In situ vaccination against a non-immunogenic tumour using intratumoural injections of liposomal interleukin 2

Cancers appear to escape surveillance by the immune system at least in part because they fail to induce a protective immune response. Therapeutic vaccines based on specific tumour antigens and tumour cells modified ex vivo by genetic techniques are but two strategies being used to circumvent this problem. In this report, we describe a simple, yet effective alternative in which tumour-specific responses are induced by in situ administration of a well-characterized liposomal formulation of the cytokine interleukin 2 (IL-2). Using the non-immunogenic B16 melanoma model, intratumoural injections of liposomal IL-2 L(IL2), were shown to induce a long-lived immune response specific for the injected tumour. In conjunction with subsequent removal of the primary tumours by surgery, the injections increased mean survival to 57 days from a control value of 32 days and partially protected surviving mice against re-challenge with B16. L(IL2) induced an early infiltration of inflammatory cells within the tumours which was followed several days later by an influx of CD3+ T cells. The cellular influx and a coincident decrease in tumour growth were noted in both injected tumours and a second non-injected tumour on the same animal, thereby demonstrating the systemic nature of the immune response. Intratumoural injections of soluble IL-2 at the same dose failed to induce B16-specific cellular immunity or to prolong survival of the mice. Thus, liposomal formulation of the cytokine was fundamental to successful induction of immunity by this in situ vaccination regimen.     

Regulation by interleukin 2 of interleukin 2 receptors and gamma-interferon synthesis by human thymocytes: augmentation of interleukin 2 receptors by interleukin 2

The role of interleukin 2 (IL 2) on the expression of IL 2 receptors and on the synthesis of gamma-interferon (gamma-IFN) by human thymocytes was investigated. Human thymocytes isolated from specimens obtained during cardiac surgery of infants and children were induced with one or all of the following agents: IL 2, concanavalin A (Con A), and 12-O-tetradecanoylphorbol 13-acetate (TPA). The expression of IL 2 receptors and gamma-IFN titers were determined. The results indicate that thymocytes cultured in complete medium do not express receptors for IL 2, nor did IL 2 by itself induce the expression of IL 2 receptors. Con A induced the expression of IL 2 receptors by a moderate number of the thymocyte population and induced the synthesis of low amounts of gamma-IFN. Preincubation of thymocytes with TPA increased the response to Con A; both the number of thymocytes expressing receptors and the synthesis of gamma-IFN were increased. Addition of IL 2 to these cultures further augmented the expression of IL 2 receptors and gamma-IFN synthesis and resulted in the optimal expression of IL 2 receptors and maximal gamma-IFN synthesis. The expression of IL 2 receptors could be detected within 24 hr and preceded the induction of proliferation; it was therefore probably not due to the clonal expansion of a population of receptor-bearing thymocytes. Conversely, inhibition of IL 2 synthesis with dexamethasone (Dex) by thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes and of gamma-IFN synthesis. Thymocytes activated with TPA and Con A were more resistant to the inhibitory effects of Dex on the expression of IL 2 receptors than thymocytes activated with Con A alone. Maximal inhibition of the expression of IL 2 receptors and of gamma-IFN synthesis was achieved as a result of the synergistic effect of anti-Tac with Dex. Therefore, when IL 2 was prevented from binding to the receptors, and IL 2 synthesis was inhibited, the number of thymocytes expressing IL 2 receptors was sharply reduced and gamma-IFN synthesis was markedly inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)     

The released interleukin 2 receptor binds interleukin 2 efficiently

The released interleukin 2 receptor (IL 2R) molecule was characterized in order to clarify its biochemical structure and to determine its functional capacity. Enzymatic digestions demonstrated that the released IL 2R, like the cell surface IL 2R, is a complex glycoprotein, modified by the addition of both N- and O-linked carbohydrates and sialic acid residues. It has a peptide backbone that is approximately 10 Kd smaller than that of its membrane-associated counterpart. Affinity chromatography demonstrated that released IL 2R from either an HTLV-I-positive T cell line (HUT-102) or PHA-activated normal peripheral lymphocytes binds efficiently to purified recombinant IL 2. Furthermore, the interaction between the growth factor and the released receptor does not appear to require any accessory molecules. These observations suggest a potentially significant role for the released IL 2R in the regulation of IL 2-dependent lymphocyte functions.     

