Murine Interleukin-2 Receptor Research Articles

Abstract 4057: Beyond IL-2: The promise of anti PD-1-attenuated IL-2 fusion protein in cancer immunotherapy

Abstract PD1+ CD8+ tumor-infiltrating T-cells have been at the forefront of cancer immunotherapies due to their antigen-specificity, cytotoxicity, proliferative capacity, and stem-like/memory properties. IL-2 is essential for T cell growth and proliferation, however, its systemic administration at a high dose was associated with intolerable toxicities, while low-dose treatment contradicted the intended therapeutic effect by activating regulatory T cells.To overcome the limitations of IL-2, we fused modified IL-2 sequences with reduced affinity for the murine IL-2 receptor to a PD1 antibody designed to target the PD1+ T cells found in tumors. The resulting murine PD1 antibody-attenuated-IL-2 (mAnti-PD1-IL2Att) demonstrated similar IL-2 potency on PD1+ cells as native IL-2, but diminished potency by orders of magnitude on PD1- cells. The agent was furthermore designed to bind PD1 non-competitively with known PD1-blocking antibodies, so that it could be used in combination with them to achieve effective anti-tumor response. We demonstrated that mAnti-PD1-IL2Att exhibits potent anti-tumor activity, resulting in complete tumor regression in a syngeneic MC38 colorectal cancer model, and significant anti-tumor activity in other models of different tumor types. The mechanism was evaluated by immunophenotyping and single-cell RNAseq. Shortly after the first administration of mAnti-PD1-IL2Att, CD8+ T cells, rather than Tregs, emerged as the primary responding cells; these CD8+ T cells exhibited increased proliferation and activation with reduced expression of exhaustion markers (PD1+Tim3+). Following subsequent doses, there was a notable increase in the number of effector cells, followed by a prolonged generation of effector and memory cells at a later stage. Myeloid cells exhibited increased transcriptional programs related to chemotaxis, co-stimulation, and co-inhibition, including the upregulation of the PD1-PDL1 pathway. By contrast, blockade of PD1 with a naked PD1 antibody led to distinct cellular responses and less pronounced anti-tumor activity, and a combination of the blocking antibody with PD1-attIL-2 was superior to either agent alone. Together, our findings suggest that mAnti-PD1-IL2Att potent anti-tumor activity was driven by its direct targeting of PD1+ on tumor-infiltrating CD8+ T-cells, shedding light on the specific changes in their dynamic differentiation trajectory and their impact on the tumor microenvironment. Citation Format: Sarah Amar, Yulia Liubomirski, Eilam Yeini, Michael Monsonego, Tomer Weiss, Galia Tiram, Anshika Katyal, Opal Avramoff, Anne Krinsky, Keren Reshef, Shai Dulberg, Ayelet Kaminitz, Dana Bar On, Inbal Ben Eliezer, Ortal Iancu, Tetsuya Taura, David Wilson, Paul Ayton, Ronit Satchi-Fainaro, Asaf Madi. Beyond IL-2: The promise of anti PD-1-attenuated IL-2 fusion protein in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4057.

T Cells Integrate Local and Global Cues to Discriminate between Structurally Similar Antigens.

T lymphocytes’ ability to discriminate between structurally related antigens has been attributed to the unique signaling properties of the T cell receptor. However, recent studies have suggested that the output of this discrimination process is conditioned by environmental cues. Here, we demonstrate how the IL-2 cytokine, collectively generated by strongly activated T cell clones, can induce weaker T cell clones to proliferate. We identify the PI3K pathway as being critical for integrating the antigen and cytokine responses and for controlling cell-cycle entry. We build a hybrid stochastic/deterministic computational model that accounts for such signal synergism and demonstrates quantitatively how T cells tune their cell-cycle entry according to environmental cytokine cues. Our findings indicate that antigen discrimination by T cells is not solely an intrinsic cellular property but rather a product of integration of multiple cues, including local cues such as antigen quality and quantity, to global ones like the extracellular concentration of inflammatory cytokines.

Diphtheria toxin-based recombinant murine IL-2 fusion toxin for depleting murine regulatory T cells in vivo.

Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells which suppress immune responses of effector cells and are known to play a very important role in protection against autoimmune disease development, induction of transplantation tolerance and suppression of effective immune response against tumor cells. An effective in vivo Treg depletion agent would facilitate Treg-associated studies across many research areas. In this study, we have developed diphtheria toxin-based monovalent and bivalent murine IL-2 fusion toxins for depleting murine IL-2 receptor positive cells including CD25(+) Treg in vivo. Their potencies were assessed by in vitro protein synthesis inhibition and cell proliferation inhibition assays using a murine CD25(+) CTLL-2 cell line. Surprisingly, in contrast to our previously developed recombinant fusion toxins, the monovalent isoform (DT390-mIL-2) was approximately 4-fold more potent than its bivalent counterpart (DT390-bi-mIL-2). Binding analysis by flow cytometry demonstrated that the monovalent isoform bound stronger than the bivalent version. In vivo Treg depletion with the monovalent murine IL-2 fusion toxin was performed using C57BL/6J (B6) mice. Spleen Treg were significantly depleted with a maximum reduction of ∼70% and detectable as early as 12 h after the last injection. The spleen Treg numbers were reduced until Day 3 and returned to control levels by Day 7. We believe that this monovalent murine IL-2 fusion toxin will be an effective in vivo murine Treg depleter.

Augmented adenovirus transduction of murine T lymphocytes utilizing a bi-specific protein targeting murine interleukin 2 receptor

Adenoviruses are currently used in a variety of bench and bedside applications. However, their employment in gene delivery to lymphocyte lineages is hampered by the lack of coxsackie virus and adenovirus receptor (CAR) on the cell surface. Exploitation of an alternative receptor on the surface of T lymphocytes can allow for utilization of adenovirus in a variety of T lymphocyte-based diseases and therapies. Here, we describe how resistance to infection can be overcome by the utilization of a bi-specific fusion protein, soluble CAR murine interleukin 2 (sCAR-mIL-2), that retargets adenovirus to the murine IL-2 receptor (IL-2R). Infection of a murine T-cell line, CTLL-2, with a sCAR-mIL-2/Adenovirus conjugate provided a ninefold increase in both green fluorescence protein-positive cells and luciferase expression. In addition, this increase in infection was also seen in isolated primary murine T lymphocytes. In this context, the sCAR-mIL-2 adapter provided a fourfold gene transduction increase in activated primary murine T lymphocytes. Our results show that recombinant sCAR-mIL-2 fusion protein promotes IL-2R-targeted gene transfer to murine T lymphocytes and that alternative targeting can abrogate their native resistance to infection.

Cloning of rat thymic stromal lymphopoietin receptor (TSLPR) and characterization of genomic structure of murine Tslpr gene

Thymic stromal derived lymphopoietin receptor (TSLPR) is a novel receptor subunit that is related in sequence to the interleukin (IL)-2 receptor common gamma chain. TSLPR forms a heterodimeric complex with the IL-7 receptor alpha chain to form the receptor for thymic stromal derived lymphopoietin, a cytokine involved in B- and T-cell function. We have cloned the TSLP receptor from rat and find that the WSXWX motif commonly found in extracellular domains of cytokine receptors is conserved as a W(T/S)XV(T/A) motif among TSLP receptors from mouse, rat and human. As in the mouse, TSLP receptor is widely expressed in rats suggesting that TSLPR may have roles in signaling outside the hematopoietic system. A zooblot analysis revealed that TSLPR is expressed in all vertebrate species examined. The absence of TSLPR in Saccharomycescerevisiae, Drosophilamelanogaster and Caenorhabditiselegans genomes is similar to the expression of several other cytokine receptors that have been characterized thus far. We have also characterized the genomic structure of the murine Tslpr gene which shows that in addition to primary sequence homology, it shares a common genomic organization of coding exons with the murine IL-2 receptor common gamma chain (Il2rg). Use of an alternative splice acceptor site leads to two alternatively spliced transcript variants of murine TSLPR, both of which are functional receptors. Finally, using linkage analysis, we mapped the murine Tslpr gene to mouse chromosome 5 between the Ecm2 and Pxn genes.

Murine IL-2 receptor beta chain blockade improves human leukocyte engraftment in SCID mice

Severe combined immunodeficient (SCID) mice accept human xenografts and can act as a model for human immune functions. Murine natural killer cells (NK), however, represent an important barrier for the reconstitution of SCID mice with human peripheral blood leukocytes (Hu-PBL). We investigated the effect on Hu-PBL survival of pretreatment with TM-beta1, a rat monoclonal antibody for the mouse IL-2 receptor beta chain. TM-beta1 greatly improved the survival of Hu-PBL. Human lymphocytes, predominantly T cells, survived in the peritoneum and infiltrated spleen and lungs already 1 week after engraftment and liver and thymus from 2 weeks on. Secondary humoral responses were evaluated with Hu-PBL from a donor immune to hepatitis-B surface Ag (HBsAg) and tetanus toxoid (TT). TM-beta1 pretreatment enhanced the recall Ig response to HBsAg and did not affect the baseline anti-TT Ig production. In conclusion, TM-beta1 pretreatment of SCID mice significantly improves the survival and functionality of the Hu-PBL graft.

