Signaling thresholds govern heterogeneity in IL-7-receptor-mediated responses of naïve CD8(+) T cells

Abstract

Variable sensitivity to T-cell-receptor (TCR)- and IL-7-receptor (IL-7R)-mediated homeostatic signals among naïve T cells has thus far been largely attributed to differences in TCR specificity. We show here that even when withdrawn from self-peptide-induced TCR stimulation, CD8(+) T cells exhibit heterogeneous responses to interleukin-7 (IL-7) that are mechanistically associated with IL-7R expression differences that correlate with relative CD5 expression. Whereas CD5(hi) and CD5(lo) T cells survive equivalently in the presence of saturating IL-7 levels in vitro, CD5(hi) T cells proliferate more robustly. Conversely, CD5(lo) T cells exhibit prolonged survival when withdrawn from homeostatic stimuli. Through quantitative experimental analysis of signaling downstream of IL-7R, we find that the enhanced IL-7 responsiveness of CD5(hi) T cells is directly related to their greater surface IL-7R expression. Further, we identify a quantitative threshold in IL-7R-mediated signaling capacity required for proliferation that lies well above an analogous threshold requirement for survival. These distinct thresholds allow subtle differences in IL-7R expression between CD5(lo) and CD5(hi) T cells to give rise to significant variations in their respective IL-7-induced proliferation, without altering survival. Heterogeneous IL-7 responsiveness is observed similarly in vivo, with CD5(hi) naïve T cells proliferating preferentially in lymphopenic mice or lymphoreplete mice administered with exogenous IL-7. However, IL-7 in lymphoreplete mice appears to be maintained at an effective level for preserving homeostasis, such that neither CD5(hi) IL-7R(hi) nor CD5(lo) IL-7R(lo) T cells proliferate or survive preferentially. Our findings indicate that IL-7R-mediated signaling not only maintains the size but also impacts the diversity of the naïve T-cell repertoire.

Figure 1

CD5 expression levels stratify a hierarchy in the IL-7-induced proliferation capacities of CD8⁺ T cells. (a) CD5 surface expression of freshly isolated OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells (top panel) and their proliferation when cultured in vitro at low density (∼2 × 10⁵ cells per ml) with 0.1 or 10 ng ml⁻¹ of IL-7, as assayed by CFSE dilution after 7 days (middle panels), and quantified as the fraction of cells divided (bottom panel). (b) Proliferation of OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells when cultured in vitro at low density with 0.1 or 10 ng ml⁻¹ of IL-7 as measured by the fraction of cells expressing the nuclear proliferation antigen Ki67 for cells either freshly isolated (top panels) or rested overnight (16 h) in cytokine-free media before stimulation (bottom panels). ^*P<0.05 between 0.1 and 10 ng ml⁻¹ conditions and between fresh and rested cells. (c) As described in a, except for OT-1 and F5 TCR-tg CD8⁺ memory-like cells generated in vitro through activation of naïve (CD44^lo) OT-1 and F5 TCR-tg CD8⁺ T cells with plate-coated anti-CD3 and 20 ng ml⁻¹ IL-2 for 3 days, followed by incubation with 40 ng ml⁻¹ IL-15 for 3 days and overnight rest in cytokine-free media before IL-7 stimulation. (d) Decay of CD5 surface expression of freshly isolated OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells cultured with 0, 0.1, 1 or 10 ng ml⁻¹ IL-7, normalized to initial expression for each cell type (top panel), or to initial CD5 expression on OT-1 cells (bottom panel). (e) As described in a, except for freshly isolated C57BL/6 naïve (CD44^lo) CD8⁺ T cells, sorted into CD5^hi and CD5^lo expressing fractions. (f) As described in a, except for freshly isolated naïve H-2K^b−/−D^b−/− (CD44^lo) CD8⁺ T cells from C57BL/6.Rag1^−/− mice transplanted with C57BL/6.H-2K^b−/−D^b−/− bone marrow, sorted into CD5^hi and CD5^lo expressing fractions. Data (a–f) are representative of two independent experiments (error bar=±1s.d.).

