To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.

We have purified the human T-cell growth factor (interleukin-2) receptor and have cloned, sequenced and expressed cDNAs corresponding to this receptor. We identify one gene, but two interleukin-2 receptor mRNAs which differ in their polyadenylation signals. We have isolated an additional cDNA that may correspond to an alternatively spliced mRNA that lacks a 216 base segment and appears to encode an altered membrane protein which cannot bind interleukin-2.

Interleukin 2 promotes proliferation of T cells by virtue of its interaction with a high-affinity cell surface receptor. This receptor is a 55,000 mol wt glycoprotein that is also recognized by the murine monoclonal antibody, anti-Tac. Quantitative binding studies with radiolabeled IL-2 and anti-Tac, however, initially indicated far more antibody binding sites per cell than IL-2 binding sites. Extension of the IL-2 binding analysis to concentrations several thousand-fold higher than that necessary for the T cell proliferative response demonstrated the existence of a class (or classes) of low-affinity IL-2 binding sites. Inclusion of the low-affinity IL-2 binding greatly reduced the quantitative discrepancy in the ligand binding assays. That the low-affinity binding, as well as the high-affinity interaction, was associated with the Tac molecule was indicated by the finding that the antibody could substantially or totally block the entire spectrum of IL-2 binding and by the finding that IL-2 could in turn block all radiolabeled anti-Tac binding. The low-affinity sites were found on activated T cells, several human and murine T cell lines and two examples of Tac-positive B cells. The physiological role of the low-affinity IL-2 binding sites and the molecular changes in the Tac protein that give rise to the affinity differences remain open to investigation.

Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (1L-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human L-2. The level of inhibition was dependent on both antibody and IL-2 concentration. Cell distribution studies using a fluorescenceactivated cell sorter showed that the antigen identified by 7D4 is expressed at a high density on, HT2 cells and on concanavalin A (Con A)-induced T-cell blasts and at a substantially lower density on lipopolysaccharide-induced B-cell blasts; 7D4 (5) and CTLL (6)-that solely depend on an exogenous source of IL-2 for growth has provided a definitive assay cell population to assess the presence of IL-2 in crude culture fluids containing many biologically active molecules and has greatly aided the purification and biochemical characterization of this lymphokine. IL-2 exerts its growth-promoting properties after interaction with specific membrane binding sites (IL-2 receptors) expressed on activated, but not on resting, T cells (7-9). After IL-2 associates with its receptor, it is internalized and undergoes lysosomal degradation (9). The relationship between the degradation and biological function of IL-2 is unclear. Further delineation of the molecular structures and pathways involved in the IL-2 growth mechanism should be facilitated as monoclonal antibodies reactive with the specific components of the hormone-receptor complex become available. Recently, monoclonal antibodies to IL-2 (10, 11) and the human IL-2 receptor (12) have been described. In the present paper, we report the characterization of a rat monoclonal antibody (7D4) that is reactive with an epitope either on the murine IL-2 receptor distal to the IL-2 hormone binding site or to another molecule physically complexed with the IL-2 receptor.MATERIALS AND METHODS Animals. Inbred strain 2 guinea pigs, C57BL/6 and BALB/ c mice, and Lewis rats were obtained from the Animal Production Section, Division of Research Services, National Institutes of Health (Bethesda, MD).Cell Lines. The properties of the murine IL-2-dependent HT2 cells and of CTLL cells have been described by Watson (5) and by Baker et al. (6), respectively. The nonsecretor hybridoma cell line SP2/0-Ag-14 (SP2/0) was used for all fusions.Antisera and Monoclonal Antibodies. Heterologous classspecific antisera to rat IgM, IgG, and IgA were obtained from Cappel Laboratories (Cochranville, PA) and used to determine the isotypes of rat monoclonal antibodies by Ouchterlony analysis of concentrated culture supernatant fluids. The following culture supernatants containing rat monoclonal antibodies to murine lymphocyte surface antigens were used: 53.7.3 (anti-Lyt-1) and 53.6.7 (anti-Lyt-2), developed by Ledbetter and Herzenberg (13) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...

No abstract