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The Journal of Immunology, 1998, 161: 3474-3483.
Copyright © 1998 by The American Association of Immunologists

Molecular Mapping with Functional Antibodies Localizes Critical Sites on the Human IL Receptor Common {gamma} ({gamma}c) Chain

Natalya Raskin1, Aniela Jakubowski1, IreneDougas Sizing, Dian L. Olson, Susan L. Kalled, Catherine A. Hession, Christopher D. Benjamin, Darren P. Baker and Linda C. Burkly2

Biogen, Inc., Cambridge, MA 02142


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The IL receptor common {gamma} ({gamma}c) chain is required for the formation of high affinity cytokine receptor complexes for IL-2, IL-4, IL-7, IL-9, and IL-15, and for signals regulating cell survival, growth, and differentiation. Our current understanding of how {gamma}c chain associates with multiple ligands and receptor subunits is drawn largely from its structural homology to the human growth hormone (hGH) receptor and known structure of the hGH/hGH receptor complex. These receptors share distinct features in their extracellular portions and are believed to function by a mechanism of ligand-induced association of receptor subunits. Here, we report the first directed mutational analysis of the human {gamma}c chain by alanine scanning conducted across seven regions likely to contain residues required for intermolecular contact. Functionally distinct, neutralizing anti-{gamma}c mAbs were employed to define critical residues. One particular mAb, CP.B8, unique in its ability to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced proliferation and high affinity cytokine binding of normal T cells as an intact mAb and as a Fab fragment, localized critical residues to four noncontinuous stretches, namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Notably, these residues form a contiguous patch on the {gamma}c chain surface in a three-dimensional structural model. These results provide functional evidence for the location of contact points on {gamma}c chain required for its association with multiple ligands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
X-linked SCID (XSCID)3 is a disease characterized by profoundly diminished cellular and humoral immunity (1, 2). In XSCID patients, mature T cells and NK cells are greatly reduced or absent due to impaired development of these cell types. Peripheral B cells are found at normal or relatively high numbers although specific Ab production is defective. XSCID is caused by mutation in the gene encoding the IL receptor common gamma chain, referred to hereafter as the {gamma}c chain (3, 4).

The {gamma}c chain is a subunit of the IL-2R (5) and of other cytokine receptors, including those for IL-4, IL-7, IL-9, and IL-15 (6, 7). The IL-2R is composed of two (IL-2Rß/{gamma}c) or three (IL-2R{alpha}/IL-2Rß/{gamma}c) chains, whereas the IL-4, IL-7, and IL-9 receptors each have a unique {alpha}-chain that associates with {gamma}c. The IL-15R also utilizes three chains, a unique {alpha}-chain, IL-2Rß, and {gamma}c (8, 9). Partnering of {gamma}c with other subunits increases receptor affinity for cytokine. In addition, it is absolutely required for many ligand-induced signals (10, 11, 12). This critical role in cytokine signaling is mediated by association of the {gamma}c cytoplasmic portion with Janus kinase 3 (Jak3) (13, 14). In addition to regulating T and NK cell development, the {gamma}c chain may regulate the activation, differentiation, and death of peripheral T cells (15, 16, 17), as well as that of a variety of other mature cell types, including monocytes, macrophages, granulocytes, and certain intestinal epithelial cells (18, 19). Thus, it is important to define both structurally and functionally the role of the {gamma}c chain in the association and activation of the {gamma}c chain-dependent cytokine receptors.

The {gamma}c chain and many receptor subunits that associate with the {gamma}c chain belong to a large family of structurally related receptors that mediate cell growth and differentiation, known as the Class I cytokine receptor family or hemopoietin receptors (20, 21, 22). These receptors are type I transmembrane glycoproteins that have a distinct structure, consisting of about 200 amino acids that form two fibronectin (FN) type III domains in their extracellular portion. Their hallmark is the presence of canonical motifs, a set of four conserved cysteines in the membrane distal domain and a WSXWS motif in the membrane proximal domain. In addition, each domain is comprised of seven ß strands whose sequences are conserved between members of the family, while loop sequences connecting the ß strands vary between family members and putatively contain residues that mediate distinct intermolecular contacts. These receptors are believed to function by a mechanism of ligand-induced association of receptor subunits, the best characterized example of which is provided by the homodimeric receptor for hGH (23). By contrast, the IL-2R{alpha}- and IL-15R{alpha}-chains are structurally distinct from this superfamily, though related to each other. Soluble ligands for these receptors are also structurally related to each other and belong to the four-helix bundle family of cytokines (24).

Hypothetical molecular models have been constructed of the {gamma}c chain complexed with IL-2 and the IL-2Rß-chain, and of the {gamma}c chain complexed with IL-4 and the IL-4R{alpha}-chain (25, 26). These models are based on a structural paradigm provided by the crystal structure of hGH bound to its receptor. In this case, two identical receptor molecules are known to complex with a single molecule of hGH (27). This structure, together with independent mutational analysis of the hGH receptor, have defined the hormone/receptor and interreceptor chain contacts and their relative contribution to complex formation (28). However, the extracellular residues on the {gamma}c chain, which are critical for interacting with various cytokines and receptor subunits, are still poorly defined. DNA sequence analysis of naturally occurring {gamma}c chain mutations have made only limited contributions to delineating this structure/function relationship since many XSCID patients harbor nonsense mutations, frame-shift mutations, or splicing defects (as reviewed in 6 . While at least some point mutations have been identified that abrogate or diminish high affinity IL-2 binding (4, 29, 30, 31, 32) and many other point mutants have been identified (33), cell surface expression and/or function of these mutants is not well characterized.

