1. Volume 47, Issue 1, Pages 171-182.e4 (July 2017)
Interleukin-7 Availability Is Maintained by a Hematopoietic Cytokine Sink Comprising Innate Lymphoid Cells and T Cells 
Christopher E. Martin, Darina S. Spasova, Kwesi Frimpong-Boateng, Hee-Ok Kim, Minji Lee, Kwang Soon Kim, Charles D. Surh 
Immunity 
Volume 47, Issue 1, Pages e4 (July 2017)
DOI: /j.immuni
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2. Figure 1
High Levels of Accumulated IL-7 in Il7r−/− Hosts Drive Division of Adoptively Transferred Il7r+/+ T Cells
(A) Proliferation of donor T cells in the LNs of the indicated hosts (day 7 [d7] after transfer, unless indicated). Gates indicate the frequency of undivided donor cells.
(B) Total TCRβ+CD8+ or TCRβ+CD4+ T cells in the LNs and spleen of the indicated mice.
(C) Relative geometric mean fluorescent intensity (RFI) of IL-7R expression on donor cells after 12–18 hr of incubation in the indicated hosts (normalized to that in B6 hosts). Error bars indicate standard error of the mean (SEM).
Data represent two independent experiments with two hosts per group (A and C) or with five (B6 and Il7r−/−Il7−/−) or six (Il7r−/−) mice (B). For (B and C), significance in relation to other mouse strains or B6 is shown as ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < ; ns, not significant.
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3. Figure 2
Stromal-Derived IL-7 Accumulates in Il7r−/− Hosts and Drives Division of Adoptively Transferred Naive T Cells
(A) Il7 transcript levels in the LNs of the BM chimeras defined in (B) but without donor T cell transfer (np, no qPCR product was generated).
(B) Donor T cell proliferation in the indicated BM chimera hosts without (black histograms) or with (gray histograms) A7R34 treatment to block IL-7R.
(C and D) Division indices of (C) donor T cells in the LNs of individual hosts or (D) the total number of donor T cells recovered from host LNs and spleen without (filled triangles) or with (empty triangles) A7R34 treatment; horizontal lines indicate the mean division index for the group.
Data were combined from two to four experiments with three to eight biological replicates per group, and error bars indicate SEM. ∗∗p < 0.005; ∗∗∗p < ; ns, not significant.
Immunity  , e4DOI: ( /j.immuni )
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4. Figure 3
IL-7 Availability in the T Cell Niche Is Regulated by IL-7R Expression on Cells of the Hematopoietic Lineage
Representative CellTrace Violet (CTV) histograms (left) or division index scores (right) for the indicated donor T cells in host LNs. To block IL-7R, A7R34 was injected into some hosts (gray histograms or empty triangles). Division index data are combined from two experiments, and horizontal lines mark the average for the group of two to seven biological replicates. ∗∗∗p <
Immunity  , e4DOI: ( /j.immuni )
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5. Figure 4 Characterization of IL-7R+ ILCs in Mouse LNs
(A) Representative data for LN ILCs from Rag1−/− or B6 mice. Gates are indicated above each panel.
(B) Total numbers of CD3−IL-7R+ ILCs in the LNs of individual mice. Horizontal bars indicate the average for the group, and the fold increase for the average ILC numbers in Rag1−/− LNs over those in B6 LNs is indicated above each graph.
(C) T-bet expression by CD3−IL-7R+ cells from the indicated Rag1−/− lymphoid tissues is further subdivided according to CD90 and NK1.1 expression, as indicated.
(D) Eomesodermin and IL-7R expression on gated CD90+NK1.1+ ILCs from the indicated Rag1−/− lymphoid tissues.
Data represent two experiments with three separately analyzed mice per group (A, C, and D) or were combined from multiple experiments with a total of 10 B6 and 13 Rag1−/− mice (B). p < 0.005; ∗∗∗p < ; ns, not significant.
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6. Figure 5 IL-7R+ Host ILCs Compete for IL-7 In Vivo
(A) Proliferation of donor T cells in the indicated hosts injected with PBS or anti-CD90.2 mAb on d0 and d3; mice were analyzed on d8.
(B) Rag1−/− mice received one (3 days before d0) or two (4 and 1 day before d0) injections of PBS or anti-CD90.2 mAb. Shown are d0 Il7 transcript levels from whole inguinal LNs.
