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Blood, Vol. 113, Issue 23, 5793-5800, June 4, 2009
Regulation of T cell-dendritic cell interactions by IL-7 governs T-cell activation and homeostasis
Blood Saini et al.
113: 5793
Supplementary materials for: Saini et al
Files in this Data Supplement:
- Figure S1. IL7 does not affect proximal T-cell signaling (JPG, 104 KB)
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(A) Total LN cells from F5 Rag1−∕− mice were cultured (106/ml) with 10nM NP68 in the presence or absence of IL7 (10ng/ml). Histograms are of IL7R expression by CD8+ gated T cells cultured with (solid line) or without (broken line) NP68 for the time in minutes indicated, in the presence (lower row) or absence (upper row) of IL7. IL7R staining of DP F5 thymocytes is shown as negative control. (B) Histograms are of pSTAT5 staining of CD8 T cells culture with (solid line) or without (grey fill) NP68 for the time in minutes indicated in the absence of IL7. (C) Total LN cells from F5 Rag1−∕− mice were cultured exactly as in (A). At 24h, Bcl-2 and pSTAT5 expression was determined. The upper row of histograms show Bcl-2 expression by F5 T cells cultured with (solid line) or without (broken line) IL7 and either stimulated with (right) or without (left) NP68 peptide. Grey fill shows isotype negative control staining. The bottom row shows pSTAT5 staining by cells from the same cultures. The left histogram shows pSTAT5 by F5 T cells cultured in the absence of NP68 but with (solid line) or without (grey fill) IL7. The right histogram shows pSTAT5 by F5 T cells stimulated with NP68 and with (solid line) or without (broken line) IL7. Grey fill shows pSTAT5 in cells from unstimulated, IL7 free cultures. Data are representative of two experiments.
- Figure S2. F5 T cells from dox free F5 TetIL7R mice do not express IL7R (JPG, 75.8 KB)
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(A) F5 TetIL7R mice were taken off dox food for 7 days and then proximal IL7R signalling function in CD8+ splenocytes assessed by measuring pSTAT5 induction following 30 minutes stimulation with IL7 (10ng/ml) in vitro (107 splenocytes/ml in PBS). Histograms are of pSTAT5 in CD8+ T cells from F5 TetIL7R mice (right) compared with control F5 mice (left) cultured with (solid line) or without (grey fill) the indicated cytokine. (B) Bar chart shows relative expression of Il7r by purified F5 T cells from control mice, dox fed F5 TetIL7R mice (ON), F5 TetIL7R mice taken off dox food 3 days previously (OFF 3d) as compared with total splenocytes from Il7r−∕− mice as control. IL7R mRNA levels were determined by quantitative PCR. CD8+ T cells were purified >98% by high speed sorting on Moflow cytomter (Dako Cytomation). RNA was isolated by Trizol extraction (Invitrogen) according to manufacturers instructions and DNAase treated (Ambion). cDNA was produced by reverse transcription (Invitrogen) using oligo dT 14-18 (GE Healthcare) and standard protocols. Expression of Il7r was determined by real time PCR using an Applied Biosystems ABI Prism 7000 Sequence Detection System and commercial FAM labelled probes (Applied Biosystems). mRNA levels were normalised against Hprt mRNA. Data are representative of two experiments.
- Figure S3. Long term survival of F5 T cells is dependent on both IL7 and IL15 (JPG, 115 KB)
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Although naïve IL7R- F5 T cells do not persist long term following transfer to Rag1−∕− hosts (Fig. S3A and S3B), experiments with naïve cells showed that IL15 had a limited capacity to maintain IL7R- F5 T cells (Fig. S3C) in Rag1−∕− hosts such that there was no detectable cell death over a 3 day duration (Fig. S3D), the same period over which the flu challenge was assessed. Furthermore, we could find no evidence that death of activated IL7R- F5 T cells could account for the observed triggering defect since both Bcl-2 expression (Fig. S4A) and Caspase 3 activation (Fig. S4B) were identical amongst IL7R- and IL7R+ F5 T cells at d3 in flu challenged hosts, suggesting that activated IL7R- F5 cells were no more susceptible and were not undergoing a greater rate of apoptosis than control F5 T cells. F5 T cells from Ly5.1+ control and dox free Ly5.1− F5 TetIL7R donors were labelled with CFSE, mixed approximately 1:1 ratio and 3 × 106 total CD8+ T cells transferred into with Rag1−∕− or Il15ra−∕−Rag1−∕− recipients. In the absence of Il15ra expression, IL15 producing cells in