Herein we report the first mutational analysis of the human {gamma}c chain, providing functional evidence for the location of critical sites required for {gamma}c chain activity. This structure/function analysis was accomplished through the use of distinct {gamma}c chain-specific mAbs as probes and a panel of alanine substitution mutants of the {gamma}c chain. Alanine substitutions were targeted to interconnecting loop residues in each of the two fibronectin type III domains. Mutations that selectively impaired the binding of a unique neutralizing mAb, CP.B8, were of particular interest, given the ability of the Ab to inhibit high affinity cytokine binding and cytokine-dependent cell proliferation as a Fab fragment. Residues critical for CP.B8 binding were localized in the AB and EF loops of the N-terminal domain, the interdomain (ID) segment, and the FG loop of the membrane proximal domain. These results provide experimental evidence for the identity of amino acid residues comprising the cytokine binding interface on the {gamma}c chain, or in close proximity to those comprising this interface, which are required for effective recruitment of the {gamma}c chain and formation of a signal-transducing complex. Common residues required for the interaction of the {gamma}c chain with IL-2, IL-4, IL-7, and IL-15 are indicated. In addition, the potential utility of the distinct anti-{gamma}c chain mAbs as tools for mutational diagnosis and for defining the nature of defects caused by XSCID genotypes is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary human cells

Human PBMC were isolated from healthy donors by Ficoll-Paque density gradient centrifugation (Pharmacia Biotech, Piscataway, NJ). PBMC were cultured in a 37°C, 5% CO2 humidified incubator at 106 cells/ml in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 µg/ml PHA (Difco Laboratories, Detroit, MI) for 3 days to polyclonally activate T lymphocytes. Where indicated, PBMC were T cell enriched before PHA activation by depleting monocytes, B cells, and NK cells with magnetic beads coated with anti-CD14 and anti-CD19 mAbs (Dynal, Lake Success, NY) and with an anti-CD56 mAb (Perseptive Diagnostics, Cambridge, MA).

Cell lines

Stable transfectants expressing the human {gamma}c chain on their surface (L929/{gamma}c) were generated by electroporation of the murine L929 fibroblast cell line (American Type Culture Collection (ATCC), Manassas, VA) with a full length human {gamma}c chain cDNA in the pMDR901 expression vector, and a plasmid containing the neomycin resistance gene. L929 cells were maintained in Eagle’s MEM supplemented with 10% FBS, 1 mM nonessential amino acids, and 4 mM L-glutamine, while L929/{gamma}c cells were grown in the same medium supplemented with 0.27 µg/ml of geneticin (Life Technologies, Grand Island, NY).

DNA constructs

The full-length human {gamma}c chain cDNA was cloned from a Jurkat T cell cDNA library constructed in the pCDM8 vector (34), using an oligonucleotide probe matching nucleotides 592–620 of the published sequence (5). The isolated clone, p4A1, contained a NotI insert encoding the {gamma}c cDNA with a single nucleotide difference from that of Takeshita et al. (5); a T in the coding region of clone p4A1 replaced a C at nucleotide 673 of the published sequence. Thus the protein encoded by p4A1 contains a methionine instead of a threonine in the extracellular portion at position 220, located in predicted ß strand F of domain 2, numbering from the signal sequence (5). However, this encoded form of the {gamma}c chain was functionally competent as measured by its ability to mediate high affinity IL-4 binding when cotransfected into COS-7 cells with the IL-4R{alpha}-chain, resulting in a dissociation constant (KD) of 200 ± 100 pM,4 an affinity approximately threefold higher than that mediated by the IL-4R{alpha}-chain alone, and in agreement with Russell et al. (35). In addition, the encoded {gamma}c chain mediated IL-2 binding when cotransfected with the IL-2Rß-chain, with a KD of approximately 1 nM, also in agreement with published results (7). Similar cytokine binding data were obtained with a myc/{gamma}c cDNA expression construct (D. Olson et al., manuscript in preparation) engineered to encode the full-length {gamma}c chain with a nine-residue myc peptide tag (EQKLISEEDL) fused to the 5' end of the mature protein, thereby allowing detection of cell surface-expressed {gamma}c chain and mutants thereof with the anti-myc mAb 9E10. This expression construct was made as follows. A blunt end/BsaHI fragment encoding the myc tag, and the first six residues of the mature {gamma}c chain protein (the terminal L of the myc peptide being the initial {gamma}c chain residue) was formed by annealing complementary oligos. This fragment was then fused downstream of a NotI/blunt end fragment encoding the VCAM-1 gene signal sequence and upstream of a 1361-bp BsaHI/NotI fragment encoding the {gamma}c cDNA from residue seven of the mature protein to the 3' end. The resulting NotI fragment was inserted into the NotI site of the pCDM8 expression vector (34) for transient expression studies and into the NotI site of the pMDR901 vector for stable expression. A {gamma}c-Ig fusion protein was constructed by PCR amplification of the {gamma}c chain extracellular sequence from template plasmid p4A1, using the following 5' primer containing a NotI cloning site, 5'-AACTGCAGCGGCCGCCATGGTGAAGCCATCATTACC-3', and the following 3' primer that contains a SalI cloning site, 5'-GACTTTGTCGACATTCTCTTTTGAAGTATTGC-3'. The resulting 789-bp PCR fragment was cut with NotI and SalI and ligated 5' of a 693-bp SalI/NotI fragment encoding the ten amino acids of the human IgG1 hinge region and full sequence of the human IgG1 CH2 and CH3 regions isolated from pSAB144 (36). This NotI fragment was then cloned into a NotI cleaved expression vector pSAB132. The resulting {gamma}c-Ig fusion construct, pLB001, encodes the amino terminal 254 amino acids of the mature human {gamma}c chain fused to ten amino acids of the hinge region of human IgG1 and the CH2 and CH3 constant domains of IgG1.

{gamma}c chain mutagenesis

Alanine substitutions were targeted to interconnecting loop residues based on sequence homology between the {gamma}c chain and the hGH receptor (25, 26). For a given interconnecting loop, double and triple point mutations were made, thereby changing two or three contiguous amino acid residues to alanine. Successive mutations were designed so as to scan through the loop sequences. Mutations were made in the myc/{gamma}c cDNA sequence carried in a sequencing vector, pLB013, with the USE mutagenesis kit according to manufacturer’s specifications (Pharmacia Biotech). The mutations were confirmed by the presence of a novel internal restriction site and by DNA sequencing. DNA fragments containing the mutant sequence were subcloned into the myc/{gamma}c cDNA expression vector, replacing the corresponding wild-type sequence, and were confirmed by presence of the novel restriction site.

