[Show abstract][Hide abstract] ABSTRACT: Late-phase human T-cell lymphotropic virus I-associated adult T-cell leukemia cells express IL-2 receptors (IL-2R) but no longer produce IL-2. We have reported that the IL-2-independent adult T-cell leukemia line HuT-102 secretes a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and lymphokine-activated killer cell activity. Stimulation of proliferation of the cytokine-dependent human T-cell line Kit-225 mediated by HuT-102-conditioned medium or by 3200-fold-purified IL-T was not blocked by the addition of antibodies against IL-2 or IL-2R alpha subunit. However, IL-T-mediated stimulation of this human T-cell line was inhibited by addition of Mik-beta 1, an antibody that binds specifically to IL-2R beta subunit. In addition, the activation of large granular lymphocytes to lymphokine-activated killer cells mediated by IL-T-containing conditioned medium was not blocked by antibodies directed toward IL-2 or IL-2 alpha but was inhibited by an antibody to IL-2R beta, suggesting the requirement of this receptor subunit for IL-T action. This conclusion was confirmed using an IL-3-dependent murine myeloid precursor cell line, 32D, that expresses IL-2R alpha and IL-2R gamma, but not IL-2R beta. Neither IL-2 nor IL-T stimulated 32D cell proliferation. However, after transfection with the gene encoding human IL-2R beta, 32D beta cells proliferated on addition of either cytokine. The IL-T-mediated stimulation of 32D beta proliferation was inhibited by an anti-IL-2R beta antibody but not by an anti-IL-2 antibody. Thus, the IL-T-mediated stimulation of T-cell and lymphokine-activated killer cell activation requires the expression of the IL-2R beta subunit.
Proceedings of the National Academy of Sciences 06/1994; 91(11):4940-4. DOI:10.1073/pnas.91.11.4940 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In early phases of human T-cell lymphotrophic virus I-induced adult T-cell leukemia (ATL), the malignant cell proliferation is associated with an autocrine process involving coordinate expression of interleukin (IL) 2 and its receptor. However, during late-phase ATL, leukemic cells no longer produce IL-2 yet continue to express high-affinity IL-2 receptors. During studies to define pathogenic mechanisms that underlie this IL-2-independent proliferation, we demonstrated that the ATL cell line HuT-102 secretes a lymphokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Conditioned medium from HuT-102, when added to the IL-2-dependent CTLL-2 line, yielded a stimulation index of 230. Since CTLL-2 was purported to be IL-2-specific, we performed a number of studies to exclude IL-2 production by HuT-102. Stimulation of CTLL-2 cells by HuT-102-conditioned medium was not meaningfully inhibited by addition of an antiserum to IL-2. Furthermore, uninduced HuT-102 cells did not express mRNA encoding IL-2 as assessed by Northern blot analysis. No biological activity on CTLL-2 cells was mediated by purified IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, or granulocyte/macrophage colony-stimulating factor, thus differentiating these factors from IL-T. Based on preliminary biochemical data, IL-T is a protein with a pI value of 4.5 and a molecular mass in SDS gels of 14 kDa. In addition to its action on CTLL-2 cells, 3200-fold-purified IL-T stimulated proliferation of the human cytokine-dependent T-cell line Kit-225. Furthermore, addition of IL-T enhanced cytotoxic activity of large granular lymphocytes (i.e., induced lymphokine-activated killer cells). Thus, IL-T is a lymphokine that plays a role in T-cell proliferation and induction of lymphokine-activated killer cells. Furthermore, IL-T may contribute to IL-2-independent proliferation of select ATL cells and lines.
Proceedings of the National Academy of Sciences 06/1994; 91(11):4935-9. DOI:10.1073/pnas.91.11.4935 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interleukin 2 (IL-2) receptor (IL-2R) is a multisubunit receptor that includes three major IL-2 binding subunits, the IL-2R alpha, beta, and gamma chains. We have detected and analyzed cooperative interactions between the IL-2R alpha and beta chains (IL-2R alpha and IL-2R beta, respectively) in COS cells transfected with cDNAs encoding the IL-2R alpha, the IL-2R beta, or both cDNAs. We demonstrated that IL-2 F42A, an analog that fails to bind to the isolated IL-2R alpha subunit and would be predicted by the hierarchical affinity-conversion model to have impaired binding to cells expressing both chains, instead readily binds to the IL-2R alpha/beta heterodimer in COS cells. Furthermore, this binding is abolished by the antibody HIEI that separates the two IL-2R subunits. The monoclonal antibodies anti-Tac and Mik-beta 1 directed at the IL-2-binding sites on IL-2R alpha and IL-2R beta, respectively, block ligand binding to the heterodimer. This binding pattern is inconsistent with the strict hierarchical affinity-conversion model that mandates an initial binding of IL-2 to IL-2R alpha followed by binding of the IL-2/IL-2R alpha complex to IL-2R beta. Instead, our results support an alternative model of preformed complexes of IL-2R beta with other IL-2R subunits. In this alternative model, IL-2R alpha and -beta exist in part as preformed complexes in which the affinity of IL-2R beta for IL-2 is altered by the proximity of IL-2R alpha, through mechanisms that do not require the prior binding of IL-2 to IL-2R alpha.
