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Blood, Vol. 106, Issue 9, 3097-3104, November 1, 2005
Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo
Blood Siegmund et al.
106: 3097
Supplemental materials for: Siegmund et al, Vol 106, Issue 9, 3097-3104
Files in this Data Supplement:
- Table S1. Significance of differences found in the suppressive effect of Treg subsets (PDF, 54 KB) -
This table summarizes the statistical significances of suppressive effects of indicated Treg subsets on the Th1-mediated footpad swelling in 5 different experiments. P values, which were determined by repeated measure analyses, are shown.  E–CD25– cells were used as control.
- Table S2. Frequency of antigen-specific Tregs and responder cells in the antigen-draining lymph node after in vivo stimulation (PDF, 48 KB) -
This table shows the percentage of KJ1.26– T cells in the antigen-draining lymph node derived from the experiment described in Figure 4. To distinguish between responder cells and adoptively transferred Treg subsets, total PI– lymphocytes were gated on CFSE– and CFSE– KJ1.26– cells, respectively. E–CD25– control cells were transferred at 2 different cell numbers.
E–CD25– control cells transferred at high cell numbers differentiated into effector cells, resulting in an increased DTH response compared with PBS control (footpad thickness, 2.6 ± 0.1 mm vs 2.1 ± 0.2 mm, respectively). This expansion and the proinflammatory activity of E–CD25– control cells was accompanied by a partial inhibition of the proliferation of naive CFSE-labeled responder cells transferred two days later by competition. This was not observed when E–CD25– control cell numbers were reduced to a level resulting in equal frequencies (compared with Tregs) at the time of measurement.
- Figure S1. In vitro generated Th1 cells mediate a strong DTH response (JPG, 186 KB)
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BALB/c mice received 5 × 10– in vitro–generated Th1 cells. One day later, the DTH reaction was induced by injecting OVA peptide into the footpads. At 24 hours and at 14 days after antigen injection, footpads were taken for histological examination. Untreated BALB/c mice served as control. Representative photomicrographs (original magnification ×20, upper row; and ×200, lower row) show severe cellular infiltrates, which were dominated by granulocytes 24 hours after immunization and by lymphocytes 14 days after immunization.
- Figure S2. Characterization of Treg subsets from DO11.10 and DO11.10xFucTVII– mice (JPG, 100 KB)
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FACS analysis of pooled spleen and lymph node CD4– T cells shows staining with the clonotypic antibody KJ1.26, as well as stainings for the adhesion molecules L-selectin (CD62L),  1-integrin (CD29) and LFA-1 (CD11a) on indicated Treg subsets from DO11.10 and DO11.10xFucTVII– mice. Representative histogram plots from 3 independently analyzed mice are shown. Numbers indicate frequency of positive cells or mean fluorescence intensity (MFI) among indicated CD4– T cell subsets.
- Figure S3. Homing behaviour of Treg subsets and in vitro–generated Th1 cells. (JPG, 25 KB)
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(A) In vitro–preactivated Treg subsets from DO11.10 (WT) and DO11.10xFucTVII– mice were radioactively labeled with –In and injected intravenously into BALB/c mice in which a DTH response had been induced 24 hours before, followed by the determination of radioactivity in the antigen-draining lymph node (dr LN) after 24 hours using a  -counter. The percentage of total recovered radioactivity is shown (n = 12; mean ± SD; data pooled from 2 independent experiments). (B) In vitro–generated Th1 cells were radioactively labeled with –Cr and injected intravenously into BALB/c mice in which a DTH response had been induced 24 hours before. Twenty-four hours later, radioactivity in both OVA/IFA– (black bars) and PBS/IFA (white bars)–injected feet, as well as in the corresponding draining lymph nodes (dr LN), was determined using a -counter. The percentage of total recovered radioactivity is shown (n = 4; mean ± SD; one representative out of 3 independent experiments). Pathogenic Th1 effector cells displayed efficient migration into the inflamed footpad and accumulated at the peak of the DTH response, which increased as much as threefold compared with E– Tregs (Figure 2). Thus, it is not surprising that in order to achieve significant in vivo suppression, transfer of a rather high number of Tregs was required (Figure 1 and Table S1), since the local regulator-target ratio at the site of immune regulation is predicted to be even lower.
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