Actions of recombinant interleukin 1, interleukin 2, and interferon-gamma on bone resorption in vitro

Human peripheral blood cells, when cultured in vitro, release bone-resorbing factors, which have been called osteoclast-activating factors (OAF) but remain unidentified. We showed previously that a monocyte product, similar to interleukin 1 (IL 1), is a powerful stimulator of bone resorption in vitro. However, the possibility remained that other immune cell products may contribute to OAF activity. We have therefore tested three recombinant cytokines; IL 1, interleukin 2 (IL 2), and interferon-gamma (IFN-gamma) for their activity in a neonatal mouse bone resorption assay. We report here that purified recombinant murine IL 1 is a potent and powerful stimulator of bone resorption in vitro, active over a concentration range of 0.14 to 33 U/ml (1.3 X 10(-12) to 3.1 X 10(-10) M). IL 1-stimulated bone resorption was unaffected by cyclooxygenase inhibition but was inhibited by calcitonin and IFN-gamma. IL 2 had no effect on bone resorption.     

Induction of interleukin 2 responsiveness in thymocytes by synergistic action of interleukin 1 and interleukin 2

Thymocyte cultures from C3H/HeJ mice were stimulated for proliferative responses with purified preparations of interleukin 1 (IL 1) and interleukin 2 (IL 2). Synergistic responses were obtained in the absence of mitogen. In the presence of excess IL 2, the thymocyte proliferation response was strictly dependent on the amount of IL 1 in the cultures. Antibodies to IL 1 inhibited the response in a dose-dependent manner. The combination of IL 1 plus IL 2 induced the appearance of IL 2 receptors on murine thymocytes as detected with a monoclonal antibody directed against the IL 2 receptor. Neither IL 1 nor IL 2 alone had this effect. The thymic subpopulation found to become IL 2 responsive upon IL 1 stimulus was the peanut agglutinin-negative (PNA-) medullary fraction.     

Expression of interleukin 2 and the interleukin 2 receptor in aging rats

Lymphocytes of aged animals exhibit a marked decrease in proliferative capacity in response to mitogen stimulation when compared to those of younger animals. In humans and mice the decreased proliferation is due at least in part (i) to the inability of lymphocytes to synthesize sufficient interleukin 2 (IL-2) and (ii) to decreased expression of IL-2 receptors (IL-2R) on the surface of aged lymphocytes. We compared proliferative abilities, IL-2 production, and IL-2R expression in splenocyte cultures of 4- to 5- and 22- to 24-month-old Fischer 344 rats stimulated with either concanavalin A (Con A) or A23187 and phorbol myristate acetate (PMA). Proliferation was significantly decreased in aged lymphocytes (30-50%) with both treatment protocols. However, unlike mice and humans we observed no difference in IL-2 activity, IL-2 mRNA levels, or IL-2R cell surface expression of lymphocytes from young and aged rats stimulated with either Con A or A23187 and PMA. These results indicate that factors other than decreased expression of IL-2 and IL-2R are responsible for the diminished proliferative capacity of aged rat lymphocytes following mitogen stimulation.     

Production of interleukin 2, interleukin 3, and interferon by mouse T lymphocyte clones of Lyt-2+ and -2- phenotype