Generation of anti-{gamma}c chain mAbs

mAbs specific for the human {gamma}c chain were generated by immunizing female RBF mice with a {gamma}c-Ig fusion protein transiently expressed after electroporation of COS-7 cells with pLB001. The {gamma}c-Ig, affinity purified from a 2- to 3-day culture supernatant, was applied to protein A-Sepharose 4B Fast Flow resin (Pharmacia Biotech), washed with 25 mM Na2HPO4 (pH 5.0) and 100 mM NaCl to remove any bound bovine IgG derived from the culture medium, and eluted with 25 mM NaH2PO4 pH 2.8 with immediate postelution neutralization. Mice were immunized with {gamma}c-Ig bound to protein A-Sepharose resin, and fusion of spleen cells to the FL653 myeloma (Fisher Scientific, Pittsburgh, PA) was conducted according to standard procedures. Clones were screened for binding to the immunogen by solid phase ELISA and by differential immunofluorescent staining of L929 cells vs L929/{gamma}c cells. All of the selected mAbs were of the mouse IgG1 subclass and were affinity purified with protein A-Sepharose 4B resin, with elution at pH 5.0 with immediate neutralization.

Reagents

Recombinant human cytokines were purchased as follows: IL-2 and IL-15 (R&D Systems, Minneapolis, MN), IL-4 (BioSource International, Camarillo, CA), and IL-7 (Genzyme, Cambridge, MA). The anti-myc mAb 9E10 (mouse IgG1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-IL-2Rß-chain mAb Mik-ß2 (mouse IgG2a) was purchased from PharMingen (San Diego, CA). MOPC 21, a murine IgG1 control protein, was purified from a myeloma culture supernatant using protein A-Sepharose 4B Fast Flow resin (Pharmacia Biotech), and UPC 10 (mouse IgG2a) was purchased from ICN Pharmaceuticals (Costa Mesa, CA). Fab fragments of the anti-{gamma}c chain mAbs and MOPC 21 control IgG1 were generated proteolytically and purified with the ImmunoPure Fab Preparation Kit (Pierce, Rockford, IL). Biotin-conjugated mAbs and Fab fragments were prepared using ImmunoPure NHS-Biotin according to the manufacturer’s recommendations (Pierce).

Immunofluorescent staining

Murine anti-{gamma}c mAbs, anti-myc mAb 9E10, and MOPC 21 control IgG1 were added at specified concentrations to 3 x 105 cells suspended in PBS containing 1% BSA and 0.02% NaN3. In the case of L929 lines or COS-7 transfectants, adherent cell monolayers were recovered from culture by washing with PBS and treatment with 5 mM EDTA, 1% BSA for 10 min at 37°C. Cells were incubated with primary Ab for 30 min at 4°C and washed three times in the same buffer. Bound mAb was detected by incubation with phycoerythrin-conjugated goat anti-mouse IgG H+L (Jackson ImmunoResearch Labs, West Grove, PA). Cells were washed three times and fixed in 2% paraformaldehyde. Fluorescence was measured with a FACScan (Becton Dickinson, San Jose, CA). Cross-competition of biotin-conjugated mAbs was conducted by incubation of 5 x 105 PHA-activated PBMC with 10 µg/ml of unconjugated mAb for 45 min at 4°C, and then 2 µg/ml of biotinylated mAb was added. The mixture was incubated for an additional 30 min at 4°C and washed three times; then phycoerythrin-conjugated streptavidin (Jackson ImmunoResearch) was added to detect binding of biotinylated mAb. After 30 min at 4°C, cells were washed three times, and fluorescence was measured.

Analysis of {gamma}c mutants

Plasmid DNA prepared from CsCl gradients for each of the mutant constructs, along with the parental myc/{gamma}c cDNA (positive) and pCDM8 vector (negative) controls, were electroporated into COS-7 cells. After 3 days, cells were recovered and analyzed by immunofluorescent staining with 10 µg/ml of anti-{gamma}c mAbs, anti-myc mAb 9E10, and MOPC 21 control IgG1. Using mean fluorescence intensity (MFI) values as a measure of binding, specific anti-{gamma}c mAb binding was obtained by subtracting background binding obtained with MOPC 21 IgG1. Similar background values were obtained for binding of anti-{gamma}c mAbs to mock transfected cells. MAb binding to mutant {gamma}c molecules is presented as a percentage of the positive control (% binding) and normalized for expression level based on mAb 9E10 staining. A value of 100% indicates that mAb binding to a mutant {gamma}c chain molecule is unaffected by the mutation.

Cell proliferation assays

The MLR of freshly isolated human PBMC was assessed by culturing them with irradiated (3000 rad) allogeneic PBMC in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured in flat-bottom 96-well plates, with responder cells at 2 x 105 cells/well, and responder/stimulator cell ratios of 1:1 and 2:1. Cells were cultured in the presence and absence of mAb, with triplicate wells for each culture condition, and assessments employed at least four independent donor/stimulator pairs. Cultures were maintained for a total of 4 to 5 days and pulsed with 2 µCi of [3H]thymidine during the last 16 h. Proliferation was measured by [3H]thymidine incorporation using an LKB plate reader (Pharmacia LKB, Gaithersburg, MD). Data are expressed as % inhibition of proliferation in cultures treated with mAb or control IgG relative to untreated cultures.

Cytokine-dependent proliferation assays were conducted using human T cell-enriched PBMC after 3 days of stimulation with PHA, followed by an overnight incubation at 106 cells/ml in medium without PHA. Recovered cells were then cultured in 96-well round-bottom plates at 5 x 104 cells/ well in the presence or absence of anti-{gamma}c chain mAbs or control IgG at specified concentrations. After 30 to 45 min at 37°C, recombinant human cytokines, IL-2, IL-4, IL-7, or IL-15, were added at specified concentrations ranging from 1.1–3.3 ng/ml, generally supporting 50 to 80% of the maximal growth response. Cells were cultured with cytokine for 40 to 44 h, and growth was measured by [3H]thymidine incorporation during the final 8 to 16 h of culture. Data are expressed as the mean cpm of triplicate wells. The specificity of these cytokine-dependent assays was established previously with neutralizing Abs against individual cytokines. Neutralizing Abs employed were the anti-IL-2 mAb clone 5334.21 (mouse IgG1), goat anti-IL-4 Abs, goat anti-IL-7 Abs, and anti-IL-15 mAb clone 34593.11 (mouse IgG1) (R&D Systems). Abs directed against the particular cytokine that was added exogenously to the culture system resulted in >90% inhibition of cell proliferation, whereas Abs against other cytokines whose receptors employ the {gamma}c chain resulted in <10% inhibition.

