As early as 10-15 min after the start of a 30 min interleukin-2 (IL-2) infusion, a rapid, virtually complete disappearance of all natural killer (NK) lymphocyte subpopulations (including both CD3- CD56+ and CD3+ CD56+ cells with either alpha/beta or gamma/delta T-cell receptor) was observed from peripheral blood. In contrast, the number of T lymphocytes (CD3+ CD56-) was unmodified for at least 2 h after IL-2 injection. The IL-2-induced, rapid disappearance from peripheral blood of NK and NK-like lymphocytes may be related to their massive adherence to the activated endothelium. In this regard, IL-2 infusion caused a very rapid rise of tumour necrosis factor-alpha (TNF-alpha) plasma concentration, whereas other cytokines, such as interferon-gamma (IFN-gamma), were induced only at later times. In vitro experiments indicated that IL-2, either alone or better combined with TNF-alpha, exerts a rapid and selective stimulatory effect on NK adhesion to endothelial cells. On the basis of these findings, we suggest that the activation of NK lymphocytes induced by IL-2, alone or combined with TNF-alpha, plays a key role in mediating the massive and selective adherence of NK and NK-like cells following IL-2 bolus infusion.

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.

Adoptive cancer immunotherapy consists of in vitro activation of natural killer (NK) cells and non-major histocompatibility complex (MHC)-restricted cytotoxic T cells by recombinant interleukin-2 (IL-2) and subsequent administration of the lymphokine-activated killer (LAK) cells to a tumor-bearing host [1]. LAK cells are routinely generated in short-term cultures of peripheral blood mononuclear cells (PBMC) in the presence of IL-2 [1]. They are distinguished from NK cells and MHC-restriced cytotoxic T cells by their ability to lyse not only NK-sensitive target cells, but also NK-resistant lines and fresh tumor cells in a non-MHC-restricted fashion [1]. Due to their ability to lyse fresh tumor cells, LAK effectors have been used in adoptive immunotherapy of advanced cancer; indeed, Rosenberg et al. have recently reported that infusion of LAK cells with high-dose rIL-2 induced a marked or complete reduction of the tumor mass in a minority of patients with otherwise refractory metastatic malignancies (particularly melanomas and renal carcinomas) [2–4]. These findings were recently extended using in vitro expanded tumor infiltrating lymphocytes (TIL) instead of IL-2-stimulated PBMC [5].

Four patients out of twenty with renal cancer and melanoma undergoing cancer immunotherapy with interleukin 2 (IL-2) and interferon alpha-2 (IFN-alpha-2) had laboratory evidence of hypothyroidism starting at cycle three to six, with a decline in serum thyroxine below normal and, in three cases, a rise in serum thyrotropin and thyroglobulin. One hypothyroid patient had elevated serum antimicrosomal antibody titres before the start of treatment and two others responded similarly during therapy. Three of the sixteen euthyroid patients also developed elevated titres of this antibody. Partial or complete remission was observed in seven of the patients-three of the four with hypothyroidism showed tumour regression. Thus IL-2 and IFN-alpha-2 can cause hypothyroidism, presumably via induction or exacerbation of autoimmune thyroid reactions. The occurrence of hypothyroidism may be mediated by high-dose IL-2 (rather than by LAK cell therapy as previously suggested) and potentiated by IFN-alpha-2.

