1. Volume 40, Issue 6, Pages 910-923 (June 2014)
Activated T Cells Secrete an Alternatively Spliced Form of Common γ-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation 
Changwan Hong, Megan A. Luckey, Davinna L. Ligons, Adam T. Waickman, Joo-Young Park, Grace Y. Kim, Hilary R. Keller, Ruth Etzensperger, Xuguang Tai, Vanja Lazarevic, Lionel Feigenbaum, Marta Catalfamo, Scott T.R. Walsh, Jung-Hyun Park 
Immunity 
Volume 40, Issue 6, Pages (June 2014)
DOI: /j.immuni
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2. Immunity 2014 40, 910-923DOI: (10.1016/j.immuni.2014.04.020)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 Soluble γc Proteins Are Products of Alternative Splicing
(A) Activated T cells express soluble γc proteins. T cells were stimulated with α-TCR and α-CD28 in the presence or absence of the metalloprotease inhibitor TAPI2. Culture supernatants were harvested at indicated days and assessed for IL-2, TNF-α, and soluble γc. Results are the summary of three independent experiments.
(B) Immunoblot detection of sγc proteins in WT and Il2rg−/− sera. Serum samples were immunoprecipitated (IP) with γc-ED-specific antibodies and probed with the same antibodies in immunoblots (IB). Data are representative of five independent experiments.
(C) Immunoblot detection of the sγc C-terminal 9 aa epitope. α-γc-ED immunprecipitated samples were probed with custom-generated sγc C-terminal 9 aa-specific antibodies. Data are representative of five independent experiments.
(D) Generation of T cells that exclusively produce membrane γc proteins. LN T cells from Il2rg−/− and mγcTg were assessed for γc surface (left) and mRNA expression (right). Cell surface γc expression (open histogram) versus control antibody staining (shaded histogram) is shown for Il2rg−/− and mγcTg LN T cells. Relative sγc and mγc mRNA expression were determined by qRT-PCR and normalized to expression in WT LN T cells, which was set to 100%. Data are summary of three independent experiments; ND indicates not detectable.
(E) Serum sγc levels in WT, γcTg, Il2rg−/−, and mγcTg mice. Data are the summary of three independent experiments.
Error bars represent mean and SEM.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2 sγc Expression Is Induced by T Cell Activation In Vivo
(A) Serum sγc levels in BALB/c mice injected overnight with α-CD3 antibodies. ELISA data are summary of two independent experiments with two mice per group.
(B and C) Serum sγc levels in Ctla4−/− and Ctla4−/−Cd28−/−, and in Il2−/− and Il2ra−/− mice. ELISA data are summary of three independent experiments.
(D) sγc protein expression in WT and sγcTg mice sera. Serum samples were immunoprecipitated with γc-ED-specific antibodies and probed with the same antibodies in immunoblots. Numbers in box indicate densitometric quantification of sγc expression. Data are representative of five independent experiments.
(E) Relative sγc levels in WT and sγcTg serum. ELISA data are summary of five independent experiments.
(F) LN cell profiles of WT and sγcTg mice. Data are representative of 17 independent experiments.
(G) IFN-γ and IL-17A expression in sγcTg CD4+ LN T cells. CD4+ LN T cells from WT and sγcTg mice were stimulated for 3 hr with PMA and ionomycin and assessed for IFN-γ and IL-17A expression by intracellular staining. IFN-γ, IL-17A profile is representative of six independent experiments (left). Bar graph shows percent IFN-γ- or IL-17A-producing CD4+ T cells.
Error bars represent mean and SEM.
Immunity  , DOI: ( /j.immuni )
Copyright © 2014 Elsevier Inc. Terms and Conditions

5. Figure 3
sγc Is Proinflammatory and Exacerbates EAE Disease Progression
(A) Serum sγc in EAE-induced mice. sγc was assessed by immunoprecipitation and immunoblot before (day 0) and after (day 15) EAE induction in WT mice. Blot shows representative serum sγc expression of four independent experiments.