IL-7-dependent growth of freshly isolated PBMC (37, 38) also was assessed using T cell-enriched PBMC cultured at 5 x 104 cells per round-bottom well with 1.1 ng/ml of IL-7 for 5 days, with growth measured by [3H]thymidine incorporation during the final 8 to 16 h of culture and data expressed as the mean cpm of triplicate wells.

Cytokine binding assays.

PHA-activated PBMC were recovered after 3 days of culture, washed with PBS, and resuspended in PBS, 1% FCS. Cells were added to Falcon 2052 tubes (Becton Dickinson Labware, Lincoln Park, NJ) with 1.5 x 106 cells per tube and incubated with and without mAbs in a total volume of 150 µl for 1 h at 4°C on a rotating platform. mAbs employed were anti-{gamma}c chain mAbs, anti-IL-2Rß-chain mAb Mik-ß2, MOPC 21 and UPC 10 control IgG, and Fab fragments of CP.B8 and MOPC 21. 125I-labeled IL-2 (New England Nuclear, Boston, MA) was then added in 50 µl to achieve a final concentration of 10 pM radiolabeled IL-2, designed as such to assess the effect of mAbs on high affinity IL-2 binding (7). The cells were incubated for 30 additional min while shaking at ambient temperature and then washed twice with 2 ml of PBS, 1% FBS; the cell pellet was counted in a Wallac (WALLAC, Gaithersburg, MD) 1470 gamma counter. Each condition was assessed in duplicate tubes in a given experiment; the cpm values were averaged and corrected for nonspecfic IL-2 binding by deducting the amount of cpm bound from a sample that contained 10 pM radiolabeled IL-2 and a 100-fold molar excess of nonradioactive IL-2 according to standard methods. Data are expressed as percent inhibition of IL-2 binding relative to that obtained in the absence of mAb.

Statistical analysis

For the MLR cultures and cytokine binding assays, comparisons over several groups were made using a one-way ANOVA, followed by Dunnett’s test for multiple comparisons to a control group (two-tailed). P values < 0.05 were taken to be statistically significant. For the analysis of mAb binding to {gamma}c chain mutants, two group comparisons were made using an unpaired two-tailed Student t test, comparing each percent binding value to 100%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Functional inhibition by distinct mAbs specific for the human cytokine receptor {gamma}c chain

mAbs specific for the human {gamma}c chain bind to the surface of L929/{gamma}c transfectants (Fig. 1GoB) and show no detectable binding to the parent L929 cell line (Fig. 1GoA) relative to an isotype-matched IgG control. Profiles for three such mAbs are shown and are representative of the results obtained with a broader panel of anti-{gamma}c chain mAbs. These mAbs also detect the naturally expressed molecule, as shown by immunofluorescent staining of human PHA-activated T lymphocytes (Fig. 1GoC). The specificity of these mAbs was also confirmed by their ability to immunoprecipitate the full-length {gamma}c chain from the surface of the HUT 78 cell line (data not shown).



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  		FIGURE 1. Cell surface binding of mAbs specific for human {gamma}c chain. Immunofluorescent staining of (A) L929 cells, (B) L929/{gamma}c stable transfectants, and (C) human PHA-activated T lymphocytes with 10 µg/ml of anti-{gamma}c chain mAbs CP.B8, AF.F4, and CQ.C11 (each mAb indicated by a solid line) and MOPC 21 control IgG1 (dotted lines).

 
The ability of individual mAbs to cross-block binding of other mAbs was determined to establish the relationship between their epitopes. The results are summarized in Table IGo. Binding of each biotin-conjugated mAb was inhibited specifically by its respective unconjugated form with only 0 to 11% maximal inhibition by the unconjugated IgG1 control. The pattern of cross-competition between different mAbs revealed five distinct epitope groups. One epitope group is comprised of mAbs AF.F4, AK.F12, and CQ.C11, given nearly complete cross-competition between members of this group but not with other mAbs, except BI.B12. Similarly, mAbs AE.F8, CJ.F4, and AH.H8 are related to each other but are distinct from other mAbs, except BI.B12. By contrast, mAb CP.B8 exhibits a unique specificity, only partially cross-blocking with other mAbs, AK.F12 and CQ.C11 in particular. Likewise, mAb AE.C9 constitutes a unique specificity. The effect of mAb BI.B12 indicates some overlap or spatial proximity between the BI.B12 epitope and epitopes recognized by two other epitope groups, represented by AF.F4 and AE.F8, respectively.


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  		Table I. Cell-based inhibition of binding of biotinylated anti-{gamma}c chain mAbs by unconjugated mAbs

 
Functional activity of the {gamma}c chain-specific mAbs was assessed by measuring their effect on proliferation of freshly isolated human PBMC cultured with allogeneic targets, a response known to be highly IL-2 dependent. Representative MLR data for several mAbs are shown in Figure 2Go, indicating that different {gamma}c chain-specific mAbs were able to inhibit significantly the proliferative response. On average, partial but significant inhibition was observed with mAbs AF.F4 and CQ.C11, i.e., 30% inhibition was achieved by 1 µg/ml of either mAb. A plateau or bell-shaped inhibition curve was observed as mAb dose increased further. Similar patterns of inhibition were observed with other mAbs in the panel. By contrast, inhibition by mAb CP.B8 tended to increase with increasing mAb dose, an average of 60% inhibition observed at the highest dose tested (100 µg/ml). Greater inhibition was observed when different noncross-blocking mAbs were used in combination; for example, 78% blocking was achieved by the CP.B8/AF.F4 pair with 50 µg/ml of each mAb.



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  		FIGURE 2. Inhibition of MLR of human PBMC by anti-{gamma}c chain mAbs. Values shown are the mean ± SEM of 4 to 6 determinations for mAbs CP.B8, CQ.C11, AF.F4, and for MOPC 21 control IgG1. The percent inhibition of proliferation by the anti-{gamma}c chain mAbs CP.B8 and AF.F4 is significant at doses of 1, 10, and 100 µg/ml as compared with MOPC 21 control IgG1, p < 0.05. The percent inhibition of proliferation by the anti-{gamma}c chain mAb CQ.C11 is significant at doses of 1 and 10 µg/ml as compared with MOPC 21, p < 0.05.

