclear cells can express LAK activity

Peripheral blood mononuclear cells (PBMC) irradiated with high dose gamma-radiation (1000-5000 rad) are commonly used as feeder cells during the cloning of T lymphocytes, natural killer (NK) and lymphokine activated killer (LAK) cells. We report here that such gamma-irradiated PBMC can be stimulated with interleukin 2 (IL-2) to express the ability to lyse a variety of tumor cell targets. The non-major histocompatibility complex (MHC) restricted cytotoxicity demonstrated by irradiated PBMC is, however, lower than that expressed by their non-irradiated counterparts. The numbers of viable, gamma-irradiated LAK cells are significantly increased by the addition of the mitogen, phytohemagglutinin (PHA). Purification of the gamma-irradiated cells expressing cytotoxic activity by flow cytometry determined that the effector cells were predominantly CD3- cells, although some CD3+ cells also expressed moderate LAK activity. The ability of gamma-irradiated cells to proliferate in the presence of PHA alone, or with IL-2 + PHA, was maximal at day 4-5; but proliferation, as detected by 3H-thymidine uptake, was not detectable beyond 12-15 days of in vitro culture. Because many of the LAK, T cell and NK cell cloning procedures require the presence of feeder layers, growth factors (usually IL-2) and mitogens, the presence of residual feeder cells expressing cytotoxic activity may affect the specificity of such clones. Thus, efforts should be made to ensure that such gamma-radiation-resistant cells capable of expressing cytotoxic activity are completely eliminated before the cloned cells are used for further experiments.     

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.     

The interaction of recombinant IL-2 with human resting lymphocytes: blocking effects of monoclonal antibodies to IL-2 receptors

Several lines of evidence suggest that subsets of resting lymphocytes naturally express interleukin-2 receptors (IL-2.R). Recombinant IL-2 (rIL-2) induced the enhancement of natural killer (NK) activity, the generation of activated killer (AK) cells, the proliferation of resting lymphocytes, and the production of interferon-gamma (IFN-gamma) in lymphocyte cultures. The subsets of lymphocytes mediating these responses appeared to be heterogeneous, but reside predominantly in nylon wool-passed non-T, non-B cells ("null cells" or T3- cells); in response to rIL-2, only Leu 11+T3- cells showed enhanced NK activity, and both Leu 11+T3- and Leu11-T3- cells showed predominantly AK activity, proliferation and production of IFN-gamma. These findings suggest that the T3- fraction (null cell fraction) contains predominantly cells expressing IL-2.R at the resting state. Unlike the case with activated T cells, however, none of these responses was blocked by any of three monoclonal antibodies to IL-2.R, including anti-Tac antibody at any dilution. These results indicate that IL-2.R on the resting T3- cells possess unique biological features compared to those on activated T or B cells. A most likely explanation is that T3- cells possess higher affinity IL-2.R than activated T or B cells. Other possibilities are also discussed.     

Cloned allospecific human helper T cell lines induce an MHC-restricted proliferative response by resting B cells

To analyze helper T (Th) cell-induced B cell proliferation in man, we have cloned allospecific Th cells, grown them as long-term IL 2-dependent T cell lines (TCL), and analyzed their phenotypic and functional properties. The two TCL described in this report, A-7 and A-57, are both composed exclusively of T3+, T4+, T8- T cells blasts. In proliferative assays, with a panel of x-irradiated allogeneic stimulator cells, A-7 was found to proliferate in response to DR3-bearing cells, whereas A-57 responds to DR2-positive stimulators. Both TCL are capable of providing MHC-restricted polyclonal help for allogeneic B cells, as measured in the reverse hemolytic plaque assay. Of greater interest, x-irradiated A-7 and A-57 cells are capable of inducing a proliferative response by allogeneic B cells that is absolutely MHC restricted at the inductive (Th-APC) level. Thus, x-irradiated A-7 cells only trigger proliferation by DR3+ B cells, whereas A-57 cells selectively activate DR2+ B cells. In contrast, after antigen-specific activation, x-irradiated A-7 and A-57 cells can recruit a significant proliferative response by allogeneic B cells bearing "irrelevant" DR antigens. The possibility that Th-induced B cell proliferation may be restricted at the effector (Th-B cell) level was addressed by fractionating B cell populations into "activated" and "resting" subsets by discontinuous Percoll density gradient centrifugation and further purification by employing a monoclonal antibody directed against an antigen expressed on activated B cells (4F2). These studies demonstrate that activated B cells are readily and nonspecifically recruited to proliferate by activated Th cells, whereas optimal proliferative responses by resting B cells require MHC restricted Th-B cell interaction.     

