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. 2012 Dec;13(12):1187-95.
doi: 10.1038/ni.2449. Epub 2012 Oct 28.

Mechanistic and structural insight into the functional dichotomy between IL-2 and IL-15

Aaron M Ring  1 , Jian-Xin Lin, Dan Feng, Suman Mitra, Mathias Rickert, Gregory R Bowman, Vijay S Pande, Peng Li, Ignacio Moraga, Rosanne Spolski, Engin Ozkan, Warren J Leonard, K Christopher Garcia
Affiliations

Mechanistic and structural insight into the functional dichotomy between IL-2 and IL-15

Aaron M Ring et al. Nat Immunol. 2012 Dec.

Abstract

Interleukin 15 (IL-15) and IL-2 have distinct immunological functions even though both signal through the receptor subunit IL-2Rβ and the common γ-chain (γ(c)). Here we found that in the structure of the IL-15-IL-15Rα-IL-2Rβ-γ(c) quaternary complex, IL-15 binds to IL-2Rβ and γ(c) in a heterodimer nearly indistinguishable from that of the IL-2-IL-2Rα-IL-2Rβ-γ(c) complex, despite their different receptor-binding chemistries. IL-15Rα substantially increased the affinity of IL-15 for IL-2Rβ, and this allostery was required for IL-15 trans signaling. Consistent with their identical IL-2Rβ-γ(c) dimer geometries, IL-2 and IL-15 showed similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induced similar signals, and the cytokine specificity of IL-2Rα versus IL-15Rα determined cellular responsiveness. Our results provide new insights for the development of specific immunotherapeutics based on IL-15 or IL-2.

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Figures

Figure 1
Figure 1
The crystal structure of the quaternary IL-15 receptor complex. (a) Front (left) and top (right) views of the IL-15 quaternary receptor complex comprised of IL-15 (green), IL-15Rα (cyan), IL-2Rβ (blue), and γc (gold). The “site I” and “site II” interactions between IL-15 and IL-2Rβ and γc, respectively, are indicated. (b) The structure of the IL-2 quaternary complex (PDB code 2B5i; left) and the superimposition of the IL-15 and IL-2 receptor complexes (right). The IL-15 and IL-2 complexes superimpose with an r.m.s.d of 1.175Å)
Figure 2
Figure 2
Comparison of the IL-15 and IL-2 site I interfaces. (a) The site I interface of IL-15 (green cylinders and side chains) contacting IL-2Rβ (blue loops and side chains). (b) The site I interface of IL-2 (magenta cylinders and side chains) contacting IL-2Rβ (blue loops and side chains). (c) Superimposition of the IL-15 (green) and IL-2 (magenta) A and C helices showing structural conservation of D61, N65, and D8 of IL-15. (d) Superposition of IL-2Rβ bound to IL-15 (light blue) and IL-2 (blue) indicating the apparent rigidity of the interface in binding two distinct cytokines.
Figure 3
Figure 3
Comparison of the IL-15 and IL-2 site II interfaces. (a) The site II interface of IL-15 (green tubes and side chains) binding to γc (yellow surface). X-SCID associated Y103 on γc is depicted in dark yellow. (b) The site II interface of IL-2 (magenta tubes and side chains) binding to γc (yellow surface). (c) Superimposition of the IL-15 (green) and IL-2 (magenta) A and D helices. Only Q108 and I111 of IL-15 are strictly conserved at the interface. (d) Comparison of γc binding interfaces. The surface of γc, shaded according to binding to IL-15 (left; dark green) or IL-2 (right; magenta). Note the increased contact area between IL-15 and γc, particularly in the upper part of the receptor.
Figure 4
Figure 4
Enhancement of IL-15—IL-2Rβ interaction by IL-15Rα. (a) SPR sensorgrams of IL-2Rβ binding to free IL-15 (left) or IL-15—IL-15Rα complexes (right) demonstrate IL-15—IL-15Rα complexes bind to IL-2Rβ with higher affinity (3 nM) relative to free IL-15 cytokine (438 nM). (b) Top view of the IL-15 quaternary complex indicating the lack of contact between IL-2Rβ and IL-15Rα. (c) A 65 ns molecular dynamics simulation shows a global reduction of the r.m.s.d. for each indicated structural element (assignments indicated in Supplementary Table 1) of IL-15 upon binding IL-15Rα. Error bars represent the standard error of r.m.s.d. (d) The five most highly-populated states of IL-15 bound to IL-15Rα (left) and free IL-15 (right) indicate the subtle global stabilization upon binding IL-15Rα.
Figure 5
Figure 5
Signaling analysis of IL-2 and IL-15 in YT-1 human NK cells. The phospho-STAT5 dose-response relationships for IL-2 (magneta circles), the “superkine” H9 (orange squares), IL-15 (light green upward triangles), and IL-15—IL-15Rα complexes (dark green downward triangles) are shown for IL-2Rα− and IL-2Rα+ YT cells (top left and right, respectively). Signaling kinetics relationships for phospho-STAT5 (2nd row), phospho-ERK1/2 (3rd row), and IL-2Rβ receptor internalization (4th row) were determined at saturating (500 nM) and subsaturating (1 nM) concentrations (boxed columns). Differences in signaling amplitude and kinetics are concentration-dependent as cytokine signaling profiles converge at saturating concentrations. Error bars represent standard error of mean fluorescence intensity of samples in triplicate
Figure 6
Figure 6
Signaling analysis of IL-2 and IL-15 in primary mouse CD8 cells. As in Fig. 5, phospho-STAT5 dose-response relationships for IL-2 (magneta circles), H9 (orange squares), IL-15 (light green upward triangles), and IL-15—IL-15Rα complexes (dark green downward triangles) are shown for freshly-isolated CD8 T cells and CD8 T cells ‘pre-activated’ with anti-CD3 antibody. ‘Pre-activated’ cells express significantly higher levels of IL-2Rα and IL-15Rα than freshly-isolated cells (Supplementary Fig. 3b). Signaling kinetics relationships for phospho-STAT5 (2nd row) and phospho-S6kinase (3rd row) were determined at saturating (500 nM) and subsaturating (1 nM) concentrations (boxed columns). Similar to the effect of the cytokines in YT-1 cells, signaling amplitude and kinetics were readily predicted by the respective point where each cytokine resided on its dose-response relationship (top row). Error bars represent standard error of mean fluorescence intensity of samples in duplicate.
Figure 7
Figure 7
RNA-seq analysis of gene transcription regulated by IL-2 and IL-15. (a) Correlations in fold changes (log2) of IL-2 and IL-15 regulated genes. 95% confidence intervals are not shown as they almost overlap with the 95% prediction from the linear regression fit. (b) Bar graphs showing the numbers of genes that are regulated by IL-2 or IL-15 at the indicated times and concentrations. (c) and (d) Heatmaps showing genes that are preferentially regulated by (c) IL-2 or (d) IL-15. Expression of each gene is normalized to the same range [-2, 2] for color display.
Figure 8
Figure 8
qPCR validation of differentially regulated IL-2 and IL-15 target genes. (a) IL-2 and IL-15 induced expression of the indicated genes at 1 and 500 nM, at 4 and 24 hr time points. (b) Examples of genes more induced by IL-2 than IL-15 even at high dose. (c) Genes that are preferentially induced by IL-15 at low dose but similarly induced by IL-2 and IL-15 at high dose. The cDNA inputs were normalized based on the ΔCt values of Rpl7 primers. Shown are the relative expression levels of triplicate samples of a representative experiment. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
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