Molecular characterization and functional analysis of murine interleukin 4 receptor allotypes.

The murine interleukin 4 receptor (IL-4R) exists as a transmembrane protein transducing pleiotropic IL-4 functions, or as soluble (s)IL-4-binding molecule with potent immunoregulatory effects. In this study we identified and characterized a murine IL-4R allotype. Sequence analysis of the IL-4R cDNA of BALB/c mice revealed 18 base substitutions leading to three extracellular and five cytoplasmic amino acid changes when compared with the published IL-4R sequence of C57BL/6 mice. Analyses with allotype-specific mAbs revealed that AKR/J and SJL/J mice possess the newly identified BALB/c IL-4R allotype whereas the IL-4Rs of C3H, CBA, DBA-2, and FVB/N mice are identical to that of the C57BL/6 mouse. The extracellular Thr49 to Ile substitution abrogates one N-glycosylation site in the naturally occurring BALB/c IL-4R as well as in the experimentally point mutated C57BL/6-T49I sIL-4R, and both molecules display a nearly threefold reduction in IL-4-neutralizing activity compared to the C57BL/6 sIL-4R. In line with this, a significantly enhanced dissociation rate of IL-4 was detected for the BALB/c IL-4R allotype by surface plasmon resonance and in radioligand binding studies with IL-4R-transfected cell lines. These findings suggest that the altered ligand binding behavior of the newly described IL-4R allotype may influence the IL-4 responsiveness, thus contributing to the diverse phenotypes of inbred mouse strains in IL-4-dependent diseases.

Regulation of the Expression of the Soluble and Membrane Forms of the Murine IL-4 Receptor

The actions of interleukin-4 (IL-4)in vivoare likely to be positively influenced by the expression of membrane IL-4 receptors (mIL-4R) on target cells and negatively by the concentration of soluble IL-4 receptors (sIL-4R) in the extracellular environment. Inasmuch as the two forms of the mouse IL-4R are differentially encoded by alternatively spliced mRNA transcripts, the purpose of this work was to determine how their expression is regulated by IL-4 and T cell activation and whether there is preferential expression of one type of transcript over the other. In this study, the expression of sIL-4R and mIL-4R transcripts was analyzed by a semiquantitative RT-PCR method in resting and mitogen-activated splenic cells. Irrespectively of the state of cell activation, IL-4 up-regulated the levels of both types of mRNA with similar kinetics and dose–response curves. In contrast, ConA failed to enhance the steady-state levels of sIL-4R or mIL-4R transcripts despite increased expression at the protein level, suggesting that sIL-4R expression is also regulated at levels other than transcription. Western blot analysis of supernatants of IL-4- and ConA-stimulated spleen cells substantiated the presence of sIL-4R molecules derived by translation of sIL-4R-specific transcripts, thus confirming the importance of this mechanism for the generation of sIL-4R molecules in normal cells. These results indicate that the sIL-4R- and mIL-4R-specific transcripts are normally regulated in a parallel manner and further suggest that expression of both forms of the IL-4R is controlled at multiple levels (i.e., transcriptional and posttranscriptional).

The role of interleukin-5 (IL-5) in allergic airway hyperresponsiveness in mice.

In order to investigate the role of IL-5 in allergic airway inflammation and hyperresponsiveness, the effects of rat anti-IL-5 monoclonal antibody (NC-17), recombinant soluble murine IL-5 receptor, and some immunosuppressors were studied in mice model. Three inhalations of an antigen by actively sensitized animals resulted in an increase in airway reactivity to acetylcholine. Twenty-four hours after the final inhalation, the number of leukocytes (mononuclear cells and eosinophils) and the amount of IL-4 and IL-5 in bronchoalveolar lavage fluid (BALF) increased significantly. NC-17 and soluble IL-5 receptor inhibited the antigen-induced increase of eosinophils with little effect on bronchial hyperreactivity. Cyclosporin A, FK-506, and cyclophosphamide clearly inhibited the antigen-induced increase of IL-5 and eosinophils in BALF. Airway hyperreactivity is inhibited by cyclosporine A and FK-506 but not by cyclophosphamide. Furthermore, to investigate the role of mast cells in the onset of allergic airway hyperreactivity, we have examined the effect of repeated antigen provocation in WBB6F1-W/Wv mice (W/Wv), mast-cell-deficient mice. Whereas significant elevation of IL-5 level and the number of eosinophils on BALF was observed, airway reactivity to acetylcholine was not changed at all. These results indicate that IL-5 may play an important role for the antigen-induced eosinophilia in BALF but not in airway hyperresponsiveness in mice.

