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Blood, Vol. 111, Issue 7, 3546-3552, April 1, 2008
Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells
Blood Schütz et al.
111: 3546
Supplemental materials for: Schutz et al
Files in this Data Supplement:
- Figure S1. No TNF-α production was detectable in co-cultures of Mart-1 specific CTL with c.b. or КaAPC (JPG, 27 KB)
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Mart-1 specific CTL were co-cultured with CMVpp65 or Mart-1 loaded КaAPC at a 1:1 ratio. 1µg/ml  -Fas-IgM mAb, 1µg/ml Mart-1 peptide and unstimulated Mart-1 specific CTL served as controls. Supernatans were collected after 48 h and analyzed using a Luminex100IS™ (Luminex Corporation, Austin, Texas, USA). The amount of TNF- is depicted as background subtracted (unstimulated Mart-1 specific CTL) pg/ml. Data represents the mean ± s.d. of two independent experiments.
- Figure S2. Antigen specific depletion of Mart-1 specific CTL is mediated by cognate loaded КaAPC but not by TNF-α (JPG, 30.1 KB)
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Mart-1 specific CTL were co-cultured either with Mart-1КaAPC in presents of a blocking anti-TNF- mAb or with Mart-1КaAPC only. Purified IgG1 was used as matching isotype control. Samples were harvested after 48 h and immediately stained with AnnexinV and PI to determine antigen specific apoptosis. Data is depicted relative to antigen specific apoptosis induced in samples co-cultured with Mart-1КaAPC only. The mean ± s.d. of four independent experiments is shown.
- Figure S3. Elimination of CTL occurs early during interaction with КaAPC (JPG, 134 KB)
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CMVpp65-specific CTL were co-cultured with different loaded or unloaded bead preparations (unloadedКaAPC, Mart-1КaAPC, CMVpp65КaAPC). At indicated time points (30, 60 and 120 min following initiation of co-cultures) beads were magnetically removed and CMVpp65-specific CTL further incubated. After 48 h samples were immediately stained with AnnexinV and PI as described in the experimental protocols. CMVpp65-specific CTL co-cultured for 48 h with the indicated beads served as positive control. Numbers depicted in the upper left quadrants represents % of total cells. Original FACS data is derived from one representative experiment.
- Figure S4. КaAPC eliminate CTL in an antigen-specific fashion (JPG, 96.7 KB)
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PKH26-labelled activated Fas+ effector-memory CTL and PKH67-labelled CMVpp65-specific CTL from the same donor were mixed to a 1:1 ratio and co-cultured with CMVpp65КaAPC for 48 h. Control cultures were treated with unloadedКaAPC, or 1 µg/ml of soluble -Fas-IgM (clone CH11). Minimal loss of viable cells was determined in untreated mixed CTL cultures (Tmix). Original FACS data for each CTL population of the Tmix are shown. Numbers depicted in the upper left quadrants represents % of total cells. The illustrating figure is derived from one representative experiment out of three independent experiments.
- Figure S5. Elimination of CMVpp65-specific CTL by CMVpp65КaAPC (JPG, 85.9 KB)
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Elimination of CMVpp65-specific CTL in co-cultures with unloadedc.b. or КaAPC loaded with Mart-1 or CMVpp65 peptide. Co-cultures with unstimulated CMVpp65-specific CTL served as negative control. Original FACS data after 48 h of co-culture are shown. Numbers depicted in the upper left quadrants represents % of total cells. The figure is generated from one representative experiment out of five independent experiments.
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