Figure 4. The exhausted TCR_Tg101 phenotype is profound.

(A) Experimental design: 8 × 10⁶ CTV-labeled TCR_Tg101 (CD45.1.2) were transferred into B6.SJL (CD45.1) mice followed by i.v. challenge with C1498 leukemia cells 1 day later. Naive (CTV^hi) or exhausted (CTV^lo) TCR_Tg101 were isolated from livers of day-17 leukemia-bearing mice by FACS and were separately transferred into secondary B6.SJL hosts. These B6.SJL mice received a s.c. C1498 cell (10⁶) challenge the following day. On day 14, numbers and CD44 expression of TCR_Tg101 in tdLNs were analyzed by flow cytometry.

(B) Representative FACS plots showing CTV dilution and CD44 expression of TCR_Tg101 in tdLNs of secondary B6.SJL mice with s.c. C1498 tumors.

(C) Quantified data showing numbers of TCR_Tg101 in tdLNs of secondary B6.SJL mice with s.c. C1498 tumors.

(D) Expression of PD-L1 and I-A^b on C1498 cells when analyzed directly ex vivo. Shaded histograms represent staining with appropriate isotype control antibodies.

(E) TCR_Tg101 (8 × 10⁶) were adoptively transferred into C57BL/6 mice. 1 day later, mice were challenged i.v. with C1498 cells (10⁶) and were treated with anti-PD-1 and anti-LAG-3 or isotype control antibodies. Survival was assessed.

(F–I) Cytokine production by TCR_Tg101 in leukemia-bearing mice treated with 4 doses of isotype control or anti-PD-1/anti-LAG-3 antibody therapy. Mononuclear cells isolated from livers on day 16 were restimulated ex vivo with anti-CD3 and anti-CD28 antibodies and cytokine production was assessed. Gating was performed on TCRβ⁺CD8⁺CD45.1.2 cells (TCR_Tg101).

(F) Representative FACS plots showing TNFα and IFNγ production by TCR_Tg101.

Quantitative data are shown in (G)–(I) as mean ± SD. Data are pooled from 2 independent experiments with 2 to 3 mice/group. *p < 0.05; ***p < 0.001; n.s., not significant.