Figure 2

CD5^lo naïve CD8⁺ T cells have prolonged cytokine and TCR-independent survival, increased glucose uptake and lower sensitivity to PI3K inhibition. Viability, as measured by DAPI exclusion, over 3 days for: (a) OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells, and (b) C57BL/6 naïve (CD44^lo) CD8⁺ T cells sorted into CD5^hi or CD5^lo expressing fractions, for cells rested in cytokine-free media overnight (16 h) and then treated±10 ng ml⁻¹ IL-7 in vitro in low density (∼2 × 10⁵ cells per ml) culture (top panel). Mean half-lives of cytokine-deprived cells are indicated in the bottom panel. Cell size as estimated by forward scatter for (c) OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells, and (d) C57BL/6 polyclonal naïve (CD44^lo) CD8⁺ T cells sorted into CD5^hi or CD5^lo expressing fractions. (e) Radioactive glucose uptake for OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells cultured 16 h±10 ng ml⁻¹ IL-7 and incubated for 45 min with 0.1 mM ³H-2-deoxy-D-glucose (4 μCi ml⁻¹). (f) Sensitivity of viability to PI3K inhibition for OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells rested overnight (16 h) in cytokine-free media and then treated 24 h with varying doses of the PI3K inhibitor LY294002. The LY294002 IC₅₀ for the viability of OT-1 and F5 T cells is shown to be 10 and 25 μM, respectively. Data (a–f) are representative of two independent experiments (error bar=±1s.d.). ^*P<0.05.

Figure 3

CD5^hi naïve CD8⁺ T cells have higher IL-7R expression and IL-7-induced signaling. (a) IL-7-induced signaling and responses under Jak and PI3K inhibition. OT-1 TCR-tg naïve (CD44^lo) CD8⁺ T cells were cultured overnight (16 h) and then treated with±10 ng ml⁻¹ IL-7 in the presence of untreated media control (Cont), vehicle (Veh), 1 μM Jak Inhibitor I (Jak), 10 μM LY294002 (LY) or 10 μM PI-103 (PI-103). Stat5 phosphorylation (pStat5; 20 min), GSK3 phosphorylation (pGSK3; 24 h), CD8α (24 h), Bcl2 (24 h), viability (24 h), proliferation (%Ki67, 5 days) and IL-7Rα levels (24 h) were measured by flow cytometry and quantified as the population median staining normalized to the IL-7-treated control samples, except for IL-7Rα, which are normalized to the untreated control. ^*P<0.05 between Veh and Cont conditions. Surface IL-7Rα expression of (b) OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells and (c) C57BL/6 naïve (CD44^lo) CD8⁺ T cells sorted into CD5^hi or CD5^lo expressing fractions, for cells freshly isolated from lymph nodes or cells cultured overnight (O/N; 16 h)±10 ng ml⁻¹ IL-7. IL-7-induced signaling in (d) OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells and (e) C57BL/6 naïve (CD44^lo) CD8⁺ T cells sorted on CD5^hi or CD5^lo expressing fractions, as assessed by pStat5 at 20 min, and pGSK3, Bcl2 and surface CD8α expression at 24 h, in cells rested overnight and then treated with±10 ng ml⁻¹ IL-7. (f) Total expression of Stat5, GSK3 and β-actin (loading control) as assessed by SDS-poly acrylamide gel electrophoresis (top panel) and quantified by densitometry (bottom panel) in OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells rested overnight in cytokine-free media. Data (a–f) are representative of two independent experiments (error bar=±1s.d.). ^*P<0.05.

Figure 4

IL-7R expression thresholds naïve CD8⁺ T-cell responses to IL-7. Linear relationship between loss of surface IL-7Rα at 6 h with (a) Stat5 phosphorylation at 10 min, and (b) Stat5 phosphorylation integrated over 6 h, for OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells rested overnight and treated with IL-7 concentrations of 0–10 ng ml⁻¹. Relationship of IL-7-induced signaling to (c, d) viability at 24 h, and (e, f) CD8α expression at 24 h for signaling quantified as either (c, e) Stat5 phosphorylation at 20 min or (d, f) GSK3 phosphorylation at 24 h, for OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells rested overnight (16 h) in cytokine-free media, and then treated with 1 ng ml⁻¹ of IL-7 and varying concentrations of Jak Inhibitor I (0.001–1 μM) to titrate down signaling. Additional supporting data and description of the design and motivation for titrating signaling via varying the dose of a Jak Inhibitor can be found in Supplementary Figures 3b–e. Data (a–f) are representative of two independent experiments (error bar=±1s.d.).