Two distinct stimulus-dependent pathways lead to production of soluble murine interleukin-4 receptor.

The IL-4R exists in two forms, either membrane bound or as a soluble (s) molecule. Since the sIL-4R binds to its ligand with high affinity, thereby acting as an immunoregulatory molecule, we were interested in the processes leading to its release. First, the release of sIL-4R in the model of murine leishmaniosis was analyzed. Infection of mice with Leishmania major resulted in up-regulation of sIL-4R production by Ag-stimulated CD4+ T cells, with a maximum around 7 days after infection. To clarify the mechanisms underlying sIL-4R release, in vitro studies were performed. After stimulation of naive lymphoid cells with IL-4, sIL-4R release was dependent on up-regulation of spliced IL-4R mRNA, as shown by inhibition with specific antisense oligonucleotides. In contrast to this, no increase in the spliced IL-4R mRNA and no inhibitory influence of antisense oligonucleotides were observed after stimulation of T cells from IL-4-deficient mice with anti-CD3 mAb. Thus, TCR stimulation can lead to IL-4-independent sIL-4R production. Under these conditions proteolytic shedding of membrane-bound IL-4R appears to be the principal mechanism of release, since in contrast to stimulation with IL-4, iodinated sIL-4R could only be immunoprecipitated after cell surface labeling and subsequent TCR stimulation. The common gamma-chain, a component of the IL-4R complex, did not appear to be involved in the pathways leading to sIL-4R expression. This analysis suggests the existence of two differentially regulated pathways of sIL-4R release, possibly having different consequences for the regulation of IL-4 bioactivity.

Ligand-induced autoregulation of IL-2 receptor alpha chain expression in murine T cell lines.

The IL-2 receptor (IL-2R) is composed of three chains alpha, beta and gamma. In mice, contrary to the human system, we have previously demonstrated that the IL-2R beta gamma complex does not bind IL-2. Therefore, mouse IL-2 response is completely dependent on the expression of the IL-2R alpha gene product. T cell clones expressing mouse IL-2R beta gamma and the human IL-2R alpha transgene have been studied. When cells are grown in IL-4, mouse IL-2R alpha is not expressed. However, exposure to IL-2 leads to the expression of the endogenous murine IL-2R alpha subunit. The T cell line expressing mouse IL-2R gamma and human IL-2R beta can grow in IL-2 but does not express endogenous murine IL-2R alpha. Transfection of these cells with the human IL-2R alpha gene restores the capacity to induce murine IL-2R alpha. This result demonstrates that IL-2-IL-2R alpha interactions are required for induction of IL-2R alpha. The kinetics of induction and deinduction of murine IL-2R alpha have been studied using clone 18.III. From negative cells, expression of murine IL-2R alpha is a very slow phenomenon. From cells fully expressing IL-2R alpha, deinduction is a two-step process: after a rapid decrease of IL-2R alpha the cells continue to express, for a long period of time, basal levels of murine IL-2R alpha. When cells expressing basal levels of IL-2R alpha are exposed to IL-2, induction of IL-2R alpha is a very rapid phenomenon. The autoregulatory loop formed by IL-2-IL-2R alpha therefore displays different levels of functioning.

Molecular mechanisms regulating the hyaluronan binding activity of the adhesion protein CD44.