(B) EAE induction in WT and sγcTg mice. EAE clinical scores are representative of seven independent EAE experiments, each with five mice per group.
(C) EAE disease course in WT and sγcTg mice. Data are summary from seven independent experiments, each with five mice per group.
(D) Th1 and Th17 cell subset analysis of spinal-cord-infiltrating CD4+ T cells in EAE-induced WT and sγcTg mice. IL-17A, IFN-γ profile is representative of two independent experiments with two mice per group.
(E) Spinal-cord-infiltrating CD4+ T cell numbers in EAE-induced mice. On day 14 after immunization, IFN-γ- and/or IL-17A-producing CD4+ T cells were enumerated from spinal cords of WT and sγcTg mice. Data are representative for two independent experiments with two mice per group.
Error bars represent mean and SEM.
Immunity  , DOI: ( /j.immuni )
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6. Figure 4 sγc Inhibits IL-2 Signaling
(A) Dimer formation of recombinant sγc proteins. Recombinant sγc proteins were expressed in insect cells and were resolved in SDS-PAGE under reducing (+DTT) and nonreducing conditions. sγc was detected with antibodies against the γc-ED. Immunoblot is representative of four independent experiments.
(B) sγc binding affinities to IL-2Rβ determined by SPR. Binding sensorgrams of monomeric (left) and dimeric (right) sγc to immobilized IL-2Rβ. The insets show dose-response curves of the maximal responses (Rmax, depicted by the dashed boxes) for each sγc concentration. Plots of Rmax versus sγc concentrations were nonlinear fit to a single-site binding affinity model to determine Kd values.
(C) Recombinant sγc binding to mature Il2rgfl/flE8IIICre thymocytes. Il2rgfl/flE8IIICre thymocytes (1.5 × 106 cells in 100 μl) were incubated for 3 hr with 2 μg rsγc in the presence or absence of IL-2 (200 ng) and assessed with γc antibodies for surface γc staining. Histograms show surface γc staining on TCRβhi thymocytes.
(D) Titration of sγc binding to Il2rgfl/flE8IIICre thymocytes. Il2rgfl/flE8IIICre thymocytes were treated with increasing amounts of rsγc in the presence or absence of IL-2 (200 ng) as described above. Data are the summary of three independent experiments (mean and SEM).
(E) Recombinant sγc impairs proximal IL-2 signaling. CD8+ T cells were stimulated with IL-2 in the presence (4 μg/ml rsγc) or absence (Veh; vehicle) of rsγc and assessed for pSTAT5 (left) or pAKT contents (right). Line graphs show fold induction of IL-2-induced STAT5 or AKT phosphorylation over cells treated with Veh alone. Data are the summary of four independent experiments (mean and SEM).
(F) Recombinant sγc inhibits IL-2-induced T cell proliferation. CFSE-labeled WT CD8+ LN T cells were stimulated with IL-2 in the presence or absence of rsγc. CFSE dilution was analyzed at the indicated time points. Histograms are representative of five independent experiments.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5 sγc Promotes Th17 Cell Differentiation of CD4+ LN T Cells
(A) sγc promotes IL-17A expression. Sorted naive CD4+ T cells were stimulated under Th17-cell-polarizing conditions and assessed for intracellular IL-17A and IFN-γ expression. Where indicated, recombinant human IL-2 (rhIL-2), neutralizing mouse IL-2-specific antibodies (α-mIL-2), recombinant sγc (4 μg/ml rsγc), or vehicle (Veh) were added at the beginning of the culture. Data are representative of ten independent experiments.
(B) Summary of IL-17A+CD4+ T cell differentiation under modified Th17-cell-polarizing conditions. Each symbol represents an individual experiment under the indicated condition as described above.
(C) IL-17A secretion in WT and sγcTg CD4+ T cells. Culture supernatants were collected at day 4 of Th0 or Th17 cell differentiation cultures. ELISA data are representative for five independent experiments.
(D) Redundant effect of rsγc and mIL-2-specific antibodies on Th17 cell differentiation. Naive CD4+ T cells were stimulated under Th17-cell-polarizing conditions in the presence of rsγc or mIL-2-specific antibodies. Each symbol represents an individual mouse.