Induction of proliferation in vitro of resting human natural killer cells: IL 2 induces into cell cycle most peripheral blood NK cells, but only a minor subset of low density T cells

We report that resting human peripheral blood natural killer (NK) cells proliferate in response to recombinant interleukin 2 (rIL 2), and addition of irradiated lymphoblastoid B cells significantly increase their proliferative response. Interaction of IL 2 with the Tac IL 2 receptor expressed on activated NK cells is necessary to maintain continued growth of these cells. Experiments in which NK cell mitosis is prevented by colchicine show that the majority of peripheral blood NK cells are induced into the first cell cycle over a 6-day culture period in the presence of rIL 2. The addition of the irradiated lymphoblastoid B cell line, Daudi, to colchicine blocked cultures does not increase the proportion of cells entering cell cycle in response to rIL 2 alone. In limiting dilution analysis, only 1/1700 B73.1+ cells grow clonally in response to rIL 2. The frequency of clonal growth of NK cells in response to irradiated Daudi cells alone is minimal, whereas the addition of irradiated Daudi cells to rIL 2 stimulated cultures resulted in a 10-fold increase in clonal frequency compared with the cultures in rIL 2 alone. Therefore, Daudi cells may act by maintaining continuous proliferation of the NK cells originally responsive to IL 2. Unlike NK cells, only a minimal proportion of peripheral blood T cells proliferate in response to IL 2. These IL 2 responsive T cells are characterized by a lower bouyant density than the majority of peripheral blood T cells. These results indicate physiologic differences between peripheral blood resting NK and T cells in their ability to be induced to cycle. IL 2 is a growth factor for both cell types, but although the presence of the growth factor is sufficient for quiescent NK cells to be induced into cycle, T cells require antigenic or other mitogenic stimuli to respond to IL 2. The small proportion of light density IL 2 responsive T cells might represent in vivo activated T cells.     

Helper-independent CD8+ cytotoxic T lymphocytes express IL-1 receptors and require IL-1 for secretion of IL-2

The purpose of this study was to examine the role of IL-1 on the activation of CD8+/CD4- class I-restricted helper cell-independent cytolytic T cell (HITc) clones known to produce IL-2 and proliferate in vitro after Ag stimulation with a Friend retrovirus-induced leukemia (FBL). The functional role of IL-1 in Ag-specific proliferation and IL-2 secretion was assessed by stimulating the T cell clones with FBL either in the presence or absence of macrophages (M phi), rIL-1, or rIL-2. Resting cloned HITc cells, purified from residual accessory cells, failed to proliferate in response to FBL alone, but proliferated in response to FBL plus M phi, rIL-1 or rIL-2. Stimulation with FBL alone in the absence of M phi or IL-1 was sufficient for induction of IL-2R expression, and rendered cells responsive to IL-2, but M phi or IL-1 were also required to induce production of IL-2. The activity of IL-1 was further examined by measuring the binding of [125I]rIL-1 alpha, which demonstrated that resting cloned HITc cells expressed IL-1R that increased in number after activation with Ag. This expression of IL-1R and requirement for IL-1 by CD8+ HITc was surprising because previous studies examining T cell populations after mitogen stimulation have not detected IL-1R on the CD8+ population. Therefore, the role of IL-1 in the activation of CD8+ CTL that do not secrete IL-2 after activation was assessed. By contrast to HITc, CD8+ CTL required exogenous IL-2 to proliferate in vitro and did not express IL-1R. These data demonstrate that the subset of CD8+ T cells responsible for IL-2 production express IL-1R and that triggering this receptor with IL-1 after Ag stimulation results in the production of IL-2 and subsequent proliferation.     