 
These findings demonstrate that the Ag-induced expansion of mature lymphocytes from normal individuals is {gamma}c-chain dependent, with significant functional inhibition with different mAb specificities.

Anti-{gamma}c chain mAbs display broad cytokine inhibitory activity: unique mechanism of action of mAb CP.B8

To determine whether or not these {gamma}c chain-specific mAbs would inhibit the functional responses to different cytokines, we assessed the growth of PHA-activated T cell blasts in response to exogenous IL-2, IL-4, IL-7, and IL-15. Individual cytokines were added at doses sufficient to induce at least 50% of maximum stimulation, and the growth response of cells cultured in the presence or absence of anti-{gamma}c chain mAbs was measured. In the absence of added cytokine, only low levels of proliferation occurred. Representative results show that mAb CP.B8 and other mAbs inhibited the IL-2-, IL-15-, and IL-4-induced responses (Fig. 3Go, A, B, and D, respectively), although inhibition was relatively greater in the IL-15 and IL-4 cultures. As observed previously for MLR cultures, CP.B8-mediated inhibition increased with increasing mAb dose. By contrast, other mAbs, including AF.F4, AK.F12, CQ.C11, AE.F8, BI.B12, and AE.C9, achieved maximum inhibition between 1 to 10 µg/ml, with a plateau or decreased inhibition thereafter (Fig. 3Go). Similar results were obtained for IL-7-induced cellular responses (Fig. 3GoC); however, the inhibitory capability of mAb CP.B8 was not consistently apparent when assessing the IL-7-dependent response of PHA-blasts. MAb CP.B8 did significantly inhibit the IL-7-induced growth of freshly isolated PBMC (Fig. 3GoC, inset). Thus, anti-{gamma}c chain mAbs belonging to different epitope groups exhibited broad neutralizing activity against four specific cytokines whose receptors employ the {gamma}c chain.



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  		FIGURE 3. Inhibition by anti-{gamma}c chain mAbs of the cytokine-dependent proliferation of PHA-activated T cells. Exogenous cytokines added are (A) IL-2, (B) IL-15, (C) IL-7, and (D) IL-4. Inhibition of proliferation of freshly isolated PBMC in response to exogenous IL-7 is shown in C, inset. The effect of anti-{gamma}c chain mAbs CP.B8, AF.F4, AK.F12, CQ.C11, AE.F8, BI.B12, and MOPC21 control IgG1 are shown over the same dose range. Background responses in the absence of exogenous cytokine and mAb, are shown (A-D, filled diamonds). The results are representative of at least two independent experiments, with values from one experiment shown.

 
The requirements for mAb-mediated inhibition were dissected by employing Fab fragments of mAbs representative of the different epitope groups. The effects of intact mAbs and their Fab fragments on cytokine-induced growth of PHA-blasts were compared directly (Fig. 4Go, A and B). While the intact mAbs AF.F4, CQ.C11, BI.B12, and AE.C9 reproducibly inhibited IL-2-dependent cell proliferation, their Fab fragments failed to inhibit the IL-2-induced growth response even at high doses. Nonetheless, these Fab fragments were active based on the ability of each Fab preparation to inhibit completely the binding of its intact biotinylated mAb to PHA-blasts, at doses of 0.4 to 1.25 µg/ml Fab and 1 µg/ml intact biotinylated mAb. In side by side assessments, complete inhibition of binding of each biotinylated mAb was achieved by 0.4 µg/ml of its unconjugated, intact mAb (data not shown). By contrast, the ability of mAb CP.B8 to inhibit IL-2-induced responses was clearly retained by its Fab fragment (Fig. 4GoC).



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  		FIGURE 4. Effect of anti-{gamma}c chain mAbs and Fab fragments on IL-2-dependent proliferation of PHA-activated T cells. The response to exogenous IL-2 is shown in the presence of (A) anti-{gamma}c chain mAbs AF.F4, CQ.C11, BI.B12, AE.C9, and MOPC 21 control IgG1, MOPC 21 (B) their Fab fragments, and (C) mAb CP.B8 mAb and its Fab fragment. Background responses in the absence of exogenous cytokine and mAb, are shown (A–C, open diamonds). The results are representative of two independent experiments, with values from one experiment shown.

 
The ability of CP.B8 to block cytokine responses as an intact mAb or as a Fab fragment indicated that it did not require the size, cross-linking, or Fc-mediated functions of an intact mAb. Rather, the data suggest that CP.B8 mediates its effect by blocking directly a critical site on the {gamma}c chain required for the recruitment of {gamma}c chain into a productive signaling complex. Therefore, the ability of CP.B8 to interfere with high affinity IL-2 binding was examined using 125I-labeled IL-2 at a concentration of 10 pM, the KD for high affinity IL-2 binding. Our results show that the binding of radiolabeled IL-2 to PHA-blasts was inhibited in a dose-dependent manner by mAb CP.B8 and by its Fab fragment (Fig. 5GoA), with no inhibition by the IgG1 control mAb MOPC 21 or its Fab fragment. Inhibition achieved by mAb CP.B8 was comparable to that exhibited by the anti-IL-2Rß-chain mAb Mik-ß2 (Fig. 5GoA). By contrast, mAb CQ.C11 exhibited a significant but low level of inhibition whereas no significant inhibition was observed with other mAbs (Fig. 5GoB). Thus, these data indicate that mAb CP.B8 can interfere with the formation of a stable high affinity cytokine/receptor complex, apparently by blocking directly a critical site on {gamma}c chain, whereas inhibition by other mAbs requires additional steric effects and/or other mechanism(s).



