Expression and role of p75 interleukin 2 receptor on human monocytes.

We investigated the expression of IL-2R subunits in human monocytes using the TU27 mAb, which recognizes the p75 chain, and anti-Tac mAb, which recognizes the p55 moiety of the IL-2R. We found that p75 but not p55 is constitutively expressed in more than 90% of fresh human monocytes. Antibody to p75, but not to p55, inhibited the activation of monocytes to a cytotoxic stage induced by IL-2 but did not block IFN-gamma-induced cytotoxicity. Our data demonstrate that the p75 chain is expressed on human monocytes and is involved in the activation of monocytes by IL-2.

Cloning of the gamma chain of the human IL-2 receptor.

A third subunit, the gamma chain, of the human interleukin-2 receptor (IL-2R) was identified, and a complementary DNA clone encoding this member of the cytokine receptor family was isolated. The gamma chain is necessary for the formation of the high- and intermediate-affinity receptors, which consists of alpha beta gamma heterotrimers and beta gamma heterodimers, respectively. The IL-2R on murine fibroblastoid cells can be internalized after binding IL-2 only if the gamma chain is present; alpha and beta are insufficient for internalization. Thus, the gamma chain is an indispensable component of the functional IL-2R.

Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association.

In the interleukin-2 (IL-2) system, intracellular signal transduction is triggered by the beta chain of the IL-2 receptor (IL-2R beta); however, the responsible signaling mechanism remains unidentified. Evidence for the formation of a stable complex of IL-2R beta and the lymphocyte-specific protein tyrosine kinase p56lck is presented. Specific association sites were identified in the tyrosine kinase catalytic domain of p56lck and in the cytoplasmic domain of IL-2R beta. As a result of interaction, IL-2R beta became phosphorylated in vitro by p56lck. Treatment of T lymphocytes with IL-2 promotes p56lck kinase activity. These data suggest the participation of p56lck as a critical signaling molecule downstream of IL-2R via a novel interaction.

Activation of human B cells by phosphorothioate oligodeoxynucleotides.

To investigate the potential of DNA to elicit immune responses in man, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell-depleted human peripheral blood B cells. Among 47 ODNs of various sequences tested, 12 phosphorothioate oligodeoxynucleotides (sODNs) induced marked B cell proliferation and Ig production. IL-2 augmented both proliferation and production of IgM, IgG, and IgA, as well as IgM anti-DNA antibodies, but was not necessary for B cell stimulation. Similarly, T cells enhanced stimulation, but were not necessary for B cell activation. After stimulation with the active sODNs, more than 95% of B cells expressed CD25 and CD86. In addition, B cells stimulated with sODNs expressed all six of the major immunoglobulin VH gene families. These results indicate that the human B cell response to sODN is polyclonal. Active sODN coupled to Sepharose beads stimulated B cells as effectively as the free sODN, suggesting that stimulation resulted from engagement of surface receptors. These data indicate that sODNs can directly induce polyclonal activation of human B cells in a T cell-independent manner by engaging as yet unknown B cell surface receptors.

IL-15/IL-15Ralpha intracellular trafficking in human melanoma cells and signal transduction through the IL-15Ralpha.

There are two IL-15 isoforms and eight isoforms for the IL-15Ralpha chain whose biological role is poorly understood. Here, we have analysed the intracellular trafficking of IL-15 and IL-15Ralpha and tried to shed some light on their function(s). In IL-15/GFP CHO transfectants both IL-15 isoforms show nuclear localization. Two melanoma cell lines (MELP and MELREO) spontaneously expressing the IL-15 isoforms, display different intracellular trafficking of the IL-15/IL-15Ralpha complex. In MELP cells only IL-15Ralpha is detected inside the nucleus, whereas IL-15 and IL-15Ralpha assemble at the cell surface and are internalized. Moreover, the transducing molecule TRAF2 co-immunoprecipitates with IL-15Ralpha and may be deflected to TNFRI using anti-IL-15 blocking mAbs and TNF-alpha. By contrast, MELREO cells display IL-15Ralpha and IL-15 nuclear localization but only a partial co-localization of these molecules on the cell surface. In these cells, TRAF2 is strongly associated with IL-15Ralpha and cannot be deflected by any treatment. Since TRAF2 activates the transcription factor NF-kappaB, IL-15 through IL-15Ralpha, could have a role in the control of this pathway. Indeed, anti-IL-15 MaB inhibit the constitutive nuclear localization of NFkappaB and the phosphorylation of its inhibitor Ikappa-Balpha. Thus, IL-15Ralpha controls NF-kappaB activation, however differences in the intracellular trafficking of the IL-15 and/or IL-15Ralpha suggest a different biological role for this complex in MELP versus MELREO cells.