Lipopolysaccharide stimulates the proliferation of human CD56+CD3- NK cells: a regulatory role of monocytes and IL-10

NK cells recognize and kill tumor cells and normal cells, and these play an important role in immune defense in cancer, infectious disease, and autoimmunity. NK killing is regulated by positive or negative signals derived from the interaction of surface receptors with ligands on the target cells. However, the mechanisms controlling the proliferation and maintenance of NK cells in normal human individuals are less clearly defined. In this study, using an entirely autologous system, we demonstrate that human peripheral blood CD3-CD56+, killer cell-inhibitory receptor (KIR)-expressing cells proliferate and expand in response to LPS. These responses are enhanced in the presence of anti-IL-10 receptor-blocking Abs or on the removal of CD14+ cells from the cultures. This enhancement is also reflected in substantial increases in cytolytic activity and IFN-gamma production. The negative effect of CD14+ cells may also be IL-10 mediated, IL-10 being lost from the culture supernatants of CD14-depleted PBMC and rIL-10 reversing the effect of this depletion. On the other hand, mRNA for the p35 and p40 subunits of IL-12 is still induced in CD14-depleted cultures. The expansion of CD3-CD56+ cells was also inhibited by CTLA4-Ig, indicating a role for CD80/86. B lymphocytes were not required for the expansion of CD3-CD56+ cells, whereas removal of MHC class II+ cells from CD14-depleted cultures resulted in a complete abrogation of these responses. Expansion of CD3-CD56+ cells was reconstituted in MHC class II-depleted cell cultures by adding back monocyte-derived dendritic cells. These results indicate that the responses of CD3-CD56+ NK cells to LPS may be driven by a MHC class II+ B7+ CD14- peripheral population, most likely blood dendritic cells.     

The effects of IL-4 on human natural killer cells. A potent regulator of IL-2 activation and proliferation

NK cells are directly activated by rIL-2 and subsequently undergo rIL-2-dependent proliferation in vitro. Herein, we report that rIL-4 is a potent regulator of human NK cells. Although rIL-4 had no effect on the cytotoxic activity of resting NK cells, it was capable of inhibiting in a concentration-dependent manner the rIL-2-induced cytolytic activation of NK cells against NK cell-resistant tumor cell targets. rIL-4 acted directly on NK cells and did not require accessory cells. rIL-4-induced inhibition of NK cell activation was specific for rIL-2 in that activation of NK cell cytolysis by IFN-alpha was not affected. These results represent the first direct evidence that rIL-2 and IFN-alpha activate NK cells by different pathways. rIL-4 also effectively blocked the rIL-2-dependent proliferation of NK cells. The results presented in this study clearly demonstrate that rIL-4 is a potent regulator of IL-2-dependent mechanisms of NK cell activation and proliferation and thus may play an important physiologic role in vivo.     

Generation of lymphokine-activated killer (LAK) cells from tumor-infiltrating lymphocytes

Culture of tumor-infiltrating lymphocytes (TIL) containing about 20% BMC2 tumor cells with recombinant human interleukin 2 (rIL-2) resulted in the diminish of tumor cells and the growth of lymphocytes. These IL-2-activated lymphocytes showed a strong cytotoxic activity against not only syngeneic tumor cells but also allogeneic tumor cells. Such broad-reactive killer cells, termed lymphokine-activated killer (LAK) cells, are also inducible from spleen cells by in vitro activation with IL-2. However, LAK cells generated from TIL (TIL-LAK) showed higher cytotoxic activity against BMC2 than LAK cells generated from spleen cells (S-LAK). Furthermore, it was demonstrated that TIL-LAK cells revealed marginal cytotoxic activity against normal Con A blasts and YAC-1 cells as opposed to S-LAK. Flow cytometric analysis of TIL-LAK indicated that TIL-LAK cells mainly consisted of Thy 1.2+, Ly 2+, asialo GM1+ cells. TIL-LAK cells displayed not only in vitro cytotoxicity but also in vivo anti-tumor activity. Furthermore, it was also confirmed that TIL-LAK cells could be induced in autochthonous mouse tumor systems and human gastric tumor systems.     