Il15ra−∕−Rag1−∕− are unable to trans-present IL15 cytokine and are therefore functionally IL15 deficient. (A) At days 7 and 16, lymph node and spleen were recovered and phenotype of donor cells determined by FACS. Dot plots show CFSE staining vs Ly5.1 amongst CD8+ F5 T cells recovered from Rag1−∕− or Il15ra−∕−Rag1−∕− hosts at the days indicated. (B) Bar charts show absolute (left) representation of CD45.1+ control IL7R+ F5 T cells (filled bars) compared with CD45.1− IL7R- F5 T cells at d1, d7 and d16 following transfer to Rag1−∕− (upper) and Il15ra−∕−Rag1−∕− (lower) hosts. The size of precursor populations was calculated from their CFSE profiles and the representation of IL7R+ and IL7R- F5 T cells re-calculated to exclude expansion due to division. Bar charts on the right show representation of IL7R+ and IL7R- F5 T cells that exclude the expansive effects of cell division in Rag1−∕− (upper) and Il15ra−∕−Rag1−∕− (lower) hosts, thereby reflecting differential survival in the different hosts. We have previously shown that there is no detectable cell death among IL7R+ F5 T cells transferred to Rag1−∕− hosts over a three week period31 and similar studies of IL7R+ F5 T cells transferred to Il15ra−∕−Rag1−∕− hosts also reveal no survival defect but rather an increase in donor population entirely accounted for by the cells CFSE profile (T. Hogan and B. Seddon, unpublished). (C) Survival of IL7R- F5 T cells is expressed as adjusted representation (from RH panels in B) of IL7R- F5 in co-transfers of IL7R+ and IL7R- F5 T cells to Rag1−∕− (filled circles) and Il15ra−∕−Rag1−∕− (empty circles) hosts, normalised to representation at d1 post transfer. (D) Graph shows cell recoveries of IL7R+ and IL7R- F5 T cells at days 1-3 following transfer to Rag1−∕− recipients normalised to recoveries at d1 as a baseline. There was no detectable cell loss of either population during the first 3 days post transfer. Data are representative of two.
- Figure S4. No evidence of apoptosis by IL7R- F5 T cells during acute flu responses (JPG, 124 KB)
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IL7R+ and IL7R- F5 T cells were co-transferred to Rag1−∕− hosts (3 × 106 total T/mouse) and challenged with flu (24U/mouse). (A) Histograms show Bcl-2 expression (solid line) by IL7R+ and IL7R- F5 T cells before transfer at d0 and by cells recovered from spleen of Rag1−∕− hosts 3 days later, during proliferation. (B) Induction of apoptosis was assessed by measuring caspase-3 activation. Active capase-3 is readily detectable in live FSc SSc gated F5 lymph node cells cultured 24h at 37° in the absence of IL7 (+ve control, solid line) compared with the same cells stained ex vivo (grey fill). Dot plots show FSc vs SSc of CD8+ gated Ly5.1+ IL7R+ and Ly5.1− IL7R- F5 T cells 3 days after co-transfer and flu challenge. Lower histograms show active caspase 3 in the inducated population. Data are representative of two or more experiments.
- Figure S5. Expression of CD5, TCR and I-Ab by F5 T cells in lymphopenic hosts (JPG, 56.3 KB)
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(A) T cells from F5 Rag1−∕− donors were transferred (2 × 106 per mouse) to Rag1−∕− or β2m−∕− Rag1−∕− hosts. Histograms show CD5 and TCR expression by F5 T cells 7 days after transfer to Rag1−∕− hosts (solid line) or β2m−∕− Rag1−∕− (broken line) hosts as compared with expression by F5 T cells from control F5 Rag1−∕− mice (grey fill). (B) Histograms show I-Ab expression by CD8+ T cells from I-Ab deficient hosts (solid line) and control C57Bl6/J mice (Grey fill). (C) Histograms show I-Ab expression by F5 T cells 7 days after transfer to Rag1−∕− hosts (solid line) or β2m−∕− Rag1−∕− hosts (broken line) as compared with F5 T cells from control F5 Rag1−∕− hosts (grey fill). Data are representative of three or more experiments.
- Figure S6. Normal dendritic cell phenotype and representation in Il7−∕− Rag1−∕− hosts (JPG, 80.4 KB)
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(A) Dot plots show staining of splenocytes from Rag1−∕− and Il7−∕− Rag1−∕− mice for CD11c and I-Ab expression and the gates used to define the mature DC (mDC) compartment. (B) Bar chart indicates total numbers of mDC in spleen of Rag1−∕− and Il7−∕− Rag1−∕− mice (n=4 each). There was no statistically significant difference in numbers. (C) Histograms show identical expression of I-Ab and CD86 by mDC from Rag1−∕− (broken line) and Il7−∕− Rag1−∕− (solid line) mice.
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