Proceedings of the National Academy of Sciences 05/1994; 91(8):3344-7. DOI:10.1073/pnas.91.8.3344 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotrophic virus-I (HTLV-I). It is an aggressive leukemia with an overall mortality rate of 50% within 5 months; no conventional chemotherapy regimen appears successful in inducing long-term disease-free survival in ATL patients. However, ATL cells constitutively express high-affinity interleukin-2 receptors (IL-2Rs) identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference in receptor expression, we administered anti-Tac intravenously (IV) to 19 patients with ATL. In general the patients did not suffer untoward reactions, and in 18 of 19 cases did not have a reduction in normal formed elements of the blood. Seven patients developed remissions that were mixed (1 patient), partial (4 patients), or complete (2 patients), with partial and complete remissions lasting from 9 weeks to more than 3 years as assessed by routine hematologic tests, immunofluorescence analysis, and molecular genetic analysis of T-cell receptor gene rearrangements and of HTLV-I proviral integration. Furthermore, remission was associated with a return to normal serum calcium levels and an improvement of liver function tests. Remission was also associated in some cases with an amelioration of the profound immunodeficiency state that characterizes ATL. Thus the use of a monoclonal antibody that blocks the interaction of IL-2 with its receptor expressed on ATL cells provides a rational approach for treatment of this aggressive malignancy.
[Show abstract][Hide abstract] ABSTRACT: Mik beta 1 is a mouse mAb directed at the beta-subunit of the human IL-2R (Tac) that inhibits IL-2 binding and inhibits IL-2 induction of large granular lymphocytes (LGL). Mik beta 1 alone does not inhibit IL-2-induced T-cell proliferation, but acts synergistically with anti-Tac to inhibit IL-2-induced proliferation of activated T cells. To evaluate these effects for possible therapy in humans, we constructed two humanized Mik beta 1 antibodies by combining the complementarity-determining regions of the murine antibody with human framework and constant regions. Compared with murine Mik beta 1, the two humanized Mik beta 1 antibodies, which differ in their degree of humanization, had similar affinities for IL-2R beta. The murine Mik beta 1 and one of the humanized Mik beta 1 antibodies were equivalent in competing for IL-2 binding to IL-2R beta and inhibiting IL-2 induction of LGL cytotoxicity. The activity of the second humanized antibody was significantly reduced. The three Mik beta 1 antibodies act synergistically to varying degrees with humanized anti-Tac to prevent IL-2-induced proliferation of activated T cells. This capacity to synergize paralleled their abilities to inhibit IL-2 binding. In addition, both humanized antibodies directed antibody-dependent cell-mediated cytotoxicity. We hope that humanized Mik beta 1 in combination with humanized anti-Tac will provide a new immunosuppressive therapy for the treatment of autoimmune diseases, graft-versus-host disease, and prevention of allograft rejection.
The Journal of Immunology 08/1993; 151(2):1075-85. · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin 2 (IL-2)-mediated signaling through its high-affinity receptor involves a complex interrelationship between IL-2 and two IL-2-binding chains, IL-2R alpha and beta chains. Previously with the reagents available it was difficult to define functional interactions between these two IL-2R subunits involved in IL-2 binding and signal transduction. To extend our understanding of the interplay between the two binding subunits we have done studies with the monoclonal antibody HIEI, which interferes with interaction of IL-2R alpha and beta chains (IL-2R alpha and IL-2R beta, respectively). Furthermore, we used two forms of IL-2, recombinant native IL-2 and F42A, an IL-2 analog (Phe-42----Ala substitution) that binds only to IL-2R beta. Analog F42A manifested 75-100% of the bioactivity of wild-type IL-2. This observation is inconsistent with the strict hierarchical IL-2-binding affinity conversion model previously proposed by Saito and coworkers [Saito Y., Sabe, H., Suzuki, N., Kondo, S., Ogura, T., Shimizu, A. & Honjo, T. (1988) J. Exp. Med. 168, 1563-1572] that predicted an ordered sequence of events in which IL-2 must first bind to IL-2R alpha before its interaction with IL-2R beta. Previous investigations using IL-2 variants were interpreted to show that IL-2R alpha merely acts to concentrate IL-2 to the cell surface and that no other meaningful interaction occurred between IL-2R alpha and IL-2R beta. However, our data are inconsistent with this view. We draw this conclusion on the basis of our observation that antibody HIEI, which reacts with an epitope of IL-2R alpha and interferes with interaction of this chain and IL-2R beta, inhibits the IL-2-dependent proliferative effects mediated by analog F42A. Furthermore, by blocking interaction of IL-2R alpha and IL-2R beta with the antibody HIEI, a decrease in the affinity of radiolabeled analog F42A for IL-2R beta was seen. In our proposed model IL-2R alpha contributes several functions to IL-2-mediated signaling through the high-affinity IL-2R. These functions include concentration of IL-2 within the two-dimensional surface of the plasma membrane as well as alteration of the functional capacity of IL-2R beta, an effect that does not require prior binding of IL-2R to IL-2R alpha. The IL-2R alpha-mediated augmentation of IL-2R beta functions involves affinity conversion of IL-2R beta, increasing its affinity for IL-2, and may involve facilitation of Il-2-mediated signaling after binding of IL-2 to this IL-2R beta.
Proceedings of the National Academy of Sciences 04/1992; 89(6):2165-9. DOI:10.1073/pnas.89.6.2165 · 9.67 Impact Factor