Phenotyping for effector/memory markers as well as adhesion molecules unravels a high degree of heterogeneity among Treg subsets. FACS^® analysis of pooled spleen and lymph node cells shows the expression of several effector/memory markers and adhesion molecules on indicated CD4⁺ T cell subsets. Representative histogram plots from three independent experiments were selected showing stainings (gray) plus isotype controls (white). The graphs summarize data from one representative experiment and show frequency or mean fluorescence intensity of the indicated subsets as mean ± SD from three independently analyzed animals.

α_E-expressing subsets have a higher migratory capacity toward inflammatory chemokines. The chemotactic response of pooled spleen and lymph node T cells to 30 nM CCL19, 100 nM CXCL9, 100 nM CCL17, and 10 nM CCL20 was analyzed in an in vitro chemotaxis assay. The number of migrating cells of each subset was measured by flow cytometry. Results are expressed as the percentage of the indicated subset that migrated to the lower chamber and where normalized to the mean migration rate of all subsets. Shown is the mean ± SD of three (CCL17, CCL20) or four (CCL19, CXCL9) experiments (*, P < 0.05; **, P < 0.01). Basal migration toward medium alone (dotted lines) showed no significant differences between the subsets.

α_E-expressing subsets show the strongest suppression of inflammatory reactions in antigen-induced arthritis. (A and B) mBSA-immunized C57Bl6 mice received 10⁵ FACS^®-sorted and polyclonally preactivated cells 24 h before intraarticular mBSA injection (α_E⁺CD25⁺, n = 4; α_E⁺CD25⁻, n = 4; α_E⁻CD25⁺, n = 11; α_E⁻CD25⁻, n = 11; and PBS alone, n = 10). (A) The progression of arthritis was monitored by the measurement of knee joint swelling (mean ± SD; one representative out of two independent experiments). α_E⁺CD25⁺ and α_E⁺CD25⁻ cells showed a higher suppressive capacity than CD25 single positive cells (P < 0.01 and P = 0.085, respectively). (B) At day 14, tissues were taken for histological examination. The final arthritis score was evaluated for each animal (mean ± SEM; one representative out of two independent experiments). α_E⁺CD25⁺ cells showed a significantly lower arthritis score than CD25 single positive cells (*, P < 0.05). (C) Radioactively labeled T cell subsets were injected i.v. in mBSA-immunized mice that had received an intraarticular mBSA injection 4 d before the homing experiment. Recovered radioactivity from the inflamed knee was determined after 24 h using a γ-counter. Percentage of total recovered radioactivity is shown (n = 6; mean ± SD; one representative out of two independent experiments). α_E⁺CD25⁺ and α_E⁺CD25⁻ cells showed a significantly higher migration into the inflamed knee joint compared with CD25 single positive cells (*, P < 0.05).

Foxp3 expression in Treg subsets. Foxp3 mRNA expression was determined in sorted Treg subsets by real-time quantitative RT-PCR. Obtained values were normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase and expressed as percentage of the level found in CD25 single positive cells (mean ± SD of three independent experiments).

α_E-expressing Treg subsets have undergone excessive proliferation. Sorted Treg subsets from pooled spleen and lymph node cells were analyzed for their TREC content. Shown is the TREC number per 1,000 cells of the indicated subsets as mean ± SD of three parallel samples from one representative out of six independent measurements. Significance (*, P < 0.05) was computed from the six means. The average TREC number per 1,000 cells in CD4⁺CD8⁻ thymocytes (“high control”) and cultured Th1 cells (“low control”) was 80 and 1, respectively (not depicted).

CD25 single positive cells display a stronger migration into lymph nodes, but α_E single positive cells can efficiently enter inflamed sites. (A) Radioactively labeled T cell subsets were injected i.v. into naive Balb/c mice followed by the determination of radioactivity in the indicated organs after 24 h using a γ-counter. Percentage of total recovered radioactivity is shown (n = 4; mean ± SD; one representative out of two independent experiments). (B) Radioactively labeled T cell subsets were injected i.v. in Balb/c mice that had been sensitized and challenged with DNFB. Recovered radioactivity from control and inflamed ear pinna was determined after 24 h (n = 6; mean ± SD; *, P < 0.05; **, P < 0.01).

CD25 single positive cells represent natural Tregs, whereas α_E-expressing subsets are prototypes of adaptive Tregs.