In the present study, we describe the isolation and characterization of a cDNA clone designated B6F1.3, that appears to 'activate' the hyaluronan-binding capacity of CD44 upon transfection into the murine fibroblastoid cell line MOP8. Sequence analysis indicates that the putative regulatory molecule encoded by this clone is identical to the murine interleukin-2 receptor gamma chain (mIL-2R gamma), a recently described type 1 transmembrane protein that constitutes an integral component of the cell surface receptors that bind a number of cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and perhaps also IL-13. Mutations in this molecule have been shown to be responsible for X-linked severe combined immunodeficiency (XSCID) in humans. With the exception of bone marrow, the mIL-2R gamma chain was found to be expressed at high levels on all hemopoietic cell lines and tissue types examined. Non-hemopoietic tissues are generally negative. FACS analysis and Western blot analysis indicated respectively that B6F1.3 does not mediate its effects by upregulating the expression of CD44 or by altering the alternative splicing of the molecule. Removal of the cytoplasmic tail of the mIL-2R gamma chain, including a Src homology region 2 (SH2) subdomain, abolished its ability to enhance CD44-mediated binding to hyaluronan suggesting the involvement of signal transduction events triggered via the cytoplasmic domain in the 'activation' process. Determining whether activating molecules such as B6F1.3 are co-expressed within tumor cells may help improve the potential value of CD44 as a diagnostic marker of metastatic disease.

Species-specific Agonist/Antagonist Activities of Human Interleukin-4 Variants Suggest Distinct Ligand Binding Properties of Human and Murine Common Receptor γ Chain

Interleukin-4 (IL-4) is a pleiotropic cytokine eliciting various responses in target cells upon binding to its receptor. Activation of the IL-4 receptor probably involves interaction of the ligand with both the IL-4 receptor alpha subunit (IL-4R alpha) and the common gamma chain (c gamma). Although human and murine IL-4 receptor alpha chains are specific for IL-4 from the same species, murine c gamma can form a signal-competent complex with human IL-4R alpha (hIL-4R alpha) and human IL-4 (hIL-4). We have generated a hIL-4 responsive murine myeloid cell line (FDC-4G) expressing a chimera comprising the extracellular domain of human IL-4R alpha and the intracellular domain of human granulocyte colony-stimulating factor receptor (hG-CSFR). This hybrid receptor was shown to form a complex with hIL-4 and the murine c gamma-chain. Biological activities of human IL-4 variants on murine FDC-4G cells and on the human erythroleukemic cell line TF-1 displayed a strikingly different pattern. Single amino acid replacements at two different positions in the C-terminal helix of hIL-4, the region of the previously defined "signaling site," lead to an inverse agonist/antagonist behavior of the resulting cytokines in the two cellular systems. From these findings we conclude that upon formation of the activated IL-4 receptor complex murine and human c gamma interact with hIL-4 in a geometrically different fashion.

Therapy of streptozotocin induced diabetes with a bifunctional antibody that delivers vinca alkaloids to IL-2 receptor positive cells.

IVA039.1 is a bifunctional antibody with specificity for the murine IL-2 receptor and vinca alkaloids. Biodistribution studies show that IVA039.1 can target and deliver vinca alkaloids to tissues that contain IL-2 receptor positive cells. Vinca alkaloids are lymphocytotoxic. Therapy of diabetic mice with IVA039.1 plus vincristine results in a significant decrease in the glucose levels of diabetic compared to untreated mice. The therapeutic effect of IVA039.1 plus vincristine therapy was additive but surprisingly not synergistic. The binding of IVA039.1 to vincristine has moderate affinity with a slow off rate. In vitro studies suggest that, when bound to IVA039.1, the vincristine is inactivated. We attribute the lack of an enhanced therapeutic response to bifunctional antibody therapy using IVA039.1 plus vincristine to the inaccessibility of the drug to the target cells.

Characterization of cDNAs encoding the murine interleukin 2 receptor (IL-2R) gamma chain: chromosomal mapping and tissue specificity of IL-2R gamma chain expression.

The IL-2R gamma chain (IL-2R gamma) is an essential component of high- and intermediate-affinity IL-2Rs, playing critical roles for ligand binding and internalization. Recently, our laboratory has demonstrated that IL-2R gamma mutation results in X chromosome-linked severe combined immunodeficiency in humans, suggesting that IL-2R gamma plays a vital role in thymic maturation of human T cells. We now report the isolation and characterization of cDNAs encoding murine IL-2R gamma. The open reading frame encodes 369 aa, identical in length to that encoded by the human IL-2R gamma cDNA. Murine IL-2R gamma and human IL-2R gamma have 69% and 70% identity at the nucleotide and amino acid levels, respectively. As expected, the murine IL-2R gamma retains the WSXWS motif and four cysteine residues characteristic of cytokine receptor superfamily members. IL-2R gamma mRNA distribution shows significant tissue specificity, with particularly high-level expression in spleen and thymus, and higher expression in single-positive (CD4+8- or CD4-8+)-enriched thymocytes than in double-negative (CD4-8-) thymocytes. Finally, we have localized the murine IL-2R gamma gene, Il2rg, to the X chromosome between Rsvp and Plp and demonstrated that a defect in IL-2R gamma is not responsible for the X chromosome-linked xid mutation, which maps to this same region. The cloning of the murine IL-2R gamma cDNA will facilitate the investigation of the role of this protein in lymphocyte function and thymic development.

