Fully allogeneic BALB/c (H-2d) or syngeneic C57BL/6 (H-2b) full-thickness body skin was grafted on the lateral thorax of FoxP3 GFP-KI reporter C57BL/6 (H-2b) mice. C57BL/6-KI mice reject the BALB/c skin graft on day 7. The number of FoxP3-GFP⁺TIM-3⁺ cells in (A) dLNs and (B) spleens from skin transplant recipients was determined by flow cytometry (n ≥ 8 mice). (A and B) The TIM-3⁺FoxP3⁺ T cells within dLNs and spleens peaked at the time of rejection and were more numerous in allogeneic as compared with syngeneic grafted hosts. Data are presented as mean ± SEM; *P < 0.05; **P < 0.01. (C) Gated CD4⁺GFP⁺FoxP3⁺ cells harvested from collagenase digested allogeneic skin transplants were analyzed for TIM-3 expression by flow cytometry. CD4⁺GFP⁺FoxP3⁺TIM-3⁺ Tregs comprised 40% of the graft-infiltrating Tregs on day 5 after transplantation. (D) The magnitude of TIM-3 expression on the TIM-3⁺ Tregs is depicted in comparison to the staining control. Gray shading represents fluorescence minus 1 for TIM-3 staining, and the black line represents TIM-3 expression on CD4⁺GFP⁺FoxP3⁺ cells in the graft (n = 4).

Five days after transfer of DBA/2 or BALB/c spleen cells into BALB/c-KI recipients, spleens were harvested and analyzed by flow cytometry in a tolerance-promoting DST model. Normal BALB/c-KI mice were used as controls. (A) CD4⁺GFP⁺FoxP3⁺ Tregs harvested from DST-treated or normal BALB/c-KI mice were stained for TIM-3 expression and (B) were further analyzed for donor-reactive Mlsa-specific Vβ6⁺ or donor unresponsive Vβ8⁺ CD4⁺ T cells. Numbers represent the percentage of CD4⁺GFP⁺FoxP3⁺ Tregs that are TIM-3⁺. Data are representative or mean ± SEM (n ≥ 8 animals); ***P < 0.001.

CD4⁺GFP⁺FoxP3⁺ cells (total Tregs) sorted from spleens of unmanipulated mice were cultured in vitro in the presence of anti-CD3 (plate coated) and soluble anti-CD28 with or without IL-2. (A) Cells were analyzed for TIM-3 expression by flow cytometry on day 4. (B) TIM-3 expression is not induced in TIM-3^–GFP⁺FoxP3⁺ cells (TIM-3^– Tregs) obtained from spleens of unmanipulated mice when cultured in vitro in the absence of anti-CD3, as indicated by flow cytometric analysis on day 4 (n = 3). Sorted GFP⁺(FoxP3⁺)TIM-3^– cells (TIM-3^– Tregs) from spleens of DBA/2 DST–infused BALB/c-KI mice were injected i.v. into (C) DBA/2 skin graft recipient BALB/c-Rag2^–/– mice or (D) ungrafted BALB/c-Rag2^–/– mice. Three weeks after transfer, CD4⁺GFP⁺(FoxP3⁺) spleen cells were analyzed for TIM-3 expression (n = 6). Numbers represent the percentage of CD4⁺GFP⁺FoxP3⁺ Tregs that are TIM-3⁺.

(A) Cells were analyzed from C57BL/6-KI recipients grafted with full-thickness body skin of BALB/c mice on day 7 after transplantation. Proliferation of TIM-3⁺ Tregs was compared with that of TIM-3^– Tregs in dLNs on day 7, as deduced via staining for BrdU (injected i.p. 24 and 12 hours prior to harvesting of cells). Representative plots of n = 4 animals are depicted. (B) The proportion of PD-1–expressing TIM-3⁺ Tregs in comparison with that of TIM-3^– Tregs and TIM-3⁺ Teffs in dLNs. In the histogram, 1 represents fluorescence minus one for PD-1 staining; 2 represents FoxP3⁺TIM-3^– cells; 3 represents FoxP3⁺TIM-3⁺ cells; and 4 represents FoxP3^–TIM-3⁺ cells. Data are representative of n = 5 animals. (C) TIM-3⁺ Tregs were analyzed in comparison with TIM-3^– Tregs and TIM-3⁺ Teffs from dLNs for their ability to stain cell surface phosphatidyl serine with annexin V and exclude the LIVE/DEAD blue viability dye. Representative plots of n ≥ 8 animals are shown. (D) Susceptibility of TIM-3⁺ Tregs to galectin-9–induced cell death was tested on GFP⁺FoxP3⁺ cells obtained by sorting from spleens of mice grafted with allogeneic skin on day 7. Cells were cultured with 0.5 μM galectin-9/PBS for 4 hours ex vivo. TIM-3⁺ Tregs were assessed for staining with annexin V and LDB by flow cytometric analysis. Numbers represent the percentage of TIM-3⁺ or TIM-3^– Tregs or TIM-3⁺FoxP3^– cells as indicated in each panel. Data are representative of n = 3 experiments.

(A) In an MLR culture, naive CD4⁺GFP^– Teffs from spleens of BALB/c-KI mice were stimulated with irradiated CD90^– splenocytes from BALB/c-KI mice for 3 days in the presence or absence of Tregs. Sorted CD4⁺GFP⁺TIM-3⁺/TIM-3^– Tregs were obtained from spleens of BALB/c-KI mice injected with DBA/2 DST 5 days prior to harvesting. T cell proliferation in these cultures was analyzed by the mean values of incorporated thymidine of duplicate/triplicate wells and compared with that of MLR cultures with Teff alone (normalized as 100%). Data are represented as mean ± SEM; n = 3 independent experiments done in duplicate, triplicates, or quadruplicates leading to a total of ≥ 8 independent values for each condition. **P = 0.002; ***P < 0.001. (B) TIM-3⁺GFP⁺FoxP3⁺ and TIM-3^–GFP⁺FoxP3⁺ cells were sorted from harvested dLNs of 30 to 35 C57BL/6-KI mice grafted with BALB/c skin on day 7. RNA was isolated from sorted TIM-3⁺GFP⁺FoxP3⁺ and TIM-3^–GFP⁺FoxP3⁺ populations. Gene expression of various markers was assessed by quantitative real-time PCR. Data are representative of 3 independent experiments. (C) CD4⁺TIM-3⁺ Tregs and CD4⁺TIM-3^– Tregs harvested from the dLNs on day 7 were stained and analyzed by flow cytometry for various Treg functional markers as shown. Arrows in the histograms depict TIM-3^– Tregs (a) and TIM-3⁺ Tregs (b). Numbers represent the percentage of TIM-3⁺ and TIM-3^– Tregs, respectively. n ≥ 5 mice.

BALB/c-KI-TIM-3⁺/TIM-3^– Tregs were sorted from spleens of DBA/2 DST-injected BALB/c mice. Teffs with or without TIM-3⁺/TIM-3^– Tregs (Teff/Treg, 2:1) were adoptively transferred into DBA/2-grafted BALB/c-Rag2^–/– mice, and graft survival was recorded. The survival of grafted skin was prolonged in both Treg- and Teff-injected groups, albeit graft survival was more prolonged by TIM-3^– Tregs in comparison to TIM-3⁺ Tregs. n = 2 independent experiments, total of ≥ 6 mice per group.