(A) Representative T cell subset staining from normal donor. CD4⁺ T cells isolated from human PBMC were stained with anti-CD25 and CD45RO antibodies. Naïve (T_N), memory (T_M), T_Nreg and T_reg cells were identified as shown in brackets. (B) GARP levels of recently activated T cell subsets from the microarray. (C) GARP levels of different T cell subsets 1 day post TCR activation as determined by qPCR. One representative experiment of five donors is shown. (D) Kinetics of GARP levels in different T cell subsets at different time points before and after TCR stimulation. Diamond, T_N; square, T_M; triangle, T_Nreg; cross, T_reg. d, day. One experiment representative of three donors is shown. (E) Expanded T_reg cells from T_Nreg precursors and (F) Foxp3-transduced cells upregulate GARP after TCR stimulation. Cells were in vitro expanded for two weeks before subjecting to qPCR analysis. T_eff, expanded T_N cells; Vector⁺, control vector-transduced T_N cells; Foxp3⁺, Foxp3-transduced T_N cells. One experiment representative of three donors is shown.

(A) Schematic of WT and mutant GARPs. SP, signal peptide; LRR, leucine-rich repeat; TM, transmembrane domain. (B) Both ΔN and ΔSP mutant-transduced cells do not sustain high levels of CD25 and CD62L expression and (C) Foxp3 expression post-activation as determined by staining. Cells were transduced and prepared as described in Fig. 2. M, Geometric mean. (D) Cytokine profiles of WT and mutant GARP-transduced cells. Cytokine production in the supernatant of the activation culture was measured using CBA kit (BD Biosciences) and percent secretion in Y-axis is calculated by first normalizing the secretion of cytokines from control cells to 100%, and calculating the amount of cytokine secreted from other cell conditions relative to this control. This representation allowed us to combine data from multiple donors in the same figure. This normalization Data represent the mean±SEM of experiments from at least three different donors. *, P<0.05; experimental versus control. Range of production (pg/ml): IL-2, 0–1000; IFNγ, 0–10000. (E) Proliferative capacity is shown as percentage of proliferated cells as determined by dilution of CFSE 4 or 5 days post re-stimulation. (F) Percentage of suppression was calculated based on CFSE dilution of target T_N cells in the suppression culture. The % suppression on Y-axis is calculated as follows: We first determined the percent of CFSE labeled target cells dividing in response to the stimuli alone. We then determined the proliferative percentage of cells in the presence of suppressor (T_regs or GARP expressing cells) or control effector T cells. The percent suppression was then calculated by percent reduction in proliferation of the target cells in the presence of effectors as compared to target cells alone. As an example, in one experiment 40% of the CFSE labeled T cells divided in response to SEB stimulation. In the presence of T_regs the same cells divided only 4%. Thus, we represent this as 90% suppression. Data represent the mean±SEM of experiments from at least three different donors. * P<0.05; experimental versus control.

CD4+ T cells were co-transduced with shRNA-expressing or control lentiviruses (GFP+) and Foxp3-expressing lentiviral vectors (RFP+). GFP+ RFP+ T cells expressing both shRNA for GARP and Foxp3, were first sorted to high purity through FACS. The suppressive activity of these cells were then determined using CFSE assay and represented as percent suppression as described in detail in Figure 3. Data are representative experiments from two different donors and shows independent results from two separate shRNAs against GARP. Data represent the mean±SEM.

(A) GARP-transduced cells express higher levels of CD25 and CD62L, and upregulate Foxp3 at a greater level after TCR stimulation than the control vector-transduced cells. Cells were transduced and expanded for two weeks before staining for CD25 and CD62L. RFP⁺ cells were then sorted and re-stimulated by DC plus SEB at suboptimal conditions before staining for Foxp3. Vector⁺, control vector-transduced T_N cells; GARP⁺, GARP-transduced T_N cells; Foxp3⁺, Foxp3-transduced T_N cells. M, Geometric mean. Data are representative experiments from at least three different donors. (B) GARP-transduced cells produce reduced levels of cytokines. Cytokine production in the supernatant of the activation culture was measured using CBA and data are shown by first normalizing the control geometric mean to be 100%. Range of control production (pg/ml): IL-2, 300–1000; IFNγ, 3000–10000. Data represent the mean±SEM of experiments from at least three different donors. *, P<0.05, n≥3; experimental versus control. (C) GARP-transduced cells exhibited impaired proliferative capacity and (D) intermediate suppressive activity. Data are representative experiments from at least three different healthy human donors.

(A) Efficient knockdown of Foxp3 expression in GARP-transduced and T_reg cells. T_N cells were co-infected with shRNA-expressing HDV (GFP) and control or GARP-expressing HDV (RFP), while T_regs from the same donor were transduced with shRNA-expressing HDV. Cells were cultured for two weeks before staining for CD25 and CD62L, and before sorting for experimental lines: GFP⁺RFP⁺ for control vector or GARP-expressing cells; GFP⁺ for T_regs. Foxp3 was stained after re-stimulation by DC and SEB. Vector⁺, control vector-transduced T_N cells; GARP⁺, GARP-transduced T_N cells; T_eff, expanded T_N cells. (B) Suppressive activity and (C) cytokine profiles of Foxp3 shRNA-expressing GARP-transduced and in vitro expanded T_reg cells. Data represent the mean±SEM of experiments from three different donors. Cytokine data are shown by first normalizing the control to be 100%. Range of control production (pg/ml): IL-2, 300–1000; IFNγ, 3000–10000. *, P<0.05, n≥3; experimental versus control. (D) GARP level in in vitro expanded T_reg cells was decreased by silencing Foxp3 as determined by qPCR analysis day 2 or day 4 post TCR stimulation. d, day. Two different shRNA oligos against Foxp3 were tested, both of which have been described before [12].