Cloning, sequencing and characterization of lentiviral-mediated expression of rhesus macaque (Macaca mulatta) interleukin-2 receptor alpha cDNA.

The rhesus macaque CD25 (RhCD25) cDNA isolated from rhesus PBMCs was found to share 95.5 and 91.9% homology with the human orthologue at the nucleotide and amino acid levels, respectively. Comparative sequence analyses suggest that both human CD25 (HuCD25) and RhCD25 share identity for most of the critical amino acids previously identified to be essential for viable folding and IL-2 ligand binding. The human leukemic cell line, HH, deficient for IL-2Ralpha was transduced with a lentiviral vector (LV) engineered to express RhCD25 (HH-RhCD25). RhCD25 was characterized for expression by flow cytometric analyses, ELISA, Western blotting, functional signalling, and biological assays in comparison to HuCD25. In summary, vectors expressing the RhCD25 cDNA can be used as a tool to aid in the characterization of soluble CD25 in non-human primate studies, and to provide a tempting alternative as an autologous cell surface marker in rhesus macaque gene therapy and bone marrow transplantation studies.

Soluble interleukin-2 receptors inhibit interleukin 2-dependent proliferation and cytotoxicity: explanation for diminished natural killer cell activity in cutaneous T-cell lymphomas in vivo?

In patients with cutaneous T-cell lymphomas (CTCL), soluble interleukin-2 receptor serum levels (sIL-2R) were determined by ELISA technique, and natural killer cell (NK) activity, by a 4-h chromium-51 release assay. Decrease of NK activity correlated with the augmentation of serum sIL-2R. After a 4-d stimulation with interleukin 2 CTCL patients' peripheral mononuclear cells (PMC) showed an increase of cytotoxic activity similar to that in healthy donors' PMC. Normal donors' PMC demonstrated a diminished IL-2-induced cytotoxic activity in 25% CTCL serum (sIL-2R of 3000, 7330, and 10700 U/ml, respectively) compared to control serum (sIL-2R of 400, 340, and 420 U/ml, respectively). IL-2-dependent proliferation of 2-d phytohemagglutinin (PHA) blasts was lower in CTCL serum than in control serum. sIL-2R was enriched from one CTCL patient's serum by IL-2 affinity chromatography. Transfection of the Tac gene into NIH/3T3 fibroblasts resulted in the production of a recombinant sIL-2R. The presence of enriched native or recombinant sIL-2R inhibited interleukin-2-dependent generation of cytotoxic activity and PHA blast proliferation. We suggest that elevated sIL-2R levels account for diminished NK activity by neutralizing interleukin 2 in CTCL patients.

Purification of the IL-2 receptor (TAC) by ligand-affinity chromatography and utilization of the immobilized receptor for receptor-affinity chromatography (RAC) purification of IL-2, mutant IL-2, and IL-2 fusion proteins.

Recombinant technology has facilitated the production of two soluble forms of human p55 interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two soluble forms of the IL-2R, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug-screening assays. The purified IL-2R subsequently has been immobilized on silica gel and employed for the purification of recombinant IL-2. Receptor-affinity-chromatography-purified IL-2 contains only a highly active monomeric form of the lymphokine, in contrast to immunoaffinity chromatography where several molecular forms of IL-2 with varying degrees of biologic activity are recovered. Receptor-affinity chromatography has been successfully applied to the purification of several mutant IL-2 as well as an IL-2-Pseudomonas exotoxin (IL2-PE40) fusion protein that is a 54.5-kDa chimeric protein in which the cell recognition domain is replaced by IL-2. The IL-2-PE40 is a potential cytotoxic agent for cells bearing the IL-2 receptor.

Biochemical identity and characterization of the mouse interleukin-2 receptor beta and gamma c subunits.