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  		FIGURE 5. Effect of anti-{gamma}c chain mAbs on IL-2 binding (10 pM) to PHA-activated PBMC. IL-2 binding in the presence of (A) anti-{gamma}c chain mAb CP.B8 and its Fab fragment, control IgG1 mAb MOPC1 21 and its Fab fragment, and anti-IL-2Rß-chain mAb Mik-ß2, and (B) 100 µg/ml of anti-{gamma}c chain mAb CP.B8, AF.F4, CQ.C11, BI.B12, the control IgG1 mAb MOPC 21, and mAb Mik-ß2. For (A) values shown are the mean ± SD of two independent experiments. Inhibition by mAbs Mik-ß2 and CP.B8 is significant at 1, 10, and 100 µg/ml as compared with mAb MOPC 21, and inhibition by the CP.B8 Fab fragment is significant at 100 µg/ml as compared with the MOPC 21 Fab fragment, p < 0.05. For B, values shown are the mean ± SD of two independent experiments. Inhibition by mAbs Mik-ß2, CP.B8, and CQ.C11 is significant as compared with mAb MOPC 21 control IgG1, p < 0.05.

 
mAb binding to human {gamma}c chain alanine substitution mutants

The unique specificity and functional activity of mAb CP.B8 provided a valuable tool to map regions of the {gamma}c chain critical for its function. A panel of {gamma}c chain mutants was constructed, and mAb CP.B8 binding to these mutants was assessed and compared with the binding to the wild-type molecule. Binding of other mAbs selected from three distinct epitope groups was also assessed. This potentially offered a means to discern structural changes that caused a generalized loss of mAb binding activity, such as gross conformational changes.

Alanine scanning mutagenesis was conducted through seven putative interconnecting loops based on molecular models identifying these sequences, and indicating their potential contribution to the {gamma}c interface with cytokine or other receptor subunits (25, 26). Their location is shown in the linear {gamma}c sequence (Fig. 6Go, see bolded residues) and in a three-dimensional molecular model (Fig. 7GoA). Alanine substitutions were made to reveal the contribution of interactions made by side chains while minimizing conformational effects (39). In general, loops were scanned by substituting two or three residues at a time, with an overlap of one residue in consecutive mutants, as listed in Table IIGo. Mutations that caused a significant reduction in mAb binding are boxed as shown.



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  		FIGURE 6. Residues critical for mAb binding to human {gamma}c chain. The extracellular sequence of the human {gamma}c chain starting from residue 35 of the mature protein is shown, numbering from the first residue of the mature protein as +1, with predicted ß strands underlined and designated by upper case letters. Putative interconnecting loops scanned by alanine mutagenesis are bolded. The ß strands and loop regions are designated based on homology with the hGH receptor sequence (25). Anti {gamma}c chain mAbs are listed, and mutations that significantly reduced mAb binding by at least 50% are shown as solid bars, with nd indicating not determined.

 


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  		FIGURE 7. Three-dimensional model localizes the contact surface for intermolecular interactions on the human {gamma}c chain. A, An {alpha}-carbon trace of the human IL-4/IL-R{alpha}/{gamma}c chain complex showing the location of the putative interconnecting loops targeted by alanine scanning mutagenesis, with the IL-4R{alpha}-chain (blue), IL-4 (pink), {gamma}c chain (red), and {gamma}c chain residues that were mutated to alanine (yellow) colored as indicated. ID designates the interdomain residues connecting domains 1 and 2, with the other loops labeled as in Figure 6Go. B, An {alpha}-carbon trace of the human {gamma}c chain showing those residues that specifically reduced the binding of mAb CP.B8 when mutated to alanine. The side chains of residues 43–47 of loop AB domain 1 (blue), of residues 98–102 of loop EF domain 1 (yellow), of residues 128–131 of ID (white), and of residues 205–206 of loop FG domain 2 (green) are colored as indicated. The figures were produced using the program Quanta (Molecular Simulations, Burlington, MA) using the coordinates for a model of the human IL-4/IL-R{alpha}/{gamma}c chain complex (25) deposited at the Brookhaven Protein Database (accession number 1ill.pdb).

 

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  		Table II. Mutagenesis of {gamma}c chain localizes critical residues for binding of neutralizing {gamma}c chain-specific mAbs

 
We found that mAb CP.B8 binding was completely abrogated or reduced significantly by mutations in five of the seven loops, namely loops AB and EF of domain 1, the interdomain segment, and loops BC and FG of domain 2. The reductions in mAb CP.B8 binding were not due to gross changes in {gamma}c folding since the binding of other mAbs, such as BI.B12 and CJ.F4, was generally retained. Binding of other mAbs was also retained in many instances. For example, substitutions in residues F43N44V45, N128L129V130, V130I131P132, and F205N206P207, which reduced mAb CP.B8 binding to 9%, 0%, 21%, and 28%, respectively, caused little, if any, significant reduction in the binding of AF.F4, AK.F12, CQ.C11, and AE.F8. Thus, mutations that preferentially impair mAb CP.B8 binding were identified in three noncontiguous stretches by alanine substitutions at F43N44V45, N128L129V130I131P132, and F205N206P207. In the latter two cases, mAb CP.B8 binding was retained by sequential mutants P132W133 and P207L208, indicating that changes in the N128L129V130I131 and F205N206 portions are critical.

In some instances, reduced binding of mAb CP.B8 and a subset of other mAbs was observed. For example, mutants L126Q127N128, H159C160L161, and L161E162H163 significantly reduced binding of mAb CP.B8 and other mAbs, including AE.F8, by >50%. Since there was no significant cross-blocking between mAbs CP.B8 and AE.F8 (Table IGo), these data suggest that loss of binding is due to an indirect effect of the mutation, possibly consequent to a change in folding that affected multiple but not all epitope groups. In addition, there are mutations that reduce binding of mAb CP.B8 and one or more members of the AF.F4 epitope group, i.e., AF.F4, AK.F12, and/or CQ.C11. Notable examples are mutant V45E46Y47 and mutations in residues K98E99I100H101L102 in the EF loop of domain 1. Given that partial cross-blocking was observed between mAb CP.B8 and members of this group (Table IGo), these mutations likely localize residues that are important to their respective epitopes. However, it is unlikely that identical residues are shared by the CP.B8 epitope and other mAb epitopes. Rather, considering that three residues are substituted at once in any given mutant, these mutations most likely localize one or more residues important for mAb CP.B8 binding and one or more adjacent residues important for the binding of other mAbs. As such, these mutated stretches may contain critical points of contact for ligands of the {gamma}c chain.

