Figure 1.
Adoptive immune therapy in mice requires both T-bet and Eomes.
B6-LY5.2/Cr mice were challenged with 3×105 B16F0 cells i.d. 6 days later, mice were irradiated at 500 rad. On day 7, the mice were adoptively transferred with 5×105 WT, T-bet−/−, Eomes−/−, or T-bet/Eomes DKO pmel-1 T cells which have been cultured in Th1 condition for 3 days. Tumor growth was monitored every two days. 5 mice were in each group. The no-transfer group did not receive any T cells. *P<0.05, determined by Mann-Whitney Test comparing recipients infused with DKO and WT T cells.
Figure 2.
T-bet and Eomes regulate tissue distribution and survival of the adoptive transferred T cells in tumor-bearing recipient mice.
B6-LY5.2/Cr mice were challenged with 3×105 B16F0 cells i.d. 6 days later, mice were irradiated at 500 rad. On day 7, the mice were adoptively transferred with 5×105 WT, T-bet−/−, Eomes−/−, or T-bet/Eomes DKO pmel-1 T cells which have been cultured in Th1 condition for 3 days. (A) Splenocytes (SPL), lymph node (LN) and tumor infiltrating lymphocytes (TILs) were harvested from tumor-bearing mice 20 days after T cell infusion. The percentage of donor cells (CD45.2+) cells was determined by flow cytometry and the representative dot plots were shown. (B) The average percentage of donor cells (CD45.2+) in spleen was shown (N = 4 for each genotype). (C) The average percentage of donor cells (CD45.2+) in lymph nodes was shown (N = 4 for each genotype). (D) The average percentage of donor cells (CD45.2+) in tumor was shown (N = 4 for each genotype). Data are presented as mean ± SEM of four mice for each genotype. *P<0.05, **P<0.01 by Student’s t-test.
Figure 3.
T-bet and Eomes regulate the surface marker and function and adoptive transferred T cells in tumor-bearing mice.
Pmel-1 T cells were adoptively transferred to tumor-bearing B6-LY5.2/Cr mice as in figure 1. 20 days post T cell infusion, spleen cells were harvested and analyzed. (A) Dot plots show percentage of CD44hiCD62Lhi, CD44hiCD62Llo and CD44loCD62Lhi T cells in within donor (CD45.2+) and recipient (CD45.2−) CD8+ T cells. (B) The average percentage of donor cells (CD45.2+) CD44+ CD8+ T cells in the spleen was shown (N = 4 for each genotype). (C) Sca-1 levels in the adoptive transferred CD8 T cells were shown. (D). Splenocytes were harvested from different recipient tumor-bearing mice, were subsequently stimulated with PMA and ionomycin for 4 hours and incubated for the last 1 hour with brefeldin A. The percentage of donor pmel-1 CD8+ T cells producing IFN-γ and IL-17 were examined. (E) The average percentage of IFN-γ+ donor cells CD8+ T cells in the spleen as shown in (D) are shown (N = 4 for each genotype). Data are presented as mean ± SEM of four mice for each genotype. *P<0.05, ***P<0.001 by Student’s t-test.
Figure 4.
T-bet and Eomes regulate T cell numbers in blood upon adoptive transfer into non-lethally irradiated mice.
B6-LY5.2/Cr mice were irradiated at 500 rad. 24 h post irradiation, the mice were adoptively transferred with 5×105 WT, T-bet−/−, Eomes−/−, or T-bet/Eomes DKO pmel-1 T cells which have been cultured in the Th1 condition for 4 days. (A) The recipient mice were bled at different time points and the frequency of the adoptively transferred T cells (CD45.2) among total CD45+ cells (CD45.2 plus CD45.1) was determined by flow cytometry. (B) The frequency of the adoptively transferred T cells in spleen and LN were analyzed 30 days post transfer. Data are presented as mean ± SEM of five mice for each genotype. ***P<0.001 by Student’s t-test.
Figure 5.
T-bet and Eomes regulate memory subsets of the adoptive transferred T cells.
(A) WT, T-bet−/− (TKO), Eomes−/− (EKO), or T-bet/Eomes DKO pmel-1 T cells were cultured in Th1 conditions for 4 days. Expression of CD44 and CD62L on pmel-1 T cells was examined by flow cytometry. (B) B6-LY5.2/Cr mice (CD45.1) were adoptively transferred with WT, T-bet−/−, Eomes−/−, or T-bet/Eomes DKO pmel-1 T cells (CD45.2). Blood was collected from different recipient mice and stained for CD45.2, CD8, CD44, CD122 30 days after transfer. (C) Thirty days post transfer, spleens and lymph nodes (LN) were harvested. Surface markers CD44 and CD62L were examined by flow cytometry. Host cells were CD8+CD45.1+. WT, TKO, EKO, and DKO indicate representative plots of CD44 versus CD62L of CD8+CD4.5.2+ donor CD8 T cells. (D) Thirty days post transfer, spleens were harvested. The average percentage of donor cells (CD45.2+) CD44+ CD8+ T cells among all the donor CD8+ T cells in the spleen was shown (N = 5 for each genotype). (E) Thirty days post transfer, spleens were harvested. Sca-1 expression on donor CD45.2+ CD8+ T cells was shown (representative of five independent experiments). (F) and (G) Splenocytes were then stimulated with PMA and ionomycin for 4 hours and incubated for the last 1 hour with Brefeldin A. CD45.2+ Cells producing IFN-γ (F) and IL-2 (G) were examined with intracellular cytokine staining and flow cytometry. The average percentages of IFN-γ+ and IL-2+ cells among the donor CD45.2+ CD8+ T cells in the spleen were shown (N = 5 for each genotype). Data are presented as mean ± SEM of five samples for each genotype. ***P<0.001 by Student’s t-test.
Figure 6.
T-bet and Eomes are required for the memory recall responses against tumor cell rechallenge.
(A) B6-LY5.2/Cr mice were non-lethally irradiated at 500 rad, within 24 hours of irradiation, mice were adoptively transferred with 4×106 WT, T-bet−/−, Eomes−/−, or T-bet/Eomes DKO pmel-1 T cells which have been cultured in Th1 condition for 4 days. One month later, mice were challenged with 2×105 B16F0 cells i.d. (B) Tumor growth was monitored every other day. 5 mice were in each group. ***P<0.001, determined by Mann-Whitney Test comparing recipients with DKO and WT T cells. (C) Thirty days post T cell infusion, spleens were harvested. Splenocytes were stained with CFSE and then stimulated with 0.01 µM gp10025–33 peptide for 72 h hours. Then the donor cells (CD45.2+CD8+) were analyzed by flow cytometry for CFSE dilution. Host CD8 T cells were the CD45.1+ CD8+ population. Data are representative of three independent experiments.
Figure 7.
T-bet and Eomes determine memory T cell subsets.
Upon activation, naïve T cells are destined to different cellular fates. Current data show that T-bet and Eomes are crucial for the generation of various effector, memory and exhausted T cell subsets. Higher levels of T-bet drive terminally differentiated effector T cells which eventually either become exhausted by further up-regulating Eomes or undergo apoptosis. T cell expressing lower levels of T-bet become memory T cells which also express Eomes. Activated naïve T cells can also take a fate of becoming TSCM, which maintain their “naivety” and developmental potential. Our data suggest that T-bet and Eomes regulate the balance between TEM, TCM, and TSCM.