Although the mouse IL-2 receptor (IL-2R) beta and gamma c subunits have been identified by molecular cloning, the biochemical identity of these subunits has not yet been established. In the present study, the mouse IL-2R was biochemically characterized from cell lines expressing normal and aberrant IL-2R. Using novel monoclonal antibodies specific for the beta or gamma c subunits, we established that the M(r) of the beta chain is 90,000-100,000 and that of the gamma c subunit is 75,000-80,000. Analysis of transfected EL4 cells that expressed alpha, gamma c, and truncated beta subunits or mutant EL4 cells, which selectively lacked cell surface gamma c, revealed that no other material migrated to a position on SDS-PAGE characteristic of IL-2/IL-2R beta and IL-2/IL-2R gamma c cross-linked complexes, respectively. Thus, the beta and gamma c subunits appear to be the sole IL-2R constituents of these IL-2 cross-linked complexes. The IL-2/IL-2R gamma c, but not the IL-2/IL-2R beta, complex exhibited enhanced mobility after SDS-polyacrylamide gel electrophoresis under nonreducing conditions, suggesting a more compact structure for gamma c as a result of intrachain disulfide bonds. The primary posttranslational modification of the mouse beta and gamma c subunits is N-linked glycosylation. These biochemical studies reconcile past uncertainties concerning the subunit composition of the mouse IL-2R and are consistent with a model of the IL-2R containing only three subunits.

Biotinylated recombinant interleukin-2. A tool for research on the interleukin-2 receptor.

Recombinant interleukin-2 was biotinylated using a N-hydroxyl-succinimidyl [3H]biotin ester. The biotinylated lymphokine retained full binding and growth-promoting activities when assayed on the interleukin-2-dependent murine cell line HT-2. In preliminary studies, biotinylated interleukin-2 was used in conjunction with immunogold staining to demonstrate cell surface interleukin-2 receptors using both light and electron microscopy techniques. In addition, with rabbit anti-biotin antibodies, biotin-interleukin-2 was able to precipitate the 55 kDa IL-2 receptor from murine HT-2 cells. Thus, biotin-interleukin-2 represents a useful, non-radioactive tool for studying the structure and function of the interleukin-2 receptor.

A soluble 'anchorminus' interleukin 2 receptor suppresses in vitro interleukin 2-mediated immune responses.

The immunosuppressive effects of a recombinant soluble IL-2 receptor L chain (s-IL-2R) were analyzed. S-IL-2R protein was obtained from the conditioned medium of L cells transfected with a mutant cDNA clone encoding the extracytoplasmic portion of the IL-2 receptor (IL-2R) and was purified to homogeneity by an IL-2-coupled sepharose column, following by reverse phase chromatography (HPLC). Soluble IL-2R protein thus prepared retained the ability to bind IL-2 specifically and suppressed the in vitro IL-2-mediated immune responses, including proliferation of IL-2-dependent cell line (CTLL-2), induction of secondary cytotoxic T lymphocytes (CTL) and the mixed lymphocyte reaction (MLR), but did not suppress the growth of IL-3-dependent cell line. Kinetic studies revealed that s-IL-2R exhibited the suppressive effects on the proliferative responses of alloantigen stimulated human tonsillar cells, only when added at an early stage, namely 0-48 h after culture onset, whereas cyclosporin A (CsA) exhibited an inhibitory effect only when added at between 0 and 24 h. This implies that s-IL-2R exerts its effect on an early stage of lymphocyte activation. The observed immunosuppressive effects of s-IL-2R suggest the possibility that s-IL-2R might be useful for the protection of rejection crisis in organ transplantation.

Rapid determination of gamma delta T-cell stimulation by microfluorimetry.

Activated T-cell express CD25, the p55 chain of the IL-2 receptor. Here we report a reliable procedure for rapid determination of human gamma delta T cell activation by microfluorimetric measurement of CD25. Three days after initiation of culture, CD25 expression was determined. The sensitivity of this detection method was compared with [3H]thymidine incorporation at day 8. This proliferation assay allowed 3-5-fold higher dilution of the stimulatory ligand. However, monitoring of CD25 expression speeded up screening by 5 days. Therefore, for rapid screening of gamma delta T cell stimulation, e.g. for identification of activating ligands, monitoring of CD25 at day 3 is superior to [3H]thymidine measurement.