Peripheral blood mononuclear cells (PBM) pulsed with lectin (PHA or Con A for 0.25-3 hr) show a low expression of interleukin-2 and transferrin receptors (IL-2Rs, TfRs) and a mild decline of intracellular ferritin level, compared to control cultures grown in continuous presence of mitogen. Interestingly, lectin-pulsed PBM do not release detectable amounts of IL-2 in the medium. Furthermore, expression of TfRs in these lymphocytes is not inhibited by addition of excess anti-IL-2 neutralizing monoclonal antibody, but is significantly inhibited by treatment with iron salts. These observations suggest that mitogen triggers an IL-2-independent expression of TfRs, at least in part via a decrease of intracellular iron level. Addition of either recombinant IL-2 (rIL-2) or an iron chelator (picolinic acid) to lectin-pulsed PBM induces both a marked enhancement of TfR synthesis and a sharp decline of intracellular ferritin level, which are comparable to the corresponding pattern observed in control cultures. Conversely, addition of iron salts fully inhibits the increase of TfR expression induced by rIL-2. These observations strongly suggest that the enhanced TfR synthesis elicited by rIL-2 is mediated by depletion of a regulatory intracellular iron pool. In line with these studies, greater than 99% purified T lymphocytes stimulated by lectin show a low expression of TfRs, which is markedly enhanced by addition of exogenous rIL-2. Altogether, we postulate that: (i) in resting T lymphocytes the gene encoding TfR is apparently in a 'closed' configuration; (ii) even in the absence of IL-2 activity, a mitogen pulse is sufficient to initiate the expression of TfRs, at least in part via a decline of intracellular iron level; and (iii) TfR synthesis is then largely amplified by IL-2, again via a decrease of the size of a regulatory intracellular iron pool.

Annals of the New York Academy of SciencesVolume 511, Issue 1 p. 131-137 Expression of Transferrin Receptors: Differential Regulatory Mechanisms in Monocytes-macrophages versus Other Hemopoietic Cellsa U. TESTA, U. TESTA Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorA. CAMAGNA, A. CAMAGNA II Clinica MedicaSearch for more papers by this authorG. GIANNELLA, G. GIANNELLA Chair of Hematology, Blood Transfusion CenterSearch for more papers by this authorE. PELOSI-TESTA, E. PELOSI-TESTA Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorM. PETRINI, M. PETRINI Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorP. SAMOGGIA, P. SAMOGGIA Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorE. MONTESORO, E. MONTESORO Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorL. BOTTERO, L. BOTTERO Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorN. M. SPOSI, N. M. SPOSI Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorG. SALVO, G. SALVO Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorF. MAVILIO, F. MAVILIO Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorG. ISACCHI, G. ISACCHI Chair of Hematology, Blood Transfusion CenterSearch for more papers by this authorG. MASTROBERARDINO, G. MASTROBERARDINO Istituto Patologia Medica (VI), University “La Sapienza”, 00161 Rome, ItalySearch for more papers by this authorC. PESCHLE, C. PESCHLE Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this author U. TESTA, U. TESTA Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorA. CAMAGNA, A. CAMAGNA II Clinica MedicaSearch for more papers by this authorG. GIANNELLA, G. GIANNELLA Chair of Hematology, Blood Transfusion CenterSearch for more papers by this authorE. PELOSI-TESTA, E. PELOSI-TESTA Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorM. PETRINI, M. PETRINI Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorP. SAMOGGIA, P. SAMOGGIA Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorE. MONTESORO, E. MONTESORO Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorL. BOTTERO, L. BOTTERO Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorN. M. SPOSI, N. M. SPOSI Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorG. SALVO, G. SALVO Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorF. MAVILIO, F. MAVILIO Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this authorG. ISACCHI, G. ISACCHI Chair of Hematology, Blood Transfusion CenterSearch for more papers by this authorG. MASTROBERARDINO, G. MASTROBERARDINO Istituto Patologia Medica (VI), University “La Sapienza”, 00161 Rome, ItalySearch for more papers by this authorC. PESCHLE, C. PESCHLE Department of Hematology, Istituto Superiore di Sanita, 00161 Rome, ItalySearch for more papers by this author First published: December 1987 https://doi.org/10.1111/j.1749-6632.1987.tb36243.xCitations: 3 a Supported in part by the Associazione Italiana per la Ricerca contro il Cancro (AIRC) and by Grant 85.02560.44 from the National Research Council (Progetto Finalizzato “Oncologia”), Rome. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume511, Issue1Normal and Neoplastic Blood Cells: From Genes to TherapyDecember 1987Pages 131-137 RelatedInformation

