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. 2021 Dec 17;11(12):e625. doi: 10.1002/ctm2.625

FIGURE 3.

FIGURE 3

Noncanonical Itk –/− Tregs suppress GVHD but maintain GVL effects. (A) Group 1 BALB/c recipient mice were lethally irradiated and transplanted with 10 × 106 TCDBM, alone. Group 2 BALB/c mice were transplanted with 10 × 106 TCDBM and 1 × 105 primary tumour cells (B‐ALL‐luc +). Group 3 BALB/c mice were transplanted with 10 × 106 TCDBM +1 × 106 WT CD8+ T cells and 1 × 105 primary tumour cells (B‐ALL‐luc +). Group 4 BALB/c mice were transplanted with 10 × 106 TCDBM, 1 × 106 WT CD8+T cells, and 1 × 105 primary tumour cells (B‐ALL‐luc +), and were treated with 0.5 × 106 canonical Tregs from WT C57Bl/6 mice. Group 5 BALB/c mice were transplanted with 10 × 106 TCDBM,1 × 106 WT CD8+T cells, and1 × 105 primary tumour cells (B‐ALL‐luc +), and were treated with 0.5 × 106 canonical Tregs from Itk–/– mice. Group 6 BALB/c mice were transplanted with 10 × 106 TCDBM, 1 × 106 WT CD8+T cells, and 1 × 105 primary tumour cells (B‐ALL‐luc +), and were treated with 0.5 × 106 noncanonical Tregs from Itk–/– mice. Tregs were sorted from either WT mice or Itk –/− mice using CD4, CD25, and FOXP3RFP. Recipient BALB/c mice were imaged using IVIS 200 three times a week. Recipient BALB/c mice were also monitored for (B) changes in body weight, and (C) clinical score, and (D) survival for more than 40 days post BMT. For body weight changes and clinical score, one representative of two independent experiments is shown (n = 3 mice/group for BM alone; n = 5 experimental mice/group for all five other groups). (E) Quantitated luciferase bioluminescence of tumour growth. (F) Mortality from GVHD and tumour during the experiment, as a percent of mice dead. Statistical analysis for survival and the clinical score was performed using the log‐rank test and one‐way ANOVA with Tukey's test, respectively, and analysis for weight changes was done using one‐way ANOVA with Tukey's test. One representative experiment out of 2 is shown for A, C‐E. B and F are a combination of two experiments, three‐ to five mice per group. Symbol meaning for p values are: ns, p > .05; * p ≤ .05; ** p ≤ .01; *** p ≤ .001; **** p ≤ .0001. Note: Control mouse is a recipient mouse given TCDBM only (group 1), used as a negative control for BLI (no bioluminescent tumour cells were given)