T lymphocyte clones were derived by stimulation of positively selected Lyt-2+ and Lyt-2- lymphocytes with Con A in an Interleukin 2 (IL 2)-enriched medium under conditions of limiting dilution. Forty clones were expanded and tested, after activation by Con A, for the production of IL 2, IL 3, and interferon (IFN). Thirteen Lyt-2- clones were all co-producers of IL 2 and IL 3, and 10/13 were also producers of IFN. Twenty-seven Lyt-2+ clones were much more heterogeneous, 13 being IL 2 and IL 3 nonproducers, whereas 14 produced variable and poorly correlated amounts of IL 2 and IL 3. Three Lyt-2+ clones were observed to produce IL 2 or IL 3 alone. The majority of the Lyt-2- (10/13) and of the Lyt 2+ (21/27) clones were also producers of IFN. Exogenous IL 2 added during the activation of the Lyt-2+ by Con A did not enhance IL 3 production, whereas it did enhance IFN production by some but not all Lyt-2+ clones. Thus, among the T lymphocytes of the Lyt-2+ and -2- phenotypes there are cells capable of releasing IL 2, IL 3, and IFN. This supports the concept that these phenotypes are associated with antigen recognition rather than with cell functions, but it is apparent that the functional capacities of individual T cells, if accurately represented by their clonal progeny, are far from uniform.     

Accessory cell-derived helper signals in human T-cell activation with phytohemagglutinin: induction of interleukin 2-responsiveness by interleukin 6, and production of interleukin 2 by interleukin 1 [corrected

Interleukins (IL-) 1 and 6 have been shown to represent accessory signals for T-cell activation. In the present study, we further examined the effects of both cytokines on accessory cell-depleted human T cells stimulated with phytohemagglutinin (PHA). The addition of IL-6 to the cultures resulted in T-cell proliferation; however, IL-1 was unable to support PHA-induced T-cell growth. The addition of IL-1 consistently induced a low level of IL-2 production and strongly enhanced T-cell proliferation in the presence of IL-6. Thus, the effect of IL-1 on T-cell growth becomes apparent only in the presence of IL-6. Blocking the IL-2-receptor (IL-2R) with the monoclonal antibodies anti-Tac and MikBêta 1 (directed to the alpha and bêta chains of the IL-2R, respectively) had no effect on PHA/IL-6-supported proliferation, but completely eliminated the growth-enhancing effect of IL-1. On the other hand, a neutralizing anti-IL-4-antiserum did not affect PHA/IL-6- or PHA/IL-6/IL-1-induced proliferation. Further experiments showed that IL-6 enhances T-cell responsiveness to IL-2, as evidenced by enhanced IL-2-induced proliferation. However, we could not find an effect of IL-6 on the expression of IL-2R as measured by staining with anti-Tac and with MikBêta 1 or by binding of (125I)-IL-2 to T cells. It can be concluded from these studies that IL-1 and IL-6 have different helper effects on PHA-induced T-cell activation. In the presence of PHA, IL-6 induces limited IL-2/IL-4-independent growth, and more importantly it renders T cells responsive to IL-2. IL-1 provides a signal leading to IL-2 production. The combination of IL-1 and IL-6 represents a synergistic helper signal, leading to an IL-2-dependent pathway of proliferation.     

Role of protein kinase C in interleukin 1, anti-T3, and mitogenic lectin-induced interleukin 2 secretion

Activation of T-lymphocytes by antigen, mitogenic lectins, or antibodies against the T-cell receptor complex, particularly in the presence of IL1, induces the secretion of the T-cell growth factor IL2. IL2 then has a major role in regulating the duration and magnitude of the immune response. Interaction of antigen, antibodies against the T-cell receptor complex, or mitogenic lectins with T-lymphocytes also induces hydrolysis of membrane phospholipids, leading to the production of diacylglycerol, an activator of the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Phorbol esters, potent activators of PKC, augment secretion of the T-cell growth factor, interleukin 2 (IL2). Activation of PKC may therefore serve as an important early event in the production and secretion of IL2. We have determined whether IL2 secretion can be induced in the murine cell T-lymphocyte line LBRM 331A5, where PKC is inhibited by staurosporine or sphingosine or in cells where PKC is depleted by prolonged incubation with high concentrations of phorbol esters. In cells in which PKC was either inhibited or depleted, antibodies against the T3 portion of the T-cell receptor complex and the mitogenic lectin phytohemagglutinin (PHA) still triggered IL2 secretion. In addition, the monokine IL1 augmented this IL2 secretion irrespective of whether PKC was inhibited or depleted. These data indicate that activation of PKC is not an obligatory step for IL2 secretion in LBRM 331A5 murine T-lymphocytes.     

