Qualitatively different T cell phenotypic responses to IL-2 versus IL-15 are unified by identical dependences on receptor signal strength and duration

Abstract

IL-2 and IL-15 are common γ-chain family cytokines involved in regulation of T cell differentiation and homeostasis. Despite signaling through the same receptors, IL-2 and IL-15 have non-redundant roles in T cell biology, both physiologically and at the cellular level. The mechanisms by which IL-2 and IL-15 trigger distinct phenotypes in T cells remain elusive. To elucidate these mechanisms, we performed a quantitative comparison of the phosphotyrosine signaling network and resulting phenotypes triggered by IL-2 and IL-15. This study revealed that the signaling networks activated by IL-2 or IL-15 are highly similar and that T cell proliferation and metabolism are controlled in a quantitatively distinct manner through IL-2/15R signal strength independent of the cytokine identity. Distinct phenotypes associated with IL-2 or IL-15 stimulation therefore arise through differential regulation of IL-2/15R signal strength and duration because of differences in cytokine-receptor binding affinity, receptor expression levels, physiological cytokine levels, and cytokine-receptor intracellular trafficking kinetics. These results provide important insights into the function of other shared cytokine and growth factor receptors, quantitative regulation of cell proliferation and metabolism through signal transduction, and improved design of cytokine based clinical immunomodulatory therapies for cancer and infectious diseases.

Figure 1. Mass spectrometry based phosphotyrosine profiling of F15R-Kit cells stimulated with IL-2 and IL-15

(A) Schematic diagram of mass spectrometry based quantitative phosphotyrosine profiling experimental workflow. (B) Heat maps depicting 85 phosphorylation sites on 81 proteins quantified across F15R-Kit cell stimulated with IL-2 or IL-15 for 15 min. Heat maps represent log 2 transformed iTRAQ-8plex fold change values relative to one replicate of unstimulated F15R-Kit cells. The fold changes depicted for IL-2 and IL-15 are an average of 3 technical replicates. (C) Cellular processes regulated by the 41 proteins with greater than 1.5 fold increase in phosphorylation level upon IL-2 and IL-15 stimulation. (D) The relative increase in phosphorylation levels of canonical IL-2/15R signaling proteins triggered by IL-2 and IL-15 in F15R-Kit cells.

Figure 2. Quantification of phosphotyrosine sites regulated by IL-2 and IL-15 stimulation of F15R-Kit cells

(A) Quantification of phosphotyrosine sites differentially regulated by IL-2 and IL-15 stimulation. iTRAQ fold changes in F15R-Kit cells stimulated with IL-2 and IL-15 normalized to unstimulated cells. Data represent an average of 3 biological replicates. * p-value <0.05. (B) Quantification of phosphotyrosine sites on canonical signaling proteins of the IL-2/15R signaling pathways. iTRAQ fold changes in F15R-Kit cells stimulated with IL-2 and IL-15 normalized to unstimulated cells. Data represent an average of 3 biological replicates.

Figure 3. F15R-Kit cells proliferate equivalently in response to a continuous, but not pulsed, stimulation by IL-2, IL-15, and mtIL-2

(A) Viable cell numbers over time for F15R-Kit cells cultured in the continuous presence of 500pM IL-2, IL-15, mtIL-2, or culture medium alone. (B) Viable cell numbers over time of F15R-Kit cells pulsed for 30min with 500pM IL-2, IL-15, and mtIL-2. Viable cell numbers were determined using Trypan Blue exclusion. Data represent Mean ± Standard Deviation (SD) for three independent experiments.

Figure 4. F15R-Kit cell proliferation requires continuous activation of the IL-2/15R signaling

(A) Representative flow cytometry histograms showing surface cytokine and intracellular pSTAT5 levels in F15R-Kit cells stimulated with 500pM of the indicated cytokine for 30min (solid histograms). Dotted histograms represent unstimulated cells. (B) Surface-bound cytokine levels and (C) intracellular pSTAT5 levels of F15R-Kit cells, pulse stimulated with 500pM cytokine measured at the indicated time points after cytokine withdrawal. Data represent median fluorescence intensity (MFI) of surface cytokine and pSTAT5 levels normalized to MFI of cells stimulated with IL-15 at the 30min time-point. (D) CFSE dilution values (left panel) and representative histograms of F15R-Kit CFSE levels (right panel) experiencing a defined duration of IL-2/15 receptor signaling, ***α_FW < 0.001 compared to unstimulated cells. CFSE dilution values represent the ratio of CFSE MFI levels of cells cultured with IL-2 and JI to CFSE MFI of unstimulated cells 48h after cytokine addition. Solid histograms represent CFSE values for F15R-Kit cells cultured with IL-2 and JI and dashed histograms represent CFSE values of unstimulated cells 48h after cytokine addition. Data represent average ± SD for 3 independent experiments.