Immunophysiological studies of interleukin-2 and canine lymphocytes

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrlL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrlL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dog's PBL to hrlL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.

Molecular cloning and expression of the human interleukin 5 receptor.

Human interleukin 5 (IL-5) plays an important role in proliferation and differentiation of human eosinophils. We report the isolation of cDNA clones from cDNA libraries of human eosinophils by using murine IL-5 receptor alpha chain cDNA as a probe. Analysis of the predicted amino acid sequence indicated that the human IL-5 receptor has approximately 70% amino acid sequence homology with the murine IL-5 receptor and retains features common to the cytokine receptor superfamily. One cDNA clone encodes a glycoprotein of 420 amino acids (Mr 47,670) with an NH2-terminal hydrophobic region (20 amino acids), a glycosylated extracellular domain (324 amino acids), a transmembrane domain (21 amino acids), and a cytoplasmic domain (55 amino acids). Another cDNA encodes only the extracellular domain of this receptor molecule. Other cDNA clones encode molecules having diversified cytoplasmic domains. COS7 cells transfected with the cDNA expressed a approximately 60-kD protein and bound IL-5 with a single class of affinity (Kd = 250-590 pM). The Kd values were similar to that observed in normal human eosinophils. In contrast to the murine 60-kD alpha chain, which binds IL-5 with low affinity (Kd = approximately 10 nM), the human alpha chain homologue can bind IL-5 with much higher affinity by itself. RNA blot analysis of human cells demonstrated two transcripts (approximately 5.3 and 1.4 kb). Both of them were expressed in normal human eosinophils and in erythroleukemic cell line TF-1, which responds to IL-5. The human IL-5 receptor characterized in this paper is essential for signal transduction, because expression of this molecule in murine IL-3-dependent cell line FDC-P1 allowed these cells to proliferate in response to IL-5.

The murine interleukin-4 receptor gene: genomic structure, expression and potential for alternative splicing.

The structure of the murine interleukin-4 receptor (mIL-4R) gene has been determined. The gene spans approximately 25 kilobases (kb) of DNA and is composed of 12 exons interrupted by 11 introns. The gene contains sequences accounting for all the sequences present in the functional mIL-4R cDNAs, including several exons which can encode DNA inserts found in recently cloned IL-4R cDNA variants. Thus expression of the gene may be regulated, at least in part, by alternative splicing. A combination of S1 nuclease protection and primer extension assays was used to localize the 5' end of the gene and demonstrated the use of multiple transcription initiation sites within this region. We have found that the overall intron-exon organization of the murine IL-4R gene is markedly similar to that of the murine erythropoietin receptor (m-epoR) and the human interleukin-2 receptor beta chain (hIL-2R beta), as well as the human growth hormone receptor (hGhR). This is consistent with the recent grouping of these receptors, on the basis of protein sequence homology, into the hematopoietin receptor gene superfamily. Such homology at the levels of both protein and gene structure suggest a divergent evolution of the receptors from a single primordial gene.

Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4.

DAB389-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-related fusion protein which has been shown to be selectively toxic to cells expressing the mIL-4 receptor. In this report, we have used site-directed and in-frame deletion mutagenesis to study the role of the putative C-terminal alpha-helix (helix E) of the mIL-4 component of DAB389-mIL-4 in the intoxication process. We demonstrate that deletion of the C-terminal 15 amino acids of the fusion toxin leads to loss of cytotoxicity. The substitution of Phe496 with either Pro, Ala or Tyr, results in a greater than 20-fold decrease in cytotoxic activity of the respective mutant fusion toxins. In addition, substitution of Leu497 with either Ala or Glu results in a similar loss of cytotoxic activity. All of these mutant forms of the mIL-4 fusion toxin demonstrate a significant decrease in binding affinity (Ki) to the mIL-4 receptor in a competitive radioligand binding assay. In marked contrast, however, the substitution of Asp495 with Asn results in a 4-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro.

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.