Research ArticleImmunology

A hierarchy of affinities between cytokine receptors and the common gamma chain leads to pathway cross-talk

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Science Signaling  03 Apr 2018:
Vol. 11, Issue 524, eaal1253
DOI: 10.1126/scisignal.aal1253
  • Fig. 1 Pretreatment of T cells with IL-7 suppresses subsequent responses to IL-4 and IL-21 in a dose-dependent manner.

    (A) Cytokine ligation and dimerization of the private receptor chains with the common γc lead to cross-activation of the associated Janus kinases JAK3 and JAK1. These kinases phosphorylate interaction sites on the receptors for STAT molecules that, in turn, are recruited and phosphorylated and then form STAT dimers that enter the cell nucleus to induce transcription. As discussed in the text, IL-4 specifically leads to activation of STAT6, IL-7 to activation of STAT5, and IL-21 to activation of STAT3. (B) Shifting IL-4 dose-response curves after pretreatment with varying doses of IL-7. Freshly isolated cells were stimulated for 10 min with increasing concentrations of IL-4 without IL-7 (black line) or pretreated with increasing concentrations of IL-7 (red, 50 pg/ml; blue, 200 pg/ml; green, 5000 pg/ml) for 10 min and stained with antibodies against pSTAT6. Maximum pSTAT6 response in the absence of IL-7 prestimulation was obtained from a fit for each experiment as described in Materials and Methods, and all pSTAT6 response values were normalized to it. (C) IL-21–induced STAT3 serine phosphorylation mimics STAT3 tyrosine phosphorylation. Freshly isolated cells were stimulated for 10 min with increasing concentrations of IL-21 and stained with antibodies directed against pSer727 in STAT3. Normalized pSTAT3 (Ser) values were obtained from a fit as in (B). (D) IL-7 pretreatment equally suppresses serine and tyrosine phosphorylations of STAT3. Freshly isolated cells were stimulated for 10 min with IL-21 (50 pg/ml) after pretreatment with increasing concentrations of IL-7 for 10 min. Percentage inhibition of IL-21–induced tyrosine or serine phosphorylation of STAT3 by IL-7 pretreatment was determined as described in Materials and Methods. Data presented in (B) to (D) were analyzed by flow cytometry by gating on CD4+TCR+CD44low cells and are means ± SD from three independent experiments. (E and F) Asymmetry in cross-talk among γc cytokines. (E) Spleen and lymph node cells from B10.BR mice were stimulated for 10 min with IL-7 or IL-21 (50 pg/ml) without IL-4 or with increasing concentrations of IL-4 pretreatment for 10 min. Percentage inhibition of IL-7–dependent STAT5 phosphorylation or IL-21–dependent STAT3 phosphorylation was determined as described in Materials and Methods by gating on CD4+TCR+CD44low cells. (F) Cells were stimulated for 10 min with IL-4 (50 pg/ml) or IL-7 (50 pg/ml) without IL-21 or with increasing concentrations of IL-21 pretreatment for 10 min. Percentage inhibition of IL-4–dependent STAT6 phosphorylation and IL-7–dependent STAT5 phosphorylation was determined as described in Materials and Methods by gating on CD4+TCR+CD44low cells. Data in (E) and (F) are means ± SD of three independent experiments. (G) IL-7–induced STAT5 phosphorylation is lacking in IL-7Rα-449F-KI mice: IL-7–dependent STAT5 phosphorylation was measured by stimulating splenocytes from IL-7Rα-449F-KI mice (open circles) or wild-type (WT) littermate controls (filled circles) for 10 min. (H) IL-7–induced suppression of IL-4 responses is not affected in IL-7Rα-Y449F-KI mice. Splenocytes from IL-7Rα-Y449F-KI mice or WT littermate controls were stimulated for 10 min with increasing concentrations of IL-7 followed by stimulation with IL-4 (50 pg/ml). Percentage inhibition of IL-4–dependent STAT6 phosphorylation was determined as described in Materials and Methods. Data in (G) and (H) are individual measurements from two independent experiments (see also figs. S1, S2, and S4).