The results of this mapping analysis are summarized schematically in Figure 6Go, with bars indicating sequences at which alanine substitution markedly reduced mAb binding. Notably, the residues that impair mAb CP.B8 binding and that are distant in the linear sequence are in close proximity in the context of the three-dimensional structural model (Fig. 7GoB), namely residues in loops AB and EF of domain 1, in the interdomain segment, and in loop FG of domain 2. Consistent with this, mAb CP.B8 bound to the intact {gamma}c chain but did not bind detectably to either domain 1 or domain 2 when expressed alone on the surface of COS-7 transfectants whereas other mAbs did bind to these isolated {gamma}c chain domains (A. Jakubowski, unpublished observation), indicating that residues contributed by both domains 1 and 2 form the epitope for mAb CP.B8.

In summary, mutational analysis of the {gamma}c chain and epitope mapping for a unique neutralizing mAb have identified sites on the {gamma}c chain required for mAb CP.B8 binding. The availability of multiple distinct mAb specificities has allowed us to distinguish residues that contribute uniquely to the conformational epitope of this Ab. Moreover, these data provide important functional evidence for the localization of {gamma}c residues required for functional responses to multiple cytokines.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Targeted mutational analysis of the human {gamma}c chain and use of a panel of distinct neutralizing anti-{gamma}c chain mAbs have identified residues required for mAb binding to this molecule. The ability of these mAbs to block {gamma}c chain function was established by their ability to inhibit cytokine-dependent T cell proliferation in vitro, including T cell responses to IL-2, -4, -7, and -15. Partial, but significant, inhibition was achieved by many of the anti-{gamma}c chain mAbs of four different epitope groups. In general, a maximum of 30 to 50% inhibition was observed at 1 to 10 µg/ml mAb, with a plateau or decreased inhibition at higher doses. We speculate that the decreased inhibition at higher doses reflects a shift from bivalent to monvalent binding, consistent with the lack of inhibitory activity exhibited by Fab fragments of these mAbs. While their mechanism of action is still unclear, most mAbs appear to inhibit {gamma}c chain function with little, if any, effect on cytokine binding. By contrast, one particular mAb, CP.B8, and its Fab fragment inhibited cytokine-dependent proliferation as well as high affinity IL-2 binding, the degree of inhibition increasing with mAb dose. Similarly, mAb CP.B8 and its Fab fragment inhibit {gamma}c chain-dependent IL-4 binding using 80-pM radiolabeled IL-4 (D. Olson, unpublished observations). The unique capability of CP.B8 to inhibit {gamma}c chain function as an intact mAb or as a Fab fragment, strongly supports the notion that CP.B8 acts by blocking critical sites on the {gamma}c chain. As such, mAb CP.B8 was particularly valuable for mapping contact points on the {gamma}c chain, providing evidence for their role as points of intermolecular contact for {gamma}c association with cytokines or other receptor subunits.

Critical residues for mAb CP.B8 binding to the {gamma}c chain have been localized to four noncontiguous stretches of the {gamma}c chain sequence. These residues are in loops AB and EF of domain 1, the interdomain segment, and loop FG of domain 2. While it is difficult to completely rule out possible indirect effects of mutations, changes in these sequences selectively reduced mAb CP.B8 binding with little, if any, effect on binding of other noncross-blocking mAbs. By contrast, changes in loop BC of domain 2 impaired binding of mAb CP.B8 as well as the binding of the noncross-blocking mAb AE.F8. We propose that the residues critical for mAb CP.B8 binding are identical, or are in close proximity, to those required for binding of the {gamma}c chain to cytokines or other receptor chains. This conclusion is supported further by the effects of {gamma}c chain mutation on IL-2 binding (D. Olson et al., manuscript in preparation). Interestingly, the discontinuous {gamma}c chain residues implicated by our studies map to a contiguous patch formed at the interface between domains 1 and 2 when placed in a three-dimensional structural model based on the hGH receptor paradigm. To ensure that the residues forming this contiguous patch were exposed on the surface of the {gamma}c chain and would therefore be accessible for binding to mAb CP.B8, calculations of the surface solvent accessibility were conducted using the program CHARMM (40). Of the 16 residues highlighted in Figure 7GoB as being important for mAb CP.B8 binding, only 2, V45 and V96, were buried, with all the other residues having surface accessibilities of between 7.4 and 111.3 Å2, with a mean value of 51.2 Å2. Therefore, while V45 and V96 may not make contact with mAb CP.B8, all other residues are likely to be sufficiently exposed to interact directly with the Ab. This finding is strikingly similar to that defined for the hGH receptor, wherein the 11 most important receptor residues constituting the functional epitope for binding to hGH map to a contiguous patch, comprised of residues from four interconnecting loops, namely loops AB and EF of domain 1, the ID segment, and loop BC of domain 2 (28). As such, the contiguous patch on the {gamma}c chain identified by our studies most likely constitutes a common surface for the interaction of the {gamma}c chain with its cytokines.

Thus, many of the important {gamma}c chain residues revealed by our studies are closely aligned with hGH receptor residues known to mediate contact with hGH. Notably, Y103 of the {gamma}c chain is aligned with W104 of hGH receptor, one of two tryptophan residues (W104 in loop EF of domain 1 and W169 in loop BC of domain 2) of the hGH receptor contributing more than 75% of the binding free energy for hGH (28). The importance of the {gamma}c chain Y103 residue is supported by our finding that substitution of L102Y103Q104 reduced mAb CP.B8 binding to 13%. In addition, the binding of mAbs AF.F4 and AK.F12, which partially cross-block with mAb CP.B8, was reduced by 50% or greater. Other areas of alignment include F43N44V45E46Y47 of the {gamma}c chain and R43E44 of the hGH receptor, both situated in the AB loop of domain 1, and V130I131 of the {gamma}c chain and D126E127 of the hGH receptor, both situated in the ID segment. By contrast, there is no {gamma}c chain residue homologous to W169 of the hGH receptor, and mutation in the BC loop of {gamma}c chain domain 2 diminished mAb CP.B8 binding but also impaired AE.F8 binding, suggesting that these residues are not direct contact sites on the {gamma}c chain. Alternatively, residues in the FG loop of domain 2 of the {gamma}c chain (F205N206) also were identified as critical. While there are no contributing residues in the homologous loop in the hGH receptor, the FG loop of the IL receptor common ß chain (ßc), another cytokine receptor family member, has been shown to contain a Y residue critical for binding of IL-3, IL-5, and granulocyte-macrophage (GM)-CSF (41, 42). The ßc chain also requires residues in the BC loop of domain 2, since substitution of Y, H, and I residues therein impaired the binding of GM-CSF and IL-5, but not of IL-3 (43).

