Host IL-7R signaling is required for CD8⁺ T cell-mediated tumor rejection in response to IL-7 treatment.

<p>(A-C) Rag^-/- and Rag^-/-IL-7R^-/- mice were reconstituted with 7–10 x 10⁵ CD8⁺CD90.1⁺ OT-I T cells or were left untreated (+/- OT-I). OT-I-reconstituted mice received rIL-7 (+ IL-7) or PBS (- IL-7) every 3–4 days for 18 days starting one day before T cell transfer. (A, B) 22–23 days after T cell transfer, some mice were challenged s.c. with 1 x 10⁶ ovalbumin-expressing EG7 lymphoma cells. Mice with tumors >250 mm³ were scored as tumor positive. Pooled data from 2 independent experiments with a total of 10–12 mice per group are shown. Statistical significance was calculated using the log-rank test. (C, D) Some recipients were not challenged with EG7 but analyzed for OT-I expansion and phenotype. (C) Splenic CD8⁺CD90.1⁺ OT-I T cells were quantified 21–25 days after adoptive transfer. Pooled data (±SEM) from 2 independent experiments with a total of 7–9 mice/group are shown. (D) CD127 expression of splenic OT-I T cells was determined 5 days after adoptive transfer. Data (±SEM) for 3–4 mice/group are shown.</p

IL-7R signaling in non-hematopoietic cells regulates CD8⁺ T cell differentiation in response to peptide vaccination and IL-7 therapy.

<p>(A-G) The indicated BM chimeras received CD8⁺ OT-I T cells and were treated with SIINFEKL and rIL-7 as described above. Three weeks after T cell transfer, splenic CD8⁺ OT-I T cells were analyzed by flow cytometry. Bar diagrams show pooled results (±SEM) of 2–3 independent experiments with a total of (A, B, C, E, G) 10–17 or (D) 4–10 mice/group. Histogram overlays show representative results for individual mice. Grey-lined histograms represent FMO staining controls.</p

Host IL-7R signaling modulates CD8⁺ T cell expansion and differentiation in response to peptide vaccination and IL-7 therapy.

<p>(A-J) Rag^-/- and Rag^-/-IL-7R^-/- mice were reconstituted with CD8⁺CD90.1⁺ OT-I T cells and treated with SIINFEKL +/- rIL-7 as described in Fig 4. Three weeks after T cell transfer (A, B) spleens and (C-J) peripheral blood were analyzed by flow cytometry. Shown are numbers of (A) splenocytes and (B) splenic CD8⁺CD90.1⁺ OT-I T cells. After gating on OT-I T cells, relative frequencies (frequ.) of (C) CD62L^hi, (E) KLRG-1^hi, (F) Ki67^hi, (H) IFN-γ^hi, (I) TNF-α^hi OT-I cells and relative MFIs for (D) CD127, (Γ) Bcl-2 and (J) PD-1 were determined. (H, I) Cytokine production was measured after <i>in vitro</i> stimulation of PBMCs with 1 μM SIINFEKL peptide in the presence of Rag^-/- splenocytes, brefeldin A and monensin for 6 hours. Relative (rel.) values were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159690#pone.0159690.g002" target="_blank">Fig 2</a>. Bar diagrams show pooled data (±SEM) from 3 independent experiments with a total of 15–18 mice/group. Histogram overlays show representative results for individual mice. Dashed lines in overlays represent (C-G and J) FMO control samples or (H, I) stained cells without prior SIINFEKL stimulation.</p

The combination of rIL-7 therapy and peptide vaccination impairs T cell-dependent tumor rejection in Rag^-/- mice.

<p>(A) Rag^-/- and (B) Rag^-/-IL-7R^-/- mice were reconstituted with 1 x 10⁶ CD8⁺CD90.1⁺ OT-I T cells or were left untreated (+/- OT-I). One day later, OT-I-reconstituted mice were either vaccinated with 50 μg SIINFEKL or received PBS (+/- Pep). rIL-7 or PBS (+/- IL-7) were injected every 3–4 days for 19 days starting one day before T cell transfer. Mice were challenged s.c. with 1 x 10⁶ EG7 tumor cells three weeks after T cell transfer. Mice with tumors larger than 250 mm³ were scored as tumor positive. Shown are pooled data from 2 independent experiments with a total of 12–13 T cell reconstituted mice. Primary tumor growth was analyzed in untreated Rag^-/- and Rag^-/-IL-7R^-/- mice (n = 3). Statistical significance was calculated using the log-rank test. (C) The numbers of splenic DCs were determined in tumor-bearing mice 28–37 days after tumor challenge. Pooled data (±SEM) from 2 independent experiments with a total of 6–11 mice/group are shown. Statistical significance was calculated using the Mann-Whitney test.</p

Host IL-7R signaling is required for granulocyte and DC expansion in response to rIL-7.