(E) sγc effect on IL-17A expression under IL-2-deficient conditions. Naive CD4+ T cells were stimulated under Th17-cell-polarizing conditions to assess intracellular IL-17A and IFN-γ expression. Where indicated, rsγc were added at the beginning of the differentiation culture. Contour plots are representative of four independent experiments (left). Bar graph shows percent increase of IL-17A-producing CD4+ T cells by rsγc treatment compared to vehicle control.
Error bars represent mean and SEM.
Immunity  , DOI: ( /j.immuni )
Copyright © 2014 Elsevier Inc. Terms and Conditions

8. Figure 6
sγc Deficiency Is Protective against Inflammatory Autoimmune Diseases
(A) EAE induction in WT and mγcTg mice. EAE clinical scores are summary of two independent EAE experiments, each with four mice per group.
(B) Th1 and Th17 cell subset analysis of spinal-cord- and brain-infiltrating CD4+ T cells in EAE-induced WT and mγcTg mice. Contour plots show representative IL-17A, IFN-γ profiles from four mice per group.
(C) Spinal-cord- and brain-infiltrating CD4+ T cell numbers in EAE-induced mice. On day 15 after immunization, IFN-γ- and/or IL-17A-producing CD4+ T cells were enumerated from spinal cords and brains of WT and mγcTg mice. Data are representative of four mice per group.
(D) Body weight change of IBD-induced mice. Rag2−/− mice were injected with each 0.5 million WT or mγcTg CD4+CD45RBhi T cells and monitored for change in body weights. Data show results from four mice per group.
(E) Colon histology from Rag2−/− host mice injected with naive CD4+ T cells from the indicated donor mice at 6 weeks after T cell transfer.
(F) Th1 and Th17 cell subset analysis in CD4+ LN T cells from IBD-induced WT and mγcTg mice (left). Data show results from three mice for each group (right).
Error bars represent mean and SEM.
Immunity  , DOI: ( /j.immuni )
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9. Figure 7 sγc Overexpression Impairs T Cell Development and Homeostasis
(A) Surface γc chain staining on WT and sγcTg CD4+ T cells. Data are the summary of four independent experiments.
(B) sγc binding affinities to IL-7Rα determined by surface plasmon resonance. Binding sensorgrams and Kd of monomeric (left) and dimeric (right) sγc proteins to immobilized IL-7Rα proteins.
(C) Cell surface γc staining of Il2rgfl/flE8IIICre thymocytes incubated with recombinant sγc. Il2rgfl/flE8IIICre thymocytes were incubated for 2 hr with 4 μg rsγc or Veh (BSA in PBS) and assessed with γc antibodies for surface γc staining. Histograms show surface γc staining on DP and TCRβhi thymocytes.
(D) Cell surface γc staining of Il2rgfl/flE8IIICre and Il7rafl/flIl2rgfl/flE8IIICre thymocytes incubated with rsγc. Surface γc staining was assessed as mean fluorescence intensity (MFI) in TCRβhi thymocytes. Data are summary of two independent experiments.
(E) Total thymocyte numbers in WT and sγcTg mice. Results are the summary of eight independent experiments.
(F) T cell apoptosis in WT and sγcTg LN T cells. Caspase-3 activity was determined in naive CD44lo CD4+ or CD8+ LN T cells via CaspGLOW apoptosis kits. Histograms show caspase-3 activity in indicated cells (left). Bar graphs show summary of three independent experiments (right).
(G) Naive and memory phenotype analysis of WT and sγcTg LN T cells. Contour plots show CD62L/CD44 and CD122/CD44 profiles and percentages of CD4+ and CD8+ LN T cells, respectively (left). Bar graph shows naive CD4+ and CD8+ LN T cells numbers from WT and sγcTg mice (right). Data are the summary of ten independent experiments.
Error bars represent mean and SEM.
Immunity  , DOI: ( /j.immuni )
Copyright © 2014 Elsevier Inc. Terms and Conditions