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Article Figures & Data

Figures

  • Figure 1.

    Only the CD62L+ subset of CD4+CD25+ Treg cells protects from lethal aGVHD in vivo. (A) CD4+ splenocytes from C57BL/6 donors were sorted into CD4+CD25- (not shown), CD4+CD25+CD62L+, and CD4+CD25+CD62L- subpopulations. (B) Lethally irradiated BALB/c recipients received 2 × 106 TCD BM cells from C57BL/6 mice for reconstitution plus 500 000 C57BL/6-derived CD4+CD25- T cells either alone (▪; n = 10) or together with 500 000 CD4+CD25+CD62L+ (▴; n = 10) or CD4+CD25+CD62L- (▾; n = 10) Treg cells. Additional groups were injected with TCD BM and 500 000 CD4+CD25+CD62L+ (▵; n = 5) or CD4+CD25+CD62L- (▵; n = 3) Treg cells only. Data were pooled from 2 independent experiments.

  • Figure 2.

    CD4+CD25+CD62L+ and CD4+CD25+CD62L- T cells express comparable levels of GITR on their surface but differ in the expression of other markers. C57BL/6 splenocytes were stained with anti-CD4 Cy7APC, anti-CD25 APC, anti-CD62L FITC, and PE-labeled antibody against the marker of interest and analyzed by flow cytometry. Two-dimensional dot plots of CD62L versus GITR, CD44, CD45RB, or CD69 are shown for CD4+CD25- T cells in the left and CD4+CD25+ T cells in the middle column. The histograms in the right column are overlays of the respective markers for CD4+CD25- T cells (shaded), CD4+CD25+CD62L- (bold), and CD4+CD25+CD62L+ Treg cells (fine). The gates for CD4+CD25- and CD4+CD25+ cells as well as the CD62L+ and CD62L- subsets are shown in the two top FACS plots.

  • Figure 3.

    Both CD4+CD25+CD62L+ and CD4+CD25+CD62L- T cells show Treg cell characteristics in vitro. (A) Alloresponse of C57BL/6-derived CD4+CD25- T cells and CD4+CD25+CD62L+ and CD62L- T cells toward BALB/c APC in vitro. Cultures were set up with 100 000 BALB/c-derived APC and 100 000 sorted CD4+CD25- T cells from C57BL/6 mice plus variable numbers of C57BL/6-derived CD4+CD25+CD62L+ (▨) or CD62L- T cells (▪) to obtain the indicated ratios. CD4+CD25+CD62L+ and CD62L- T cells were also stimulated alone. Proliferation was assessed by labeling the cultures with 3H-thymidine for the final 16 hours of the 5-day incubation period. Data represent mean + SD of triplicate cultures. Shown is 1 of 5 experiments with similar results. (B) Sorted CD4+CD25+CD62L- (▪), CD4+CD25+CD62L+ (▨), and CD4+CD25- T cells (□; 25 000) were stimulated for 24 hours with 50 ng/mL PMA and 1 μM ionomycin. IL-2 in the supernatant was determined by ELISA. The results represent mean + SD of triplicate cultures. (C) cDNA was prepared from cell populations sorted as in panel B and analyzed for expression of FoxP3 by real-time quantitative PCR using β-actin as normalizing gene.

  • Figure 4.

    CD4+CD25+CD62L+ T cells home significantly better to secondary lymphoid tissues than CD4+CD25+CD62L- T cells. Sorted CD4+CD25+CD62L+ (▨) and CD4+CD25+CD62L- T cells (▪) were labeled with 111In before cotransfer with unlabeled CD4+CD25- T cells and TCD BM into irradiated hosts. Mice were killed 48 hours later and radioactivity in the various organs was measured. (A) Global comparison of aGVHD target organs (liver, small bowel, large bowel) and primary lymphoid organs (spleen, peripheral LN [PLN], mesenteric LN [MLN]). Results are expressed as fraction of injected radioactive dose divided by organ weight in grams (% ID/g) representing organ-specific enrichment. Mean + SD is given. (B) Results from individual mice are shown for mesenteric LN, spleen, and liver. The horizontal line represents the group median. P values are provided above the figure. Data were pooled from 3 experiments (CD4+CD25+CD62L+ mice, n = 7; CD4+CD25+CD62L- mice, n = 8).

  • Figure 5.

    CD4+CD25+CD62L- T cells are quantitatively less efficient suppressors in vivo. Lethally irradiated BALB/c mice received TCD BM and CD4+CD25- T cells from congenic C57BL/5 without (control hosts) or together with (experimental hosts) CD4+CD25+CD62L+ or CD4+CD25+CD62L- Treg cells from wild-type (WT) C57BL/6 donors. All mice were killed 5 days after transfer. Single cell suspensions were prepared from mesenteric LN (MLN), spleen, and liver of individual mice. Viable cells were counted, stained with appropriate antibodies, and analyzed by flow cytometry. The progeny of CD4+CD25- and CD4+CD25+ T cells was identified as CD4+H-2Kb+[congenic marker]+/-. Results for individual mice are shown. The horizontal line represents the group median. P values are given above the figures. Data were pooled from 4 experiments with 9 to 11 mice per group. (A) Recovery of CD4+CD25+ T cells after transfer of the CD62L+ or CD62L- subset. (B) Expansion of CD4+CD25- T cells in mesenteric LN, spleen, or liver after cotransfer of CD4+CD25+CD62L+ or CD4+CD25+CD62L- Treg cells. (C) Fraction of CD4+CD25+ T-cell progeny among all donor CD4+ T cells in individual mice.

  • Figure 6.

    Minimal histologic damage in the large intestine 5 days after cotransfer of CD4+CD25+CD62L+ Treg cells. Lethally irradiated BALB/c hosts received TCD BM or TCD BM plus 500 000 CD4+CD25- T cells alone or together with 500 000 CD4+CD25+CD62L+ or CD4+CD25+CD62L- T cells. Animals (n = 3 each group) were killed 5 days later, and a piece of large bowel was processed for standard H/E histology. Representative sections are shown at 1:40 magnification for recipients of (A) TCD BM alone, (B) CD4+CD25- T cells, (C) CD4+CD25- plus CD4+CD25+CD62L+ T cells, and (D) CD4+CD25- plus CD4+CD25+CD62L- T cells.