The effect of aging on T cell responses in the horse.

Horses greater than 20 years of age exhibit alterations in their immune responses similar to those observed in other aged individuals. The purpose of this study was to characterize immunosenescence in a population of aged ponies. The peripheral blood mononuclear cells (PBMC) from aged ponies exhibited a decreased proliferative response to various mitogens that was not overcome by the addition of interleukin 2 (IL-2) to the cultures. No difference in overall expression of the IL-2 receptor was seen between young and aged ponies, though CD8(+) cells from aged ponies exhibited increased levels of IL-2 receptor expression. The kinetics of the response to both mitogen and IL-2 did not appear to be affected in the aged PBMCs. These results indicate that the age-related decrease in the proliferative response to mitogens is not due to a failure to produce or respond to IL-2 but probably involves some other process.

Human B lymphocytes express the p75 component of the interleukin 2 receptor.

The nature of the interleukin 2 (IL-2) receptor on purified human B lymphocytes was examined. Both normal and malignant cells showed evidence of a 70-75,000 mol. wt (p75) IL-2 binding molecule as assessed by 125I-labeled IL-2 binding and receptor cross-linking. On normal, Tac-negative B lymphocytes the estimated number of p75 binding sites was 1100 per cell and the dissociation constant (Kd) was 1.7 nM. Consistent with this, cross-linking experiments demonstrated the presence of an IL-2 binding molecule of 70-75,000 mol. wt. Purified B cells from patients with hairy cell leukemia and chronic lymphocytic leukemia (CLL) also expressed the p75 IL-2 binding molecule. In the HCL samples, a small number of high-affinity IL-2 binding sites were detected (27-90) while the majority of binding sites (2100-10,800) were typical of low-affinity p55 Tac binding. IL-2 added to the purified normal and CLL B lymphocytes led to the induction of p55 Tac expression and the generation of high-affinity IL-2 receptors. This response to IL-2 was equivalent to the response observed when normal B lymphocytes were stimulated by Staphylococcus aureus Cowan I.

Soluble IL-2 receptor beta and gamma subunits: ligand binding and cooperativity.

Biologically relevant interleukin-2 receptors (IL-2Rs) are present in two affinity states on responsive cells. High affinity receptors (HAR) apparently exist as heterotrimers (alpha, beta and gamma) while the other functional complex, the intermediate affinity receptor (IAR), is comprised of beta and gamma chains. The mechanisms by which the beta and gamma subunits contribute the formation of HAR and IAR are still unclear. Soluble forms of the beta and gamma chains were cloned, epitope-tagged, expressed in insect cells and purified. IL-2 binding and neutralization of IL-2 bioactivity by beta and gamma extracellular domains (ectodomains) was analyzed by several biochemical and biological approaches. The results indicate that beta clearly binds IL-2 with low affinity (KI-KD = 3 microM) whereas gamma binding is detectable, but of very low affinity (apparent KI > 15 microM) in the absence of beta. Interestingly, combinations of beta and gamma ectodomains interact to bind IL-2 with higher affinity and greater stability than either chain alone. An apparent stable binding complex is formed when beta, gamma, and IL-2 are combined. Ligand binding by the beta and gamma chains in solution is specific for IL-2 and is of sufficient affinity and stability to effectively neutralize IL-2 in biological assays (binding IC50 = biological IC50). Direct analyses of binding kinetics by surface plasmon resonance reveals that the increased affinity and biological neutralizing ability of beta gamma, as compared to beta, is due to a very slow dissociation rate contributed by the gamma ectodomain. While IL-2R beta and gamma cytoplasmic and transmembrane domains are not essential for interactive binding of IL-2, they may contribute to IL2 binding affinity. The recognition and association of IL-2 by the beta gamma IAR appears to be contributed primarily by the beta chain while the stability and dissociation is likely dominated by the gamma chain. It is anticipated that the gamma subunit functions in a similar manner when participating in high affinity IL-4 and IL-7 binding.

Immune response to polyglycolic acid implants.