Figure 5. F15R-Kit cell size increases with cytokine dose but proliferation remains identical

(A) Median FSC values for F15R-Kit cells cultured for 48h with the indicated doses of IL-2 or IL-15 normalized to the 100pM dose for each cytokine. *α_fw < 0.05, **α_fw < 0.01, ***α_fw < 0.001. (B) Plot showing the ratios of the CFSE values of F15R-Kit cells cultured with the indicated dose of cytokine to the unstimulated cells at 48h. ***α_fw < 0.001 compared to unstimulated cells. (C) Representative flow cytometry dot plots showing forward scatter and histograms showing CFSE levels of F15R-Kit cells 48h after treatment with the indicated dose of IL-2 or IL-15. Dotted histograms represent unstimulated cells. Data represent Mean ± SD for 3 independent replicates.

Figure 6. F15R-Kit cell size and glycolytic activity increases with increasing IL-2/15 receptor signal strength while proliferation remains identical

Cytokine starved F15R-Kit cells were pretreated with the indicated dose of JAK Inhibitor I, stimulated with a 500pM dose of cytokine, and intracellular pSTAT5 levels, forward scatter, and CFSE levels measured at the indicated time-points using flow cytometry. (A) F15R-Kit intracellular pSTAT5 levels 15min after addition of IL-2 or IL-15. (B) F15R-Kit cells forward scatter values at the indicated time-periods after the addition of IL-2 or IL-15. (C) CFSE dilution levels compared to the unstimulated control at the indicated time points for cells cultured with IL-2 or IL-15. Glucose consumption and lactate production by F15R-Kit cells treated with the indicated dose of JAK Inhibitor I and stimulated with 500pM of (D) IL-2 or (E) IL-15, *α_FW < 0.05, **α_FW < 0.01 compared to 0nM JI condition. Data represent Mean ± SD for 3 independent replicates.

Figure 7. Quantitatively distinct regulation of F15R-Kit cell size and proliferation through the IL-2/15 receptor signal strength

CFSE dilution and FSC levels plotted against intracellular pSTAT5 levels of F15R-Kit cells cultured for 48h with multiple doses of JAK Inhibitor I and 500pM IL-2 (black diamonds) or 500pM IL-15 (gray circles) (A–B). (A) CFSE dilution vs. pSTAT5 levels. The black dotted lines represent the threshold signal strength above which proliferation is identical and independent of the specific pSTAT5 level. CFSE dilution values between 0.6 and 1 represent median fluorescence for populations where > 0% but <100% of cells complete one cell division. (B) Forward scatter vs. pSTAT5 levels for cells with signal strength above the threshold indicated in figure 5A. (C) Forward scatter vs. pSTAT5 levels of F15R-Kit cells cultured with the indicated doses of JAK Inhibitor I and 500pM IL-2 (black diamonds) or 500pM IL-15 (gray circles). IL-2/15 receptor signal strength is a superior predictor of cell size compared to JAK Inhibitor I dose, cytokine dose, or cytokine identity.

Figure 8. F15R-Kit cell size vs. mTOR signaling

Forward scatter vs. 40S ribosomal protein S6 (pS6) levels of F15R-Kit cells cultured with various doses of JI. Forward scatter and pS6 values measured 96h after cytokine addition for cells stimulated with (A) 500pM IL-2 (black diamonds) or (B) 500pM IL-15 (gray circles).

Figure 9. Quantitatively distinct regulation of cell size and proliferation through the IL-2/15 receptor signal strength in primary human T lymphocytes

Primary human CD4⁺ and CD8⁺ T cells were activated with anti-CD3 and anti-CD28 stimulation, treated with the indicated JAK Inhibitor I dose, and stimulated with 1nM IL-2 or IL-15. Forward scatter of (A) CD4⁺ T cells and (C) CD8⁺ T cells at the indicated time-points after cytokine addition. CFSE dilution values for (B) CD4⁺ T cells and (D) CD8⁺ T cells at the indicated time-points after cytokine addition. Non-activated T cells were used as the negative control for measuring CFSE dilution. Forward scatter vs. intracellular pSTAT5 levels at 48h after cytokine addition for (E) CD4⁺ T cells (gray circles) and (F) CD8⁺ T cells (black squares). CFSE dilution vs. intracellular pSTAT5 levels at 48h after cytokine addition for (G) CD4⁺ T (gray circles) cells and (H) CD8⁺ T (black squares) cells.