Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling

Abstract

To drive lymphocyte proliferation and differentiation, common γ-chain (γc) cytokine receptors require hours to days of sustained stimulation. JAK1 and JAK3 kinases are found together in all γc-receptor complexes, but how their respective catalytic activities contribute to signaling over time is not known. Here we dissect the temporal requirements for JAK3 kinase activity with a selective covalent inhibitor (JAK3i). By monitoring phosphorylation of the transcription factor STAT5 over 20 h in CD4(+) T cells stimulated with interleukin 2 (IL-2), we document a second wave of signaling that is much more sensitive to JAK3i than the first wave. Selective inhibition of this second wave is sufficient to block cyclin expression and entry to S phase. An inhibitor-resistant JAK3 mutant (C905S) rescued all effects of JAK3i in isolated T cells and in mice. Our chemical genetic toolkit elucidates a biphasic requirement for JAK3 kinase activity in IL-2-driven T cell proliferation and will find broad utility in studies of γc-receptor signaling.

Figure 1

Chemical Structure of JAK3i.

Figure 2. Early and late signaling events are differentially affected by JAK3i

CD4+ T-cell blasts were pretreated for 2 hrs with indicated dose of JAK3i (a–b, e) or tofacitinib (c–d) and stimulated with IL-2 (50 u/mL). (a & c) STAT5 phosphorylation was monitored by phosphoflow 15 min after IL-2 stimulation. (c & d) Dose-response curves comparing effects on pSTAT5, proliferation, and CD25 upregulation. Proliferation was assessed by ³H-thymidine uptake after 24–48 hrs. CD25 (IL-2Rα) upregulation was quantified by FACS after 24 hrs (see Supplementary Fig. 2a for FACS histograms). e) S6 phosphorylation (Ser240/244) was monitored by phosphoflow in parallel with pSTAT5 after 15 minutes of IL-2 stimulation. See Supplementary Fig. 4c for corresponding pSTAT5 histograms. Data in a,c & e are representative of 5, 8 and 2 independent experiments. Data in b are cumulative of 6 (proliferation) and 4 (pSTAT5 & CD25) independent experiments, plotted as mean ± SEM. Data in c are cumulative of 3 (proliferation), 7 (pSTAT5) and 5 (CD25) independent experiments, plotted as mean ± SEM. See Supplementary Table 3 for average IC₅₀ values from all experiments.

Figure 3. C905S JAK3 mutant rescues JAK3i effects in primary CD4+ T cells

Rescue of JAK3i-mediated inhibition of (a) proliferation in unsorted CD4+ cells and (b) pSTAT5 (t = 15 min) in CD4+GFP+ cells. Cells were retrovirally transduced with empty vector, WT JAK3, or C905S JAK3, treated with JAK3i for 2 hrs, and stimulated with 50 units/mL rhIL-2. See Supplementary Fig. 6a for transduction efficiency. (a) and (b) are representative of two independent experiments. (c) Cellular selectivity of JAK3i in primary murine immune cells. IFN-γ-stimulated pSTAT1 (JAK1/JAK2 dependent), measured by phosphoflow, and IL-2-driven proliferation (JAK1/JAK3 dependent), measured by ³H-thymidine uptake, were assessed in CD4+ T-cell blasts. T-cell receptor (TCR)-driven IL-2 production (ITK/RLK dependent) was assessed by ELISA in naïve CD4+ T cells. Activation of naïve B cells (BTK dependent) by B-cell receptor (BCR) stimulation (α-IgM) was determined by FACS measurement of CD69 upregulation. Data are representative of 2 (IFN-γ, BCR), 9 (IL-2), or 3 (TCR) independent experiments. See Supplementary Table 4 for IC₅₀ values from all cellular selectivity experiments. Proliferation (a, c) and IL-2 ELISA (c) data are plotted as mean ± SEM of triplicates.

Figure 4. A second wave of STAT5 signaling is highly sensitive to JAK3 inhibition

(a) CD4+ T-cell blasts were pretreated with inhibitors at the EC₉₀ dose for blocking T-cell proliferation (JAK3i 15 nM, tofacitinib 50 nM) for 2 hrs and then stimulated with IL-2 (50 u/mL). pSTAT5 was monitored by phosphoflow at the indicated times. (b) pSTAT5 time course from (a), quantified by mean fluorescence intensity (MFI), normalized to the maximum value (t = 15 min, DMSO) and plotted ± SEM of 10 independent experiments. (c) Time course showing C905S JAK3 rescue of pSTAT5 inhibition by JAK3i. (d–e) Cells were pretreated with JAK3i (30 nM) and stimulated with IL-2. mRNA levels were monitored by qPCR at the indicated times. Plotted as mean fold-induction with 95% confidence intervals. Data are representative of 10 (a), 2 (c) or 3 (d–e) independent experiments.

Figure 5. Sustained JAK3 activity is required for S-phase entry

(a) pSTAT5 levels were monitored over time in T cells treated with JAK3i (15 nM) either 2 hrs before or 90 min after IL-2 stimulation. Data are normalized the maximum value (t=15 min DMSO) and plotted as mean ± SEM of 4 independent experiments. (b–c) JAK3i was added at a concentration of 15 nM (b) or 100 nM (d) at the indicated times after IL-2 stimulation. After 24 hrs, cells were labeled with EdU for 1 hr, and the percentage of EdU+ cells was measured by FACS and normalized to DMSO (± SEM of replicates, n = 3). Typically, 35–45% of cells were in S-phase 24–25 hours after stimulation. (d) Cyclin D3 was monitored by immunoblot 24 hrs after IL-2 stimulation in the presence of indicated concentrations of JAK3i. For full gel, see Supplementary Fig. 12. (e) Cells were pretreated with 15 nM JAK3i, and cyclin E1 mRNA was quantified by qPCR at the indicated times after IL-2 stimulation. (f) T cells were treated with JAK3i 2 hrs before or 1.5 hrs after IL-2 stimulation. After 24 hrs, phosphorylation of the retinoblastoma protein (Rb) at S800/S804 was quantified by FACS. Original histograms are shown in Supplementary Figure 10d. Data are representative of 4 (d), 3 (b, e) or 2 (c, f) independent experiments.

Figure 6. JAK3 inhibition blocks IL-2-driven proliferation in vivo

(a) Experimental timeline, showing adoptive transfer of CD4+ T-cell blasts (5 × 10⁶) labeled with carboxyfluorescein succinimidyl ester (CFSE), twice-daily dosing with JAK3i, and thrice-daily injections of IL-2 (20,000 units). (b) Representative CFSE dilution histograms for the indicated treatments. (c) Median CFSE intensity (plotted ± SEM for n>2) for the indicated treatments, 2–3 mice per group as indicated. (d) Congenic marking and transfer scheme for in vivo proliferation and rescue experiment. A 1:1 mixture of WT and C905S T-cells (total 5 × 10⁶) was transferred to each mouse. (e) FACS identification of WT and C905S JAK3-transduced cells from the same mouse (see complete gating in Supplementary Figure 11). (f) Representative Celltrace Violet dilution profiles. Each column depicts cells from the same mouse. (g) Median Celltrace Violet intensity for the indicated treatments and JAK3 overexpression construct (n = 3 mice per IL-2-stimulated treatment condition, n = 1 mouse for the unstimulated control). Data in a-c are pooled from two independenet experiments and data in d-g are from one experiment with indicated numbers of mice.