(C) Numbers of host CD19+IL-7R+ cells in the indicated tissues of Rag1−/− mice treated with or without anti-CD90.2 mAb for 1 week.
(D) Frequency of Ki-67+ T cells in spleens of CD90-disparate Rag1−/− chimeric mice treated, where indicated, with anti-CD90.2 mAb to deplete ILCs and/or with A7R34 to block IL-7R.
(E and F) Proliferation of sorted CD90.1+CD44lo polyclonal donor T cells in the LNs of germ-free Rag1−/− hosts treated with anti-CD90.2 mAb; CD4+ and CD8+ T cells were co-transferred and analyzed on d7.
(G and H) Representative data (G) and calculated division index scores (H) of donor T cells in Rag1−/− hosts treated with anti-CD90.2 or anti-NK1.1 mAb and analyzed on d7; gray histograms and empty triangles indicate hosts treated with A7R34.
Data represent at least two experiments with two to seven biological replicates per group. Error bars indicate SEM. ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < ; ns, not significant.
Immunity  , e4DOI: ( /j.immuni )
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7. Figure 6
RORγt+ ILC3s Effectively Consume IL-7 and Stably Express High Levels of IL-7R In Vitro
(A) Cytokine receptor expression by freshly isolated T cells from B6 LNs and by ILCs from Rag1−/− LNs (black histograms), as well as the corresponding populations from knockout mice (gray histograms; Il7R−/− on left and Il2rb−/− on right).
(B) Average geometric MFI (gMFI) for cells in the IL-7R+ bracket gate in (A).
(C and D) LN suspensions from B6 or Rag1−/− mice were cultured with or without IL-7 or, as indicated, in one condition and then the other. All samples were analyzed simultaneously for IL-7R expression by flow cytometry; ILCs and T cells were distinguished by the expression of CD3 and the indicated markers. ILC1, CD3−CD90+NK1.1+GATA-3+/loRORyt−; ILC2, CD3−CD90+NK1.1−GATA-3hiRORγt−; ILC3, CD3−CD90+ NK1.1−GATA-3+/loRORγt+.
(E–G) 3 × 104 CD90+NK1.1− ILCs from Rag1−/− mice or 4.3 × 104 combined CD44loCD4+ and CD44loCD8+ T cells from B6 mice (E); 2 × 104 (1×) or 6 × 104 (3×) CD90+NK1.1− or CD90+NK1.1+ cells from Rag1−/− donors (F); or 1 × 104 ILC2s (CD3−CD90+NK1.1−GFP−), ILC3s (CD3−CD90+GFP+), or CD44loCD3+CD4+ or CD44loCD3+CD8+ T cells (G) were sorted from B6.RORγtGFP/+ donor LNs and incubated in media with rhIL-7. The amount of IL-7 remaining after 2 days was determined by in vitro T cell survival assay.
Data represent at least two experiments with two to seven replicates per group. Error bars indicate SEM. ∗∗p < 0.005; ∗∗∗p < ; ns, not significant.
Immunity  , e4DOI: ( /j.immuni )
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8. Figure 7
IL-7 Differentially Regulates FOXO1 Signaling Pathway in ILCs and CD8+ T Cells
(A and B) Naive CD8+ T cells from B6 mice or ILCs (Thy1+B220−CD3e−) from Rag1−/− mice were sorted and then incubated in culture media overnight or were treated with IL-7 (6 ng/ml) for the indicated periods (6 or 12 hr). For groups indicated as IL-17wo 3 hr, cells were incubated with IL-7 for 12 hr and then withdrawn from IL-7 culture and further incubated for 3 hr in media. Samples designated as “fresh” were processed immediately after sorting, and other samples were processed separately at the indicated time points. cDNAs from samples were prepared for real-time PCR with SYBR or TaqMan probes. Relative gene expression was calculated after normalization with HPRT internal control. Relative expression of Il7r (A) and Foxo1 (B) is shown. Error bars indicate standard deviation.
(C) Sorted naive CD8+ T cells or ILCs were incubated with or without IL-7 (6 ng/ml) for 6 hr and processed for immunoblotting for examination of pFOXO1, pAKT, and total FOXO1.
(D) Graphs show the pFOXO1/FOXO1 ratio from cells analyzed in (C).
Data represent two or more independent experiments.
Immunity  , e4DOI: ( /j.immuni )
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