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Blood, Vol. 112, Issue 4, 1249-1258, August 15, 2008
Antigen-specific T-T interactions regulate CD4 T-cell expansion
Blood Helft et al.
112: 1249
Supplemental materials for: Helft et al
Files in this Data Supplement:
- Figure S1. Identical results obtained using positive selection or no selection of naïve and memory Marilyn T cells (JPG, 82.7 KB)
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H-Y peptide-loaded DCs were injected into the footpad of female B6 mice with or without a first cohort of naïve CD45.2 CFSE labeled Marilyn cells. Six days later, a second cohort of naïve or memory CD45.1 CFSE labeled Marilyn cells was injected, and the CFSE profile assessed after another 6 day period. (A–D) Naïve and memory Marilyn cells were positively selected with an anti-CD45.1-PE antibody associated with anti-PE magnetic beads using an Automacs apparatus just before injection into recipients. (E–F) Naïve and memory Marilyn cells were injected without any selection. Dot plots of gated TCR+CD45.1+ cells are shown. Undivided cells are boxed.
- Figure S2. Memory CD4 T cells respond more efficiently to Ag in vitro and in vivo than naïve CD4 T cells
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(A) Phenotype of naïve and memory Marilyn CD4 T cells. Naïve or memory Marilyn splenocytes were stained with mAb anti-CD45.1, anti-CD4 and the indicated markers. The histograms shown are gated on CD45.1+/CD4+ cells. CD4+ cells from C57BL/6 splenocytes are shown as control. (B) In vivo proliferation of naïve and memory Marilyn T cells. A mixture of naïve (CD45.2) and memory (CD45.1) Marilyn cells were injected into CD45.2 B6 mice that were then immunized with DCs loaded (right panel) or not (left panel) with H-Y peptide. The draining lymph node was studied 6 days later. M=memory; N=naïve. (C–F) In vitro proliferation and functional avidity measurement of naïve and memory T cells. (C) Different numbers of naïve or memory CD45.1 Marilyn cells were incubated with irradiated male splenocytes and 3H-thymidine incorporation was measured after 4 days. (D) Forty thousand un-purified naïve or memory Marilyn cells were incubated with female splenocytes loaded with different amounts of H-Y peptide as indicated, and proliferation was measured as the percentage of maximal 3H-thymidine incorporation at day 4. Maximal incorporation for naïve T cells was 52934 cpm and for memory cells was 10718 cpm (memory CD4 T cells represent 10–15% of the responding cells distributed in the well). (E–F) Forty thousand un-purified naïve or memory Marilyn cells were incubated with female splenocytes loaded with 1.5 nM peptide and the indicated concentrations of anti-CD4 (GK1.5) or anti-MHC II (Y-P) ascitis. Proliferation was measured as above. (G, H) Production of IFN-γ by memory but not naïve Marilyn T cells. (I) Naïve or memory CD45.1 Marilyn spleen cells were incubated with different amounts of H-Y peptide, and intracellular staining for IFN-γ was performed after a 5 hour incubation. (J) Dot plot of IFN-γ staining in TCR+CD45.1+ gated naïve and memory Marilyn cells stimulated by the highest dose of peptide (right panels) or not (left panels).
- Figure S3. Memory cell proliferation is more strongly inhibited than that of naïve cells when they are in the same lymph node (JPG, 63.3 KB)
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B6 mice were injected with CFSE labeled CD45.2+ Marilyn T cells and were immunized with H-Y peptide loaded DCs in the footpad. Naïve (CD45.1/2) and memory (CD45.1) Marilyn T cells were injected i.v. 6 days later. Dot plots represent the naïve (upper panels) or memory (lower panels) Tg T-cell proliferation of the second cohort in the absence (left panels) or in the presence of the first cohort (right panel). Representative of 2 experiments.
- Figure S4. Activation of naïve and memory Marilyn T cells by activated CD4 T cells that have acquired MHC-II/peptide complexes is antigen specific and MHC-II–restricted (JPG, 74.2 KB)
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(A) Naïve Marilyn (c, d, i, j) or OT-II (e, f, k, l) cells were incubated for 20h with H-Y or OVA peptide-loaded LPS-matured DCs, respectively. T cells were then FACS-sorted, incubated with either control anti–H-2-Db(c, e, i, k) or blocking anti–H-2-IAb (d, f, j, l) antibody, washed and fixed in the presence of 0.01%glutaraldehyde. After extensive washing, the fixed MHC-II/peptide complexes-presenting T cells (200 000) were used as APCs to stimulate 20 000 purified CFSE-stained naïve (a–f) or memory (g–l) Marilyn T cells. Cells were analyzed 15h later for CD69 upregulation. H-Y peptide-loaded LPS-matured DCs (1000) were used as positive control APCs (b, h). (B) Activated OT-II CD4 T cells that have acquired MHC-II/OVA peptide complexes are able to activate OT-II T cells but not Marilyn T cells. OT-II cells were incubated with OVA peptide-loaded DCs, sorted by FACS and used to stimulate either OT-II (a), or Marilyn naïve (b) or memory (c) LN cells.
- Figure S5. Phenotype and number of activated CD4 T cells used in Fig. 7 experiment (JPG, 87 KB)
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(A) Phenotype of activated Marilyn CD4 T cells. CD45.1/2 Marilyn T cells were cultured overnight with either CD45.1 LPS-matured DCs that had been loaded with H-Y peptide (left histogram) or with LPS matured DCs in the presence of an anti-TcR antibody (central histogram). Marilyn activated T cells were stained with anti-CD45.1, anti-CD45.2, anti-Vβ6, anti-CD4 mAb and the indicated markers. The histograms shown are gated on CD45.1+/CD45.2+/Vβ6+/CD4+. Naïve CD4+ T cells from Marilyn lymph node are shown as control. (B) Number of injected activated Marilyn CD4 T cells in the draining lymph node after 12 days. Naïve or memory CFSE labeled CD45.1 Marilyn T-cells were injected i.v. into CD45.2 female hosts. In parallel, CD45.1/2 Marilyn T cells were incubated in vitro overnight with H-Y peptide loaded LPS-matured DCs or with LPS-matured DCs together with an anti-TCR antibody. The next day, the 2 sets of activated Marilyn T cell were FACS sorted and injected into the footpad. 6 days later H-Y loaded DCs were used to stimulate the T cells. Absolute number of activated T cells (CD45.1+/CD45.2+/Vβ6+/CD4+) found in the DLN 6 days later is indicated.
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