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Blood, Vol. 108, Issue 6, 1949-1956, September 15, 2006
Do CD8 effector cells need IL-7R expression to become resting memory cells?
Blood Buentke et al.
108: 1949
Supplemental materials for: Buentke et al
Files in this Data Supplement:
- Figure S1. Survival of F5 TreIL-7R T cells in vitro (JPG, 77.1 KB)
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F5 T cells were purified from spleen of F5 controls, dox fed F5 TreIL-7R mice and F5 TreIL-7R donors taken off dox food 3 days previously. Graphs show cell viability of F5 T cells from F5 controls (left) and F5 TreIL-7R mice (right). F5 control T cells were cultured with IL-7 (empty square) and without (empty circle) IL-7, F5 TreIL-7R T cells from dox fed donors were cultured with dox, with IL-7 (filled squares) and without IL-7 (filled circle), while T cells from dox free donors were cultured with IL-7 (empty square) and without (empty circle). Viability was determined by FACS on the basis of FSc and 7-AAD exclusion by CD8+ gated cells. IL-7 only promoted survival of F5 TreIL-7R T cells in the continued presence of both dox and IL-7. Neither IL-7 alone nor dox alone could prevent cell death and similar levels of apoptosis were observed in all these cultures.
- Figure S2. Impaired responses by F5 TreIL-7ROFF T cells to influenza virus infection (JPG, 76 KB)
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F5 T cells from F5 TreIL-7ROFF and F5 control mice were mixed in equal numbers and 2 × 106 total T cells injected together with A/NT/60-68 influenza virus i.v. into Rag1-/- recipients. Hosts were bled at different times post transfer and analysed for CD8, CD44, TCR and Ly5.1 expression. (A) The graph shows the frequency of all CD8+CD44hiTCRhi F5 T cells detectable in peripheral blood following infection. (B) The graph shows frequency of CD8+CD44hiTCRhi F5 T cells in peripheral blood that were F5 TreIL-7ROFF T cells. The representation of F5 TreIL-7ROFF T cells reproducibly dropped early in the response, by day 6, but remained constant thereafter. It is possible that the inefficiency of IL-7R deficient F5 T cells to form long term memory was a failure of naïve T cells to survive initially and subsequently prime, a priming defect of the F5 TreIL-7ROFF T cells, an expansion defect following priming, a failure for effectors to generate memory or a combination of all these factors.
- Figure S3. Induction of TreIL-7R expression in F5 TreIL-7R effector cells does not enhance engraftment into Rag1-/- hosts (JPG, 102 KB)
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Splenocytes from F5 control mice, dox fed F5 TreIL-7R and F5 TreIL-7R donors taken off dox food three days previously were stimulated with NP68 peptide for 72h followed by a further 96h culture in IL-2. Cultures of cells from dox fed were additionally supplemented with dox (2µg/ml). After 7 day culture with peptide and IL-2, dox supplemented F5 TreIL-7R and dox free F5 TreIL-7R blasts were mixed in equal numbers with control F5 blasts and 2 × 107 total cells transferred into Rag1-/- hosts. Recipients of dox supplemented F5 TreIL-7R cells were fed dox containing food. Recipient mice were bled at different times post transfer and analysed for CD8, TCR and Ly5.1 expression. (A) Graphs show frequency of Ly5.1-Ly5.2+ F5 TreIL-7R T cells (open circles) and Ly5.1+ F5 control cells (filled circles) in PBLs of dox fed and dox free Rag1-/- hosts. (B) Graph shows proportion of donor CD8+ T cells that were of Ly5.1- Ly5.2+ F5 TreIL-7R origin in dox fed Rag1-/- (filled circles) and dox free Rag1-/- (empty circles) hosts. (C) Dot plots show IL-7R expression versus Ly5.1 35 days post transfer in PBLs from dox fed or dox free hosts. Numbers indicate MFI of IL-7R expression by the corresponding Ly5.1+ or Ly5.1- population. Administration of dox had no detectable effect on the behaviour of F5 TreIL-7R T cells compared with F5 TreIL-7R T cells in dox free hosts, consistent with the low level of transgene expression observed in peripheral F5 TreIL-7R T cells.
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