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. 2021 Mar 11;24(4):102286. doi: 10.1016/j.isci.2021.102286

Figure 3.

Figure 3

Eomes is required for cytotoxicity and GVL effect by both WT and SLP76Y145FKI T cells

(A and B) Purified WT and SLP76Y145FKI CD8+ and CD4+ T cells were examined for expression of CD44, CD122, and Eomes by flow cytometry.

(C) Purified donor CD8+ T cells from either WT or SLP76 Y145FKI Eomes-sufficient, Eomes-deficient, or Eomes-flox control mice were transplanted into irradiated BALB/c (MHC haplotype d) mice. On day seven, donor T cells were purified as described and used in an ex vivo cytotoxicity assay against B-ALL-luc cells at 40:1, 20:1, and 10:1 ratios.

(D) 1X106 purified WT or SLP76Y145FKI. Eomes-sufficient, Eomes-deficient, or Eomes-flox control CD8+ T cells and 1X106 purified CD4+ T cells were mixed and transplanted along with 2X105 B-ALL-luc cells and 10 × 106 T cell-depleted bone marrow TCDBM cells into irradiated BALB/c mice. Host BALB/c mice were imaged using IVIS 3 times a week. Group one received 10 × 106 T TCDBM alone. Group two received 10X106TCDBM along with 2X105 B-ALL-luc cells (TCDBM + B-ALLluc). Group three was transplanted with 10X106TCDBM and 1X106 purified WT CD8+ T cells +1X106 CD4+ T cells, and 2X105 B-ALL-luc cells (TCDBM + B-ALLluc+ WT CD8+CD4). Group four received 10X106TCDBM and 1X106 purified CD8+ T cells +1X106 CD4+ T cells from SLP76 Y145FKI Eomes-sufficient mice along with 2X105 B-ALL-luc cells (TCDBM + B ALLluc+ SLP75Y145FKI CD8+CD4). Group five received 10X106TCDBM and 1X106 CD8+ T cells +1X106 CD4+ purified T cells from SLP76 Y145FKI Eomes-deficient mice along with 2X105 B-ALL-luc cells (TCDBM + B-ALLluc + SLP75Y145FKI Eomes-cKO CD8+CD4). Group six received 10X106TCDBM and 1X106 CD8+ T cells +1X106 CD4+ purified T cells from WT Eomes-deficient mice along with 2X105 B-ALL-luc cells (TCDBM + B-ALLluc + WT Eomes cKO CD8+CD4).

(E–G) (E) The mice were monitored for survival, (F) body weight changes, and (G) clinical score for 50 days post-BMT. For weight changes and clinical score, one representative of 2 independent experiments is shown (n = 3 mice/group for BM alone; n = 5 experimental mice/group for all three groups). The survival groups are a combination of all experiments.

(H) We have quantitated luciferase bioluminescence of tumor growth. Statistical analysis for survival and the clinical score was performed using log calculation. Two-way ANOVA was used for statistical analysis and results were confirmed by students t-test, p values are presented. Note: Controls are naive for tumor but transplanted with 10 × 106T cell depleted bone marrow alone (TCDBM) and used as a negative control for BLI. See also Figure S4.