<p>Rag^-/- and Rag^-/-IL-7R^-/- mice were treated with rIL-7 or PBS (+/- IL-7) every 3–4 days and spleens were analyzed by flow cytometry after 10–24 days. (A-E) Shown are numbers of (A) splenocytes (Splen.), (B) CD11b⁺Gr1⁺ granulocytes (Gran.), (C) CD11c⁺MHC-II⁺ dendritic cells (DCs), (D) CD8⁺ and (E) CD8^- DCs. Shown are pooled data (±SEM) from 2 independent experiments with a total of 7–8 mice/group.</p

Host IL-7R signaling is not required for rIL-7-induced T_M differentiation.

<p>(A-E) Rag^-/- and Rag^-/-IL-7R^-/- mice were treated with rIL-7 or PBS (+/- IL-7) every 3–4 days starting one day prior to adoptive transfer of 1 x 10⁶ CD8⁺CD90.1⁺ OT-I T cells. 21–25 days after transfer, splenic OT-I T cells were analyzed by flow cytometry. After gating on CD8⁺CD90.1⁺ or CD8⁺Vα2⁺ OT-I T cells, mean fluorescence intensities (MFIs) for (A) CD127 and (B) Bcl-2 as well as the frequencies (frequ.) of (C) CD62L^hi, (D) KLRG-1^hi and (E) IFN-γ^hi cells were determined. IFN-γ production was measured after <i>in vitro</i> re-stimulation with 1 μM SIINFEKL peptide in the presence of brefeldin A for 6 hours. (A-E) Data shown in bar diagrams were normalized to the mean values determined in PBS-treated Rag^-/- mice and are shown as relative (rel.) values. Pooled data (±SEM) from 2–3 independent experiments with a total of 7–13 mice/group are shown. Histogram overlays show relative cell numbers (rel. cell #) and relative fluorescence intensities (rel. fl. int.) for individual mice after gating on OT-I cells. Dashed lines in overlays represent (A-D) FMO control samples or (E) stained cells without prior SIINFEKL stimulation.</p

IL-7R signaling in non-hematopoietic host cells contributes to granulocyte, DC and CD8⁺ T cell expansion in response to IL-7 therapy.

<p>Lethally irradiated CD45.1⁺ Rag^-/- and CD45.2⁺ Rag^-/-IL-7R^-/- mice were reconstituted with the indicated bone marrow (BM) cells (donor→recipient). After successful BM reconstitution, BM chimeras received 1 x 10⁶ CD8⁺ OT-I T cells. One day later, mice were vaccinated with 50 μg SIINFEKL. IL-7 treatment was done as described above. (A-E) Three weeks after T cell transfer, recipient spleens were analyzed by flow cytometry and the numbers of the indicated cell types were determined. Shown are pooled data (±SEM) of 3 independent experiments with a total of 10–17 mice/group. (F) BM chimeras received 1 x 10⁶ renilla luciferase-transgenic (ChRluc^tg) CD8⁺ OT-I T cells (OT-I^ChRluc) and were treated with SIINFEKL and rIL-7 as described above. Six days after T cell transfer, mice were treated with 100 μg colenterazine to determine bioluminescence intensities (BLI) as a readout for OT-I abundance and distribution. Shown are pooled results (±SEM) of 2 independent experiments with a total of 5–10 mice/group.</p

Data_Sheet_1.PDF

<p>Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor–ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4⁺ and CD8⁺ T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.</p

IL-7R signaling protects Rag⁻ mice from DSS-induced colitis.

<p>(<b>A, B</b>) WT (n = 4), Rag⁻ (n = 8), Rag⁻IL-7R⁻ (n = 7) and Rag⁻OT-I⁺ mice (n = 6) received dextran sulfate sodium (DSS) via the drinking water. From day 5 on, DSS-free drinking water was provided. (<b>A</b>) Body weight was determined every day and calculated in relation to the initial body weight. Shown are the mean relative body weight ± SEM and the time after onset of DSS treatment. (<b>B, C</b>) Colon samples were taken at day 8 and analyzed histologically. Shown are histological scores for groups of untreated (open symbols; n = 4) and DSS-treated mice (closed symbols; n = 6–8). (<b>C</b>) Shown are representative colon sections from the indicated mice. (<b>A–C</b>) Data represent one experiment.</p

IEC express functional IL-7R.

<p>(<b>A, B</b>) IEC were isolated from the colon of (<b>A</b>) Rag⁻IL-7R⁻ and (<b>A, B</b>) Rag⁻ mice. (<b>B</b>) Rag⁻ IEC were stimulated with 20 ng/ml rec. IL-7 for 15 minutes or were left untreated. The levels of IL-7R expression and Stat5 phosphorylation (p-Stat5) were determined by flow cytometry. (<b>A, B</b>) Shown are relative cell numbers and relative fluorescence intensities for (<b>A</b>) IL-7R and (<b>B</b>) p-Stat5 after gating on viable (<b>A</b>) (7AAD⁻), (<b>A, B</b>) CD45⁻, EpCam⁺ IEC. Results are representative for 2–3 independent experiments.</p