Cytological analysis of material aspirated from the effusion which occasionally develops around a polyglycolic acid (PGA) osteosynthesis implant showed a predominance of inflammatory monocytes and in particular lymphocytes. In order to discover whether PGA implants are immunologically inert, density gradient-isolated peripheral blood mononuclear cells were cultured in 0.2 ml of 10% delta FCS-RPMI 1640 culture medium supplemented with 10 mg PGA. Phytohaemagglutinin (PHA) lectin, a purified protein derivate of tuberculin (PPD) antigen and culture medium alone were used as positive and negative controls. We studied lymphocyte activation kinetics on days 0, 1, 3 and 5. Major histocompatibility complex locus II antigen (MHC locus II antigen) and interleukin-2 receptor (IL-2R) expression were analysed using the avidin-biotin-peroxidase complex (ABC) method and lymphocyte DNA synthesis by using 3H-thymidine incorporation and beta-scintillation counting. Especially on culture days 0 and 1, lymphocytes and monocytes were seen by light microscopy to be attached to PGA particles. However, our results show no PGA-induced lymphocyte DNA synthesis, but PGA-induced MHC locus II antigen and IL-2R activation marker expression was seen, greater than in negative controls, but less than that seen in PPD antigen driven lymphocyte response. This suggests that PGA is an immunologically inert implant material, but it does seem to induce inflammatory mononuclear cell migration and adhesion, leading to a slight non-specific lymphocyte activation. This activation is lower than that seen in mitogen and antigen-driven lymphocyte responses.

Demonstration of two distinct forms of released low affinity-type rat IL-2 receptors.

The release of IL-2 binding proteins, derived from the 55,000 MW low affinity IL-2R (L chain), has been observed for virtually all L chain-bearing cells in either humans, the mouse or the rat. Based on the characterization of the released human L chain as a molecule 10,000 MW smaller than the cell surface receptor, either proteolytic cleavage or differential splicing of the L chain encoding mRNA have been suggested as mechanisms underlying the receptor release. Combining affinity labelling of the L chain with [125I]IL-2 and immunoprecipitation with L chain-specific monoclonal antibody (mAb) applied for the detection of soluble rat IL-2R revealed the existence of two classes of soluble receptors, one being of the same size as cell surface-expressed L chain, the other of 40,000 apparent molecular mass. These findings raise the possibility of other mechanisms of receptor release than those discussed for human L chain.

Medium-scale ligand-affinity purification of two soluble forms of human interleukin-2 receptor.

Recombinant technology has facilitated the production of two soluble forms of human interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two IL-2Rs, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug screening assays and the receptor-affinity purification of human recombinant interleukin-2.

Four interleukin-2 surface binding proteins detected in rat spleen cells.

Four specific interleukin-2 (IL-2) surface binding proteins can be detected by covalent cross-linking of [125I]IL-2 to rat spleen cells that have been activated with various stimuli including concanavalin A (Con A), phytohaemagglutinin (PHA), calcium ionophore, and phorbol dibutyrate (PDB) with or without calcium ionophore. These four cross-linked proteins could not be demonstrated in either unstimulated T cells or in activated T cells when binding was performed in the presence of a 20-100-fold excess of unlabelled IL-2. The molecular weights of the four cross-linked proteins, after subtraction of the molecular weight contribution of IL-2 are: 53,000, 70,000, 90,000 and 118,000. The 53,000 MW protein was identified as the rat IL-2 receptor (IL-2R) alpha-chain by immune precipitation. Additionally, results suggest that the rat IL-2R alpha-chain is tightly complexed to both the 118,000 and 90,000 MW IL-2 binding proteins. Purification of surface labelled proteins from activated cells using IL-2 affinity chromatography yields four proteins with similar molecular weight to those identified by cross-linking plus an additional non-ligand cross-linked protein of 46,000 MW. The 46,000 MW band may be a non-binding associated protein since it was not seen following [125I]IL-2 binding cross-linking. Tryptic digests and two-dimensional separation of the affinity-isolated proteins indicate that unique peptide maps are generated for the 46,000, 53,000 and 70,000 MW proteins and excludes the possibility that the bands identified by cross-linking represents cross-linking of multiple ligands to the 53,000 MW subunit. However, the 90,000 and 118,000 MW bands yield peptide maps that closely resemble each other suggesting that these binding proteins may be related. These results suggest that at least four IL-2 surface binding proteins may constitute the rat IL-2R system.