  • Fig. 2 Cytokine-specific private receptor chains outnumber the γc.

    (A) γc cytokine receptor chain abundance: Freshly isolated spleen and lymph node cells were stained with cytokine receptor antibodies, and the numbers of bound antibodies on CD4+TCR+CD44low cells were calculated as described in Materials and Methods. (B) γc-YFP abundance does not affect the abundance of IL-4Rα and IL-7Rα. Preactivated AND H2a*b CD4+ T cells were transfected with the pVenus–IL-2Rγ plasmid, after which the cell surface γc cytokine receptor numbers were determined as described in (A). (C) Overexpression of γc abrogates IL-7–induced suppression of IL-4 responses: The transfected cells in (B) were stimulated for 10 min with IL-4 (50 pg/ml) with or without pretreatment with increasing concentrations of IL-7 for 10 min. Percentage of inhibition of IL-4–induced STAT6 phosphorylation by IL-7 was determined after gating on cells expressing different amounts of γc-YFP (negative, low, med, and high). Data are means ± SD of at least three independent experiments (see also figs. S3 and S4).

  • Fig. 3 Low receptor occupancy is sufficient for STAT phosphorylation and suppressive cross-talk.

    (A to C) IL-4, IL-7, and IL-21 responses are highly sensitive. Freshly isolated T cells were stimulated for 10 min with increasing concentrations of IL-4 (A), IL-7 (B), and IL-21 (C); stained with antibodies against pSTAT molecules; and analyzed by flow cytometry by gating on CD4+TCR+CD44low cells. Normalized STAT phosphorylation values were determined as described in Materials and Methods and plotted against receptor occupancy. Doses of cytokines were converted into receptor occupancies using Kd values obtained experimentally (fig. S5) or from the literature (table S1) and the receptor numbers obtained in Fig. 2A. Data are means ± SD of 11 to 20 independent experiments. (D and E) Few ligated IL-7Rs suffice to suppress IL-4 and IL-21 responses. T cells were stimulated for 10 min with IL-4 (50 pg/ml) (D) or IL-21 (50 pg/ml) (E) with increasing concentrations of IL-7 prestimulation. Percentage inhibition of IL-4 (D) or IL-21 (E) response by IL-7 pretreatment was determined as described in Materials and Methods and plotted against IL-7R occupancy obtained as described in (B). Data are means ± SD of 4 to 13 independent experiments (see also fig. S4 and table S1).

  • Fig. 4 Computational model explaining suppressive cross-talk originating at the receptor level.

    (A and B) Preassociation with IL-7Rα limits the availability of γc for signaling through IL-4Rα. (A) Computationally predicted affinities (Kd) of the γc for the private receptor chains with and without cytokine ligation. (B) Distribution of γc between unbound, IL-4Rα–bound, and IL-7Rα–bound states before cytokine stimulation. Data are means ± SD from 16 simulations using parameter sets matching the experimental constraints in table M1 (Supplementary Modeling File). (C and D) The limited number of γc results in fewer IL-4–ligated IL-4Rα–γc complexes that can induce STAT6 phosphorylation. (C) Fraction of free γc relative to unstimulated cells [corresponding to the left point in (B)] for increasing doses of IL-7 10 min after stimulation. (D) Pretreatment with increasing doses of IL-7 leads to increasingly reduced input signal for STAT6 phosphorylation. The graph shows the fraction of IL-4–ligated IL-4Rα–γc complexes relative to cells without IL-7 pretreatment. The plotted lines in (C) and (D) represent results from simulations using 16 parameter sets matching the experimental constraints in table M1 (Supplementary Modeling File; see also fig. S6).

  • Fig. 5 Suppressive cross-talk correlates with IL-7Rα abundance, and engaged IL-7Rα reduces the ability of IL-4Rα to form complexes with γc.