Figure 5

Proliferation requires higher threshold IL-7R signaling capacity than survival. (a) Comparison of IL-7-induced responses in OT-1 cells with signaling reduced to levels achievable by F5 cells. 24-h GSK3 phosphorylation and 5-day CD8α surface expression, viability and Ki67⁺ proliferating fraction following stimulation with 10 ng ml⁻¹ IL-7 for uninhibited OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells compared with OT-1 cells treated with 0.0625 μM of Jak Inhibitor I or 1 μM of the PI3K inhibitor PI-103. Relationships between IL-7-induced signaling at 24 h (GSK3 phosphorylation) and the following responses at 5 days: (b) viability, (c) CD8α surface expression and (d) proliferation as measured by % Ki67⁺ cells, for OT-1 TCR-tg naïve (CD44^lo) CD8⁺ T cells treated with 10 ng ml⁻¹ IL-7 and varying doses of Jak Inhibitor I, compared with uninhibited F5 cells. Responses are normalized to uninhibited OT-1 controls, and the dashed vertical lines indicate signaling levels in uninhibited OT-1 and F5 cells. Data (a–d) are representative of two independent experiments (error bar=±1s.d.).

Figure 6

Manipulation of in vivo IL-7 shifts the abundance of CD5^hiIL7R^hi and CD5^loIL7R^lo CD8⁺ T-cell subsets. (a) CD5 expression profiles of donor versus recipient polyclonal naïve (CD44^lo) CD8⁺ T cells followed over 3 weeks for donor C57BL/6.Thy1.2⁺ CD44^lo CD8⁺ T cells adoptively transferred into congenic age- and sex-matched C57BL/6.Thy1.1⁺ recipients. (b) CD5 expression of OT-1 TCR-tg naïve (CD44^lo) CD8⁺ T cells sorted into CD5^hi and CD5^lo expressing fractions (top panel) and their proliferation 5 days after adoptive transfer into syngeneic Rag1^−/− hosts, as assessed by CFSE dilution of donor cells recovered from recipient spleens (middle panels) and quantified as the fraction of cells divided (bottom panel). (c) CD5 expression of C57BL/6.Thy1.2⁺ naïve (CD44^lo) CD8⁺ T cells sorted into CD5^hi and CD5^lo expressing fractions (top panel) and their proliferation 7 days after adoptive transfer into lymphoreplete C57BL/6.Thy1.1⁺ hosts given 5 μg IL-7 by mini-osmotic pump infusion over 7 days, as assessed by CFSE dilution of donor cells recovered from recipient spleens (middle panels) and quantified as the percent of cells divided (bottom panel). Comparison of the (d) CD5 and (e) CD8α surface expression profiles of CD8⁺ T cells recovered from the lymph nodes of lymphoreplete C57BL/6 mice given PBS versus 5 μg IL-7 by mini-osmotic pump over 7 days. A minimum of two mice were used for each experimental condition. Data are representative of two independent experiments (error bar=±1s.d.). ^*P<0.05.

Figure 7

IL-7R level on CD8⁺ T cells suggests effective in vivo IL-7 concentrations supporting homeostasis. (a) Surface IL-7Rα expression of OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells rested overnight in cytokine-free media and treated 24 h with varying IL-7 concentrations ranging from 0.0001–100 ng ml⁻¹, compared with the IL-7Rα expression of freshly isolated cells, indicating effective in vivo homeostatic cytokine concentrations reflect in vitro IL-7 concentrations of 0.01–0.1 ng ml⁻¹ at the level of receptor suppression. (b) 24-h viability and (c) 5-day proliferation (%Ki67⁺ fraction) for OT-1 and F5 TCR-tg naïve (CD44^lo) CD8⁺ T cells treated with varying IL-7 concentrations in vitro as described in a, showing that over the effective in vivo IL-7 concentration range indicated in a, both cell types survive, but do not proliferate. Bioassay for effective in vivo IL-7 levels in which (d) 10⁶ 2C Thy1.1⁺ Rag1^−/− naïve (CD44^lo) CD8⁺ T cells were transferred into Thy1.2⁺ B6, OT-1 Rag1^−/−, 2C Rag1^−/−, F5 Rag1^−/− or Rag1^−/− hosts (minimum of three mice per recipient type), and (e) the relative effective IL-7 levels between hosts were inferred from the IL-7Rα and CD8α expression of Thy1.1⁺ donor cells. (f) IL-7Rα expression of donor cells showed a strong linear correspondence with number of CD8⁺ T cells in recipient spleens. Data are representative of two independent experiments (error bar=±1 s.d.).

Figure 8

Model for the role of IL-7 in the homeostasis of CD5 distributions in the CD8⁺ T-cell repertoire. Model depicting IL-7-mediated homeostasis in the diversity of CD5 expression among the naïve CD8⁺ T-cell repertoire. At effective IL-7 concentrations in normal lymphoreplete hosts, the diversity of CD5 expression is preserved. High [IL-7] shifts the CD8⁺ T-cell population towards high CD5 expression because of the selective proliferation of CD5^hi cells, whereas low [IL-7] may conceivably favor a population with low CD5 expression because of the selective survival of CD5^lo subsets.