Fig 2: (A) Representative Ca2+ flux as assessed by fluorometric evaluation after aCD3 stimulation of indicated transduced CD8+ T cells. Kinetic of the ratio indo-blue to indo-violet over time shown and assessed by flow cytometry. (B) Calcium responses were integrated and quantified relative to Cish WT+ empty vector. Error bars represent SD from the mean (n = 3). (C) To evaluate cells positive for intracellular expression of TNF-a or IFN-?, indicated transduced CD8+ T cells were re-stimulated with anti-CD3 coated antibody (at indicated concentration) and soluble CD28 (2 µg/ml) in presence of Golgi stop for 6 hrs. Cells were then labeled for surface Thy1.1 and intracellular TNF-a or IFN-?. The flow cytometry analysis of Thy1.1+ T cell populations is shown. (D) Absolute cell numbers of Thy1.1+ T cells positive for intracellular TNF-a or IFN-?. Bars show mean + SEM out of at least 4 independent experiments. Statistical significance was determined by two-tailed Student’s t test. *P = 0.05, **P = 0.01, ***P = 0.001. (NS = Non-stimulated).

Fig 3: Characteristics of the isolated cells. A The morphology of passage 1~3 (P1/P2/P3) cells from rats. B The adipogenic induction and oil red O staining. C The analysis of the cell cycle of the isolated cells by flow cytometry. D The expression of surface markers of the isolated cells; Typical hematologic lineage CD29 and CD90 were expressed in cells, while CD45 and CD34 were not. Scale bars, 50 µm

Fig 4: Enforcing expression of GLUT3 enhances glucose uptake by TEM cells. (A) Schematic of pMSGV retroviral vectors encoding Thy1.1 (T cell engineering control termed MOCK), and GLUT3 comprising a Flag tag. (B) Schematic of OT1 T cell isolation, activation and expansion in high-dose IL-2. (C) Evaluation of transduction efficiency for GLUT3 expression by anti-Flag antibody staining and flow cytometric analysis. (D) Viability of transduced T cells as measured by staining with a viability dye and flow cytometric analysis (days 9-12). (E) Percentage of CD44+ CD62L+ and CD44+ CD62L- T cells in culture. Left: summary of all donors tested. Right: representative flow cytometry dot plot. (F) Left: glucose uptake by T cells evaluated with the fluorescent glucose analog 2-NBDG with or without the addition of the glucose competitor 2-DG. Right: representative histograms showing 2-NBDG uptake. (G) Glucose uptake by T cells measured in a luminescence-based assay. Shown is average ± standard deviation (SD) of different cultures. For data in which fold-change is shown, values of GLUT3 expressing T cells of each culture are normalized with the values of the corresponding MOCK-T cells. Shown is average ± standard deviation (SD) of different cultures. Statistical analysis by paired, two-tailed t test (F: MOCK vs GLUT3 p=0.001, MOCK vs MOCK+2DG p= 0.0029, GLUT3 vs a GLUT3 +2DG p=0.0138). **p< 0.01; *p < 0.05.

Fig 5: Gli1-labeled meniscus cells possess mesenchymal progenitor properties.(A) Representative immunofluorescence images of Sca1, Cd90, Cd200, PDGFRa, Cd248, and Prg4 staining in 3-month-old Gli1ER/Td meniscus. Scale bars, 200 µm. Boxed areas are shown at high magnification in corresponding panels at the bottom. Dashed lines indicate meniscus surface. Yellow cells are double positive for progenitor marker and Td. F: femur; T: tibia; M: meniscus. Blue: DAPI. (B) Quantification of the expression level of mesenchymal progenitor markers in Gli1+ and Gli1- cells from meniscus. Digested meniscus cells from 3-month-old Gli1ER/Td mice were subjected to flow cytometry analysis. n = 3 independent experiments. (C) CFU-F assay of digested meniscus cells. Td+colonies and Td- colonies were counted from 1 × 104 seeded cells. n = 3 independent experiments. (D) Representative bright-field images of the scratch-wound closure in Gli1+ or Gli1- meniscus cells at 0 and 48 hr. Scale bars, 200 µm. Solid lines indicate the remaining area not covered by meniscus cells. (E) The relative migration rate was measured by the percentage of scratched area being covered by migrated cells at 48 hr. n = 3 independent experiments. (F) Representative adipogenic (AD), osteogenic (OB), and chondrogenic (CH) differentiation images of Gli1+ and Gli1- cells. Cells were stained by Oil Red, Alizarin red, and Alcian blue, respectively. (G) qRT-PCR analysis of lineage markers in Gli1- or Gli1+meniscal cells after being cultured in adipogenic, osteogenic and chondrogenic differentiation media for 1, 2, and 3 weeks, respectively. n = 4 independent experiments. Statistical analysis was performed using unpaired two-tailed t-test. Data presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001.Figure 2—source data 1.Raw data for Figure 2B,C,E,G.