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1.
Figure 5

Figure 5. Rapamycin/TGFβ induced Treg are suppressive in vivo. From: Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease.

In vitro expanded CD4+25- or CD4+25-45RA+ Rapa/TGFβ iTreg (30×106 cells) were co-transferred with allogeneic PBMCs (30×106 cells) into NOD/Scid/γc−/− mice to assess the ability to ameliorate xenogeneic GVHD. A) Characteristics of the Rapa/TGFβ iTreg cultures that were used. B) Kaplan-Meier survival curves for mice receiving PBMCs only or PBMCs plus adoptive transfer of Rapa/TGFβ Treg induced from CD4+25- or CD4+25-45RA+ T-cells.

K.L. Hippen, et al. Am J Transplant. ;11(6):1148-1157.
2.
Figure 6

Figure 6. Rapa(d7)/TGFβ induced Treg are as potent as nTreg at suppressing GVHD in vivo. From: Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease.

In vitro expanded nTreg and Rapa(d7)/TGFβ iTreg (30×106 cells) were co-transferred with allogeneic PBMCs (30×106 cells) into Rag−/−,γc−/− mice to assess the relative potency for preventing xenogeneic GVHD. A) Characteristics of the nTreg and iTreg cultures that were used. B) Kaplan-Meier survival curves for mice receiving PBMC only or PBMC plus adoptive transfer of nTreg of iTreg. C) Animals were bled on day 18, and the degree of human T-cell expansion (CD4+ or CD8+) was determined using counting beads. n=10, 8, and 7 mice for PBMC, nTreg and iTreg, respectively.

K.L. Hippen, et al. Am J Transplant. ;11(6):1148-1157.
3.
Figure 2

Figure 2. Naïve (CD4+25-45RA+) cultures treated with Rapa plus TGFβ do not express LAP, but express high levels of CD25. From: Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease.

In vitro expanded nTreg and naïve T-cells treated with TGFβ and/or Rapa were stained with antibodies to LAP and CD25 to determine potential mechanisms used for suppression. A) Representative example of LAP expression in the various cultures. Representative example (B) and summary (C) of relative CD25 expression (naïve T-cells expanded without TGFβ or Rapa was set at 100). Staining for CD25 was performed on day 14 whereas staining for LAP, which is only transiently expressed, was performed on day 7. Data are representative of at least five independent experiments.

K.L. Hippen, et al. Am J Transplant. ;11(6):1148-1157.
4.
Figure 3

Figure 3. CD4+25-45RA+ cells expanded in the presence of Rapa and TGFβ were hypoproliferative and contain very few IL-2, IFNγ or IL-17 secreting cells. From: Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease.

nTreg and naïve T-cells expanded ±TGFβ and ±Rapa for 14 days were re-stimulated for 4 hours with PMA and Ionomycin in the presence of Brefeldin followed by intracellular staining for Foxp3 and either IFNγ (A, B), IL-2 (C, D), IL-17 (E, F) or IL-4 (G, H) to quantitate the percentage of effector cells present and determine their phenotype (i.e. Th1 or Th2). Data presented are either the % total cytokine+ cells (A, C, E, G) or % of Foxp3+ cells that are cytokine+ (B, D, F, H). Data represent the average ± SEM for 3–5 independent experiments.

K.L. Hippen, et al. Am J Transplant. ;11(6):1148-1157.
5.
Figure 4

Figure 4. Rapa/TGFβ induces equivalent Foxp3 expression and suppressive function in unfractionated (CD4+25-) and naive (CD4+25-45RA+) T-cells. From: Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease.

Total CD4+25- and naïve (CD4+25-45RA+) T-cells were purified from PB using magnetic beads and expanded in vitro ±TGFβ and ±Rapa for 14 days. A) Fold expansion of total cells for cultures started with CD4+25- (gray bar) or naïve (black bar) T-cells expanded ±TGFβ and ±Rapa on day 0 or 7. B) Total calculated cell yield for CD4+25- and CD4+25-45RA+ Rapa(d0)/TGFβ iTreg cultures after 14 days, based on 4 independent experiments starting with an average apheresis unit of 10×109 cells. C) Total % of Foxp3 expressing cells in the various cultures. D) Average % of CD4+ cells that are Foxp3+ and Foxp3++ in unfractionated or naïve T-cell cultures expanded ±TGFβ and ±Rapamycin. E) Suppressive function of cultures assessed by CFSE-based proliferation assay at a 1:8 ratio of expanded cells per CFSE-loaded PBMC. n=3 independent experiments.

K.L. Hippen, et al. Am J Transplant. ;11(6):1148-1157.
6.
Figure 1

Figure 1. Rapa enhances TGFβ induced Foxp3 expression and confers in vitro suppressive function. From: Generation and large-scale expansion of human inducible regulatory T cells that suppress graft-versus-host disease.

nTreg (CD4+25++) and naïve T-cells (CD4+25-45RA-) were purified from peripheral blood using magnetic beads. A) Purified cells were stimulated on day 0 with a cell line expressing CD86 and CD64 (KT64/86) to which anti-CD3 mAb is bound. High dose IL-2 (300IU/ml) was added on day 0 for naïve T-cells and day 2 for nTreg. TGFβ (10ng/ml) was added to naïve T-cells on day 0, while Rapa (109nM) was added on either day 0 or day 7. Naïve T-cells were also re-stimulated on day 7. Cultures were phenotyped by flow cytometry on day 7 and 14, while suppressive function and cytokine secretion was tested on day 14. B) Fold expansion of total cell number over the 14 day assay. C) Representative example showing differential Foxp3 expression in nTreg and naïve T-cells cultured ±TGFβ and ±Rapa. D) Total % of Foxp3 expressing cells in the various cultures. E) Average % of CD4+ cells that are Foxp3+ and Foxp3++ in nTreg and naïve T-cells cultures expanded ±TGFβ and ±Rapamycin. F) Suppressive function of cultured cells was determined using a CFSE-based proliferation assay in which expanded nTreg or naïve T-cells were incubated with CFSE-loaded allogeneic PBMC at doses from 1:2 to 1:32 (expanded cell:PBMC) and stimulated with anti-CD3 beads for 4 days. Data shown are from the 1:8 ratio. n=3–5 experiments.

K.L. Hippen, et al. Am J Transplant. ;11(6):1148-1157.

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