Monocyte interleukin 2 receptor gene expression and interleukin 2 augmentation of microbicidal activity

Activation of human peripheral blood monocytes results in the expression of interleukin 2 (IL 2) receptors, which are absent on resting monocytes. In a population of purified monocytes, the appearance of IL 2 receptors occurs on the majority of cells in association with increased levels of HLA-DR. Lipopolysaccharide (LPS) induces maximum numbers of IL 2 receptors within 12 hr, whereas IFN-gamma requires 48 hr. We used cDNA encoding for the human IL 2 receptor to evaluate IL 2 receptor gene expression in resting and activated monocytes. Within 4 hr after LPS stimulation, IL 2 receptor mRNA species of 3500 and 1500 bases appear, reaching peak levels between 8 and 12 hr and declining thereafter. The LPS-activated monocyte IL 2 receptor protein is expressed on the cell surface within a few hours after the detection of IL 2 receptor mRNA. The addition of IL 2 to IL 2 receptor-positive monocytes augments their generation of reactive oxygen intermediates and their cytotoxic activity. Thus monocytes when activated undergo a series of morphologic, phenotypic, and functional changes, including the expression of IL 2 receptors, which may provide an important immunoregulatory pathway.     

Synergism of interleukin 7 with the thymocyte growth factors interleukin 2, interleukin 6, and tumor necrosis factor alpha in the induction of thymocyte proliferation

The role of previously defined thymocyte (Thm) growth factors in interleukin (IL)-7-induced Thm growth has not been fully elucidated. Therefore, experiments were designed to examine the capacity of IL-7 to: (i) directly induce Thm proliferation in the absence of experimental and known physiologic costimulators of Thm mitogenesis, and (ii) synergize with other Thm growth factors in supporting Thm proliferation. The data indicate that IL-7 is directly mitogenic for Thm; that is, IL-7 induces Thm proliferation in the absence of experimental comitogens such as concanavalin A, phytohemagglutinin, and phorbol myristate acetate and in the presence of neutralizing antibodies to murine IL-1 alpha, IL-1 beta, IL-2, IL-2 receptor (IL-2R)(p55), IL-2R(p70), IL-4, IL-6, and tumor necrosis factor (TNF). We also tested previously described Thm growth factors, i.e., IL-2, IL-4, IL-6, and TNF-alpha, for the capacity to synergize with IL-7 in Thm growth. Our results indicate that IL-2, IL-6, and TNF-alpha, but not IL-4, synergize with IL-7 in supporting Thm proliferation. These data suggest that IL-7 functions alone and in a synergistic fashion with other cytokines to regulate Thm growth.     

Modulation of interleukin 2 internalization and interleukin 2-dependent cell growth by antireceptor antibodies

The growth factor interleukin 2 (IL2) binds to and is internalized together with high-affinity surface receptors present on lymphoid cells. This endocytosis thus results in down-regulation of the receptors. However, it is not known if the internalization is relevant to the induction of cell growth. In the present study a rat monoclonal antibody to the P55 chain of the IL2 receptor was used to examine the role of receptor internalization in the IL2-dependent autocrine human tumor T cell line IARC 301. When given alone, this antibody did not inhibit IL2 binding, internalization, or IL2-dependent cell proliferation. However, crosslinking by anti-rat immunoglobulins, which did not affect binding of the growth factor, inhibited both IL2 internalization and cell proliferation. Besides offering a novel means for the specific inhibition of the uptake of IL2 bound to IL2 high-affinity receptors, the results are compatible with the association of this receptor-ligand uptake to the growth stimulation by IL2.     

Molecular characterization of interleukin 2

The isolation of specific T cell growth factors termed interleukin 2 (IL 2) is changing the approach to understanding T cell function. This class of growth factors has allowed te establishment of a number of human and murine T cell lines. We summarize the biochemical properties of human and murine IL 2. Studies have been initiated to isolate mRNA encoding for IL 2. Such RNA can be translated in rabbit reticulocyte lysates yielding IL. 2. This RNA may be useful for the development of probes to isolate lymphokine genes.     