It has been demonstrated that young RBCs (reticulocytes and early mature erythrocytes) possess more insulin receptors than old RBCs (late mature erythrocytes) but it is not yet known whether insulin receptors on young and old RBCs are regulated similarly. In the present investigation insulin receptors on young and old RBCs have, therefore, been studied in five normal male subjects before and after 2 days dexamethasone ingestion (0.5 mg tablet every 6 h) and, in the same subjects, before and 5 h after ingestion of 75 g glucose. The results obtained clearly demonstrate that dexamethasone increases insulin receptor concentration while glucose ingestion increases both insulin receptor affinity and concentration on young RBCs. By contrast, neither stimuli modify insulin receptors on old RBCs. Studies on RBCs are usually performed on the whole RBC population not taking into account this differential responsiveness of receptors on young versus old RBCs; consequently, this phenomenon might be responsible of the fact that some data reported on RBCs are not in agreement with those reported on monocytes or adipocytes and it should be taken into consideration when using RBCs to evaluate insulin receptor regulation.

Resting human T-lymphocytes show an elevated intracellular concentration of ferritin, whereas transferrin receptors are not detectable. Stimulation by phytohemagglutinin markedly lowers their ferritin content, while inducing the synthesis of transferrin receptors. Addition of iron salts (ferric ammonium citrate) in activated T-lymphocyte cultures causes a marked enhancement of both [3H]uridine and [3H]thymidine incorporation. Nevertheless, it also induces a concentration-dependent decrease in transferrin receptor synthesis, associated with a marked rise of ferritin production. Hemin treatment exerts the same effects. Addition of picolinic acid in phytohemagglutinin-stimulated cultures causes a decrease of [3H]thymidine incorporation, whereas transferrin expression is markedly enhanced. The action of iron salts and chelators is specific for transferrin receptors, since the expression of other membrane markers of activated human T-lymphocytes (interleukin-2 receptor, insulin receptor, and HLA-DR antigen) is not modified by treatment with iron or picolinic acid. These observations suggest that expression of transferrin receptors in activated T-lymphocytes is specifically modulated by their intracellular iron level, rather than their proliferative rate. Addition of picolinic acid to resting T-lymphocytes in the absence of mitogen induces a marked decrease of their ferritin content, but not the appearance of transferrin receptors. On the basis of these results, we suggest a three-step model: (a) in resting T-lymphocytes, the gene for transferrin receptor is apparently "closed," in that it is not expressed under both normal conditions and following iron deprivation. (b) After mitogen stimulus, T-lymphocytes are reprogrammed into cell cycle progression, which necessarily entails synthesis of transferrin receptors (c) Expression of these receptors is modulated by the intracellular iron level, rather than the rate of proliferation per se.

The present study was undertaken in an attempt to elucidate the mechanism(s) underlying transferrin (TRF) receptor expression in human erythroleukemic (K562 and HEL) lines during the exponential and the plateau phase of growth. TRF receptor synthesis is enhanced when stationary cells are subcultured at low density in fresh medium. This rise occurs in either the presence or the absence of serum, which is associated with cell proliferation or quiescence, respectively. In the presence of serum, it is not inhibited by the addition of hydroxyurea (i.e., an agent blocking DNA synthesis). Thus, the receptor synthesis is enhanced not only in subcultures of actively proliferating cells (in the presence of serum), but also in subcultures of quiescent elements (in the absence of serum or upon the addition of serum plus hydroxyurea). Conversely, the ferritin content is markedly decreased when stationary cells are subcultured at low density, in either the presence or the absence of serum. These results suggest that stationary cells subcultured in fresh medium undergo a depletion of their intracellular iron pool, which in turn may represent the stimulus triggering TRF receptor synthesis. This hypothesis is supported by two observations: both the depletion of this pool and the rise in TRF receptor synthesis are more marked in the absence than in the presence of serum; addition of excess exogenous iron fully inhibits the rise of TRF receptor synthesis in cells subcultured with fresh medium and serum.