Large granular lymphocytes are central cells in the interleukin-2-dependent differentiation pathway of natural killer cells

Peripheral blood low-density cells were sorted, with respect to their ability to accumulate the lysosomotropic agent mepacrine (Mep), into lysosome-rich (Mep+) and lysosome-poor (Mep-) cell populations. Cells of large granular lymphocyte (LGL) morphology and phenotype were found in the Mep+ but not in the Mep- cell population. The latter cells lacked any natural killer (NK) activity. Cultures of the Mep- cells resulted in the appearance of cells showing K-562 lytic activity, LGL morphology and CD16 and/or Leu-7 positivity. This process was facilitated by the supplementation of the culture with recombinant human interleukin-2 (rIL-2). Mep+ cells retested after 7 days of culture showed a decline in the fraction of granular (LGL and Mep+) cells. This decrease was less pronounced but also seen in rIL-2-supplemented cultures. In spite of the lower number of typical LGL, Mep+ cells cultured with rIL-2 were mostly large but scarcely granular; rIL-2-activated K-562 killing (rIL-2 AK) of originally Mep+ cells was much higher than K-562 lytic activity of these cells at the beginning of the culture, and as compared to rIL-2 AK of Mep- cells. From this finding it is apparent that the most active rIL-2 AK cells originate from low-density granular (Mep+) cells (LGL) and, therefore, we propose to call them 'giant' NK cells. Furthermore, in the presence of rIL-2, LGL differentiate from agranular (Mep-) low-density cells. In view of these data, LGL appear to be resting cells on the differentiation pathway of NK cells.     

Interleukin-2 reverses T cell unresponsiveness to Plasmodium falciparum-antigen in malaria immune subjects

Peripheral blood mononuclear cells (PBMC) from a large proportion of 34 healthy adult native residents in a malaria endemic area showed null or marginal proliferative response (low-responders) to schizont-enriched Plasmodium falciparum malaria antigen (M.Ag) but good response to pokeweed mitogen. In contrast, substantial proliferative response to M.Ag was observed in 8/8 adult temporary residents with a history of one to three acute malaria episodes. Purified CD4+ T cells preferentially responded to M.Ag, however in low-responders CD4+ T cell proliferation was poor. Moreover, no inhibition of CD4+ T cell proliferation was observed when graded numbers of CD8+ T cells were added in culture. The addition of recombinant interleukin 2 (rIL-2) to M.Ag restored the proliferative response of low-responders' PBMC. This response was M.Ag-specific when CD4+ T cells grown in M.Ag plus rIL-2, but not in rIL-2 alone, were tested in secondary cultures.     

Natural killer cell immunodeficiency in patients with chronic myelogenous leukemia. IV. Interleukin-1 deficiency, gamma-interferon deficiency and the restorative effects of short-term culture in the presence of interleukin-2 on natural killer cytotoxicity, natural killer-target binding and production of natural killer cytotoxic factor

We report herein that defective natural killer (NK) cell cytotoxicity, NK cytotoxic factor (NKCF) production and NK target binding ability of patients with chronic myelogenous leukemia (CML) are functionally restorable after short-term culture (less than 1 week) with recombinant interleukin-2 (rIL-2). We have previously reported that, despite normal to increased numbers of CD16+ large granular lymphocytes, fluorescence-activated-cell-sorted NK cells from CML patients are profoundly defective in NK cell activity and are unable to lyse the CML blast-crisis-derived, NK-sensitive target K562. Since we and others have also previously shown that the defective NK cytotoxicity from CML patients is restorable after 1-4 weeks of incubation with rIL-2, we therefore deemed it important to study the kinetics of IL-2-mediated NK restoration at earlier time intervals (less than 1 week). In the present report, we have demonstrated a significant restoration of NK cell cytotoxicity in CML patients against K562 after 5 days of short-term culture with rIL-2. In addition, recovery of NKCF production and restoration of target-binding capacity to normal levels by NK cells from CML patients were also observed after short-term (less than 1 week) rIL-2 treatment. Finally, we have demonstrated in the present report that adherent cells and peripheral-blood lymphoid cells from CML patients, as compared to normal controls, are unable to produce IL-1 beta and interferon-gamma, respectively, after stimulation with phorbol myristate acetate (IL-1 beta) and phytohemagglutinin-M (interferon-gamma).