Fig 1: TCRTg101 expand and acquire a dysfunctional phenotype in leukemia-bearing animals(A and B) Survival of C57BL/6 mice challenged i.v. with C1498 cells (106) and transferred or not with in vitro activated (A) or naive (B) TCRTg101 (4 × 106) 3 days later. Data are pooled from 2 independent experiments with 3–4 mice/group.(C–G) 2 × 106 CTV-labeled TCRTg101 (CD45.2) were transferred into B6.SJL mice (CD45.1) followed by an i.v. challenge with 106 C1498 leukemia cells 1 day later.(C) Representative FACS plots showing CTV dilution of adoptively transferred TCRTg101 in spleens and livers of leukemia-bearing mice at the indicated time points.(D–G) Quantitative data showing the percentage of divided TCRTg101 (D and F) and TCRTg101 number (E and G) in spleens (D and E) or livers (F and G) of leukemia-bearing mice over time. Data are pooled from 2 to 3 independent experiments with 2 to 4 mice/group including data from leukemia-free control mice.(H) Representative FACS plots showing expression of PD-1, LAG-3, TIM3, and TIGIT on TCRTg101 in livers of tumor-free or leukemia-bearing mice.(I) Quantitative data from (H) as mean ± SD.(J–Q) Mononuclear cells isolated from livers of mice 13 to 14 days (J–M) or 17 to 18 days (N–Q) after C1498 cell inoculation were restimulated with PMA + ionomycin (P+I) (J–M) or anti-CD3 and anti-CD28 antibodies (N–Q) for 4 h. Cytokine production was analyzed by flow cytometry. Gating was performed on TCRß+CD8+CD45.1 cells (endogenous CD8+ T cells) or TCRß+CD8+CD45.2 cells (TCRTg101).(J and N) Representative FACS plots showing TNFa and IFN? production by endogenous CD8+ T cells or TCRTg101.Quantitative data are shown in (K)–(M) and (O)–(Q) as mean ± SD.(R) TCRTg101 killing assay. CTVhi and CTVlo TCRTg101 (CD45.1.2) were FACS-purified from livers of mice 17 to 18 days after C1498 inoculation and were co-cultured for 20 h with equal numbers of C1498 cells and EL4 cells (both CD45.2) labeled with different CTV concentrations. Naive TCRTg101 purified from control Tg101 mice, or those activated in vitro with anti-CD3 and anti-CD28 antibodies or with C1498.B7.1 cells served as negative and positive controls for C1498 cell lysis, respectively. Specific lysis was analyzed by flow cytometry. Gating was performed on live CD8-CD45.2 cells.Data are representative (K–M) of or pooled (I and O–R) from 2 independent experiments with 2 to 4 mice/group and shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant; NT, no tumor.