Modulation of the expression of interleukin 2 receptors of human thymocytes by recombinant interleukin 2

The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-gamma synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-gamma, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-gamma was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with anti-Tac remained low throughout the first week of culture and interferon-gamma synthesis was not observed. Nonetheless, interleukin 2-induced proliferation was inhibited by anti-Tac on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-gamma synthesis, and for the growth of thymocytes in vitro.     

Expression of interleukin 2 receptors on interleukin 3-dependent cell lines

Several mouse IL 3-dependent cell lines, IC2, LT4, FDC-P2, and PB-3C, derived from spleen or bone marrow cells were shown to express low affinity receptors for IL 2 (Kd; 0.5 to 8 X 10(-8) M). High affinity receptors for IL 2 were not detected on the IL 3-dependent cells within the experimental limitation of this study. The clones did not respond to IL 2 at all at the concentration as high as 25 micrograms/ml. The number of the receptors expressed on those clones was estimated to be 0.2 to 2 X 10(5)/cell, which is comparable with the number of those on IL 2-dependent T cell clones. Expression of IL 2 receptor was confirmed in mRNA levels for both IC2 and LT4 cells. A relatively low level expression of one (4.5 Kb) of four IL 2 receptor mRNA species was observed with those IL 3-dependent clones compared with IL 2-dependent T cells. It seems that these low affinity receptors may be expressed on IL 3-dependent cells that undergo differentiation or maturation in mast cell and some myeloid cell lineages.     

Role of interleukin 2, interleukin 4 and interleukin 5 in the T helper cell-driven B cell polyclonal differentiation in the elderly.

There is evidence for an impaired T cell-mediated B cell response during senescence. In thirty aged donors, pokeweed mitogen (PWM)-driven immunoglobulin (Ig) synthesis by B cells co-cultured with autologous enriched CD4+ lymphocytes and low amounts of monocytes, was evaluated. Under such experimental conditions, elderly cultures displayed a reduced IgG and/or IgM production when compared with the younger counterpart. Moreover, interleukin (IL)-2 and/or IL-5 addition to cultures led to an enhancement of Ig release. In contrast, IL-4 supplementation failed to positively modulate B cell differentiation. At the same time, aged cells cultured in the presence of IL-2 + IL-5 exhibited an increased Ig synthesis, while the addition of IL-2 + IL-4 or IL-4 + IL-5 mixtures did not induce any significant effect in comparison with homologous untreated samples. The results suggest a critical role for IL-2, IL-4 and IL-5 in the modulation of T helper cell-driven B cell polyclonal responsiveness in the elderly.     

The murine interleukin 2 receptor. Irreversible cross-linking of radiolabeled interleukin 2 to high affinity interleukin 2 receptors reveals a noncovalently associated subunit

The murine interleukin 2 (IL-2) receptor is a 55- to 60-kDa glycoprotein (p58) that binds IL-2 at a high and low affinity. In this investigation, we have identified sublines of EL4 that vary in their capacity to express high affinity IL-2 receptors after transfection of the IL-2 receptor cDNA. These and other cell populations were used to determine whether unique membrane molecules were specifically associated with the high affinity IL-2 receptor. Irreversible chemical cross-linking of [125I]IL-2 to only high affinity IL-2 receptors resulted in detection of IL-2 cross-linked to p58 as a 70- to 75-kDa band and other complexes of 90 to 95 kDa, 115 kDa, 150 kDa, 170 to 190 kDa, and 245 kDa. Antibodies specific for p58 resulted in precipitation of each of these complexes. However, disruption of noncovalent interactions prior to immunoprecipitation resulted in an inability to detect the material at 90 to 95 kDa. Therefore, we conclude that this complex most likely represented IL-2 cross-linked to a 75- to 80-kDa subunit that was noncovalently associated with p58. The other complexes greater than 150 kDa may represent these subunits cross-linked to each other. The detection of all the cross-linked complexes larger than 75 kDa appeared to be directly related to formation of high affinity IL-2 receptors because IL-2 was cross-linked only to p58 for three cell lines that exclusively expressed low affinity IL-2 receptors. Thus, high affinity murine IL-2 receptors are comprised of at least one alpha (p58)- and beta (p75)-subunit. Our data also raise the possibility of a more complex subunit structure.     