While the ability of mAb CP.B8 to inhibit IL-2-, IL-4-, IL-7-, and IL-15-induced responses indicates a common surface for the interaction of the {gamma}c chain with multiple cytokines, it is possible that the {gamma}c chain residues required for binding to different cytokines are overlapping but not identical. Consistent with this, He et al. (44) reported mAbs specific for the murine {gamma}c chain that preferentially inhibited IL-4 vs IL-7 responses. Our studies suggest that residues required for binding IL-2 and IL-7 may be overlapping but distinct from those required for binding to IL-4 and IL-15, since we have noted that mAb CP.B8 inhibits IL-4 and IL-15 responses to a relatively greater degree than IL-2 and IL-7 responses of PHA-blasts. Alternatively, the differential activity of mAb CP.B8 could reflect quantitative differences at a variety of other levels, including the density of cytokine-specific receptors and/or the frequency of responsive cells, since IL-7-induced responses of freshly isolated PBMC were more readily inhibited than those of PHA-blasts. In addition, a given cytokine response may be preferentially inhibited due to differences in the degree to which the {gamma}c chain preassociates with other specific receptor chains in the absence of cytokine (45) and/or due to differences in the affinity of the {gamma}c chain for complexes of cytokine and cytokine-specific receptor chain.4

Our mutational analysis also explored the identity of contact residues that potentially mediate interaction of the {gamma}c chain with other cytokine receptor subunits. Given evidence that the {gamma}c chain cannot bind ligand independent of other cytokine-specific receptor chains (7), it is postulated that interchain contacts may be established, thereby stabilizing complex formation. In the hGH/hGH receptor complex, interreceptor contacts occur at the base of the membrane proximal domains, largely through residue Y200 (27). Since the {gamma}c chain is analogous to the second hGH receptor molecule, it was mutated in homologous regions of domain 2. Alanine scanning through loop residues L143S144E145S146Q147 resulted in no significant decrease in mAb binding. Furthermore, preliminary data with alanine substitutions at residues S190V191D192Q194K195R196 in the EF loop of domain 2 of the {gamma}c chain also indicated little, if any, effect on mAb binding (A. Jakubowski, unpublished observation). While we find no evidence for the existence of interreceptor chain contacts, we cannot rule them out since our mAbs did not map to this candidate region. Further studies are needed to resolve this issue.

The targeted mutagenesis of the human {gamma}c chain reported herein provides evidence delineating the structure/function relationship for this molecule. The analysis of naturally occurring {gamma}c chain mutants from XSCID patients has also made contributions to defining this relationship (6). Of particular relevance for our discussion are XSCID mutations that cause single amino acid changes in the extracellular region of the {gamma}c chain, excluding those that alter canonical cytokine receptor motifs or residues within ß strands. Several mutations have been reported to abrogate or reduce high affinity IL-2 binding based on the activity of EBV-transformed B cell lines or of cells transfected with mutant sequences derived from the XSCID patients. Interestingly, these include mutation E46K in loop AB of domain 1 (31), A134V in loop EF of domain 1 (30), and R204C in loop FG of domain 2 (29). However, expression of these XSCID {gamma}c chain mutants at the level of the cell surface was confirmed only in the case of the A134V mutant, using the nonblocking anti-human {gamma}c chain mAb TUGh4 (18). Several other key residues have also been suggested based on XSCID mutation I131N in loop EF of domain 1 and L161S in loop BC of domain 2, as reviewed by Leonard et al. (6) and, more recently, by Puck et al. (33), who have executed an extensive survey of XSCID patient sequences, reporting 18 different point mutations in the extracellular region of the {gamma}c chain, all of which are expressed at the mRNA level. However, these analyses continue to be limited by the availability of mAbs specific for the human {gamma}c chain (18, 46), none of which are reported to have blocking activity. Our panel of anti-{gamma}c chain mAbs will therefore be useful for evaluating the expression of XSCID {gamma}c chain mutants, thereby contributing to diagnosis of mutations and to defining the functional basis for these genetic defects. Taken together, our approach and the previous XSCID mutant analyses provide strong support for the critical role of the AB and EF loops of domain 1 and the FG loop of domain 2 in mediating the interaction of {gamma}c chain with cytokine. In addition, our data further address the role of the BC loop in domain 2, providing evidence that changes in this loop indirectly affect the cytokine binding site on the {gamma}c chain.

In summary, we have provided functional evidence for the critical role of four noncontiguous stretches of the human {gamma}c chain sequence that together comprise a common interface for the interaction of the {gamma}c chain with other molecules, most likely its multiple cytokines. This was achieved through the use of a panel of novel anti-{gamma}c chain mAbs representing five different epitope groups, including the unique blocking mAb CP.B8. The identification of these key sequences provides practical guidance for the development of small molecules that bind to or mimic this receptor interface, enabling inhibition of {gamma}c chain recruitment and thereby the regulation of cytokine receptor function in pathologic settings.


   Acknowledgments
 
We thank Mark Krivopal, Daisuke Tsujimoto, and Michael Cohen for excellent technical contributions, Dr. Adrian Whitty for helpful discussions during the course of this work, and Dr. Herman Van Vlijmen for determining the surface solvent accessibility of the {gamma}c chain.


   Footnotes
 
1 Equal contributions were made by these authors. Back

2 Address correspondence and reprint requests to Dr. Linda C. Burkly, Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142. E-mail address: Back

3 Abbreviations used in this paper: XSCID, X-linked SCID; {gamma}c, common {gamma} chain; hGH, human growth hormone; ID, interdomain; MLR, mixed lymphocyte reaction. Back

4 A. Whitty, N. Raskin, D. L. Olson, C. W. Borysenko, C. D. Benjamin, and L. C. Burkly. Interaction affinity between cytokine receptor components on the cell surface. Submitted for publication. Back

Received for publication March 23, 1998. Accepted for publication June 1, 1998.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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