    (A) The cell surface γc cytokine receptor numbers were determined as in Fig. 2A for freshly isolated cells from B10.BR, AND X B10.A (F1-AND H2a*b), and AND H2b mice by gating on CD4+TCR+CD44low cells. (B) Freshly isolated cells from B10.BR (B10.BR H2k), AND X B10.A (F1-AND H2a*b), and AND (H2b) mice were stimulated as described in Fig. 3D, and the percentage inhibition of IL-4 signaling by pretreatment with IL-7 in CD4+TCR+CD44low cells was determined. Data are means ± SD of three independent experiments. (C) BRET assays were performed in CHO cells after cotransfection with phRluc–IL-2Rγ (donor), pVenus–IL-4Rα (acceptor), and pcDNA3.1–IL-7Rα (see Materials and Methods). BRET between γc and IL-4Rα is expressed as the ratio between the fluorescence of the acceptor (YFP) over the luciferase activity of the donor. BRET ratios from cells not expressing YFP are also shown as a baseline. Cells were stimulated with IL-4 (50,000 pg/ml) for 10 min with or without IL-7 pretreatment with IL-7 {100 pg/ml [low dose (LD)] or 5000 pg/ml [high dose (HD)]} for 10 min. (D) Replacing IL-7Rα with the IFN-γ receptor increases BRET between IL-4Rα and γc. Data are means ± SD of 5 to 12 replicates from one of two independent experiments. Mann-Whitney tests were performed to determine statistical significance.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/524/eaal1253/DC1

    Fig. S1. Specificity, baseline, and kinetics of STAT activation downstream of γc cytokines.

    Fig. S2. Expression kinetics of γc cytokine receptors as a function of TCR activation in vivo and in vitro.

    Fig. S3. Responses in human T cells mimic those found in mouse T cells.

    Fig. S4. STAT activation at different receptor occupancies for γc cytokines and determination of IL-7 and IL-4 dissociation constants.

    Fig. S5. The cell surface abundance of γc is not affected by IL-4 or IL-7.

    Fig. S6. Computational modeling reproduces experimentally determined cytokine dose-response curves and IL-7–induced suppression of IL-4–induced STAT6 phosphorylation.

    Fig. S7. IL-7 signaling–induced phosphatase directed at STATs is not responsible for the inhibitory action of IL-7.

    Table S1. Reported affinities of γc cytokines for their receptors.

    Table S2. Parameter values used for simulating the affinity conversion model.

    Table S3. Raw MFI values for receptor expression and calculated numbers of antibodies bound.

    Supplementary Modeling File

    Simmune modeling files

    References (5263)

  • Supplementary Materials for:

    A hierarchy of affinities between cytokine receptors and the common gamma chain leads to pathway cross-talk

    Pauline Gonnord, Bastian R. Angermann, Kaitlyn Sadtler, Erin Gombos, Pascal Chappert, Martin Meier-Schellersheim,* Rajat Varma*

    *Corresponding author. Email: mms{at}niaid.nih.gov (M.M.-S.); rvarma{at}xencor.com (R.V.)

    This PDF file includes:

    • Fig. S1. Specificity, baseline, and kinetics of STAT activation downstream of γc cytokines.
    • Fig. S2. Expression kinetics of γc cytokine receptors as a function of TCR activation in vivo and in vitro.
    • Fig. S3. Responses in human T cells mimic those found in mouse T cells.
    • Fig. S4. STAT activation at different receptor occupancies for γc cytokines and determination of IL-7 and IL-4 dissociation constants.
    • Fig. S5. The cell surface abundance of γc is not affected by IL-4 or IL-7.
    • Fig. S6. Computational modeling reproduces experimentally determined cytokine dose-response curves and IL-7–induced suppression of IL-4–induced STAT6 phosphorylation.
    • Fig. S7. IL-7 signaling–induced phosphatase directed at STATs is not responsible for the inhibitory action of IL-7.
    • Table S1. Reported affinities of γc cytokines for their receptors.
    • Table S2. Parameter values used for simulating the affinity conversion model.
    • Table S3. Raw MFI values for receptor expression and calculated numbers of antibodies bound.
    • References (5263)

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    Other Supplementary Material for this manuscript includes the following:


    © 2018 American Association for the Advancement of Science

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