Experimental visceral leishmaniasis: production of interleukin 2 and interferon-gamma, tissue immune reaction, and response to treatment with interleukin 2 and interferon-gamma

During the first 2 to 4 weeks of progressive visceral infection with the intracellular protozoan, Leishmania donovani, spleen cells from BALB/c mice failed in response to leishmanial antigen to produce either of the activating T cell-derived lymphokines, interleukin 2 (IL 2) or gamma-interferon (IFN-gamma). Four weeks after infection, however, antigen-induced IL 2 and IFN-gamma secretion emerged and coincided with the onset of control over parasite replication and the subsequent killing of greater than 80% of intrahepatic L. donovani. The development of this immunosecretory activity correlated with the hepatic tissue response at the site of parasitized Kupffer cells. This response progressed from Kupffer cell fusion (week 1) to fusion plus a mononuclear cell infiltrate (week 2) to well-organized granuloma formation (weeks 4 to 8). In contrast, T cell-deficient nude BALB/c mice exerted no control over L. donovani, their spleen cells failed to generate antigen-induced IFN-gamma, and at 4 weeks, their livers were devoid of any tissue reaction. Since spleen cells from 2-week infected normal mice did not produce antigen-stimulated IL 2 or IFN-gamma, these mice were treated with recombinant (r) lymphokines. Various protocols using both high and low dose human rIL 2 had no antileishmanial effect. Hepatic parasite replication was completely halted, however, by macrophage-activating doses of murine rIFN-gamma. These results reemphasize that an intact T cell-dependent response is required for successful defense against L. donovani, indicate that this immune response can be measured at both the cellular (secretory) and tissue levels, and confirm that IFN-gamma can exert an antileishmanial effect in vivo.     

Regulation of interleukin 2 receptor expression on a human cytotoxic T lymphocyte clone, synergism between alloantigenic stimulation and interleukin 2

A human T cell clone (termed 40.2.6) established from a rejected human kidney allograft has been studied for its ability to express membrane IL 2 receptors in response to antigen (irradiated cells from the graft's donor) and recombinant IL 2 (rec-IL 2). On antigenic stimulation, the 40.2.6 clone produced low levels (0.15 U/ml) of IL 2 (peak at 24 hr) and incorporated (3H)thymidine (peak at 48 hr). This incorporation was strongly enhanced on addition of rec-IL 2 and was inhibited by the 33B31 antibody, an anti-human IL 2 receptor monoclonal antibody (Mab). The 125I-labeled 33B31 Mab has been used to quantify the density of IL 2 receptors on 40.2.6 cells. Cells not re-exposed to antigen or rec-IL 2 had a level of 33B31-binding sites which declined rapidly (10% of starting value after 2 days). This level remained much more stable when rec-IL 2 (1 U/ml) was present in the medium (80% at day 2). Antigen induced a three- to eightfold increase in the level of 33B31-binding sites which peaked at 24 hr and then declined. When a similar antigenic stimulation was performed in the presence of rec-IL 2 (1 U/ml), the level of 33B31-binding sites peaked at a higher value (eight- to 20-fold increase at day 2), and its subsequent decline was slower. These potentiating effects of rec-IL 2 were dose-dependent and occurred at low concentrations corresponding to the saturation by rec-IL 2 of high affinity IL 2 receptor sites. Finally, high affinity IL 2 receptors, as measured by the binding of 35S-labeled rec-IL 2, were found to be similarly up-regulated by antigen and rec-IL 2. Together, our results obtained on a monoclonal human T cell population with highly purified rec-IL 2 demonstrate that rec-IL 2 and antigen act in synergy to induce the expression of both high and low affinity membrane IL 2 receptors.