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

Figure 4.

Figure 4

SLP76Y145/ITK signaling is required for T cell migration to the GVHD target tissues

(A) Irradiated BALB/c mice were allo-transplanted and injected with FACS-sorted CD8+ or CD4+ T cells mixed at a 1:1 ratio from WT B6.SJL (Ly5 CD45.1) and WT C57B16 (CD45.2) mice. We also transplanted FACS-sorted CD8+ or CD4+ T cells from B6.SJL (Ly5 CD45.1) and SLP75Y145FKI (C57B16 in background, CD45.2) mice at a 1:1 ratio. FACS analysis of sorted T cells pre-transplant is shown.

(B and C) On day seven post-transplantation, the spleen, liver, and small intestine (SI) from recipients were examined for donor CD4 and CD8+ T cells by H2Kb+ CD45.1+ (LY5) or CD45.2+(B6). We also examined donor CD4 or CD8+ T cells from WT mice by H2Kb+ CD45.1+, and from SLP75Y145FKI mice by H2Kb+ and CD45.2+. The ratio of WT: SLP75Y145FKI CD8+ and CD4+ T cells in the organs was determined.

(D) We transplanted either CD4+ or CD8+ T cells separately into irradiated BALB/c mice, in separate experiments. At day 7 post-allo-HSCT, liver and small intestines were examined by H&E staining (10X) magnification).

(E and F) On Day 7 post-allo-HSCT, donor CD8+ or CD4+ T cells from separate experiments were isolated and examined for the expression of CXCr3, CX3r1, CXCr1, CCR12, s1pR1, CrTAM, CXCR6, CCR9, CXCR5, and CXCr4 using q-PCR. p values were calculated using two-way ANOVA and Student's t test, p values are listed.

(G) Irradiated BALB/c mice were BM-transplanted and injected with CD8+ T cells and CD4+ T cells from luciferase-expressing WT or SLP75Y145FKI mice (C57Bl/6 background). On day 7 post-allo-HSCT, recipient BALB/c mice were injected with D-luciferin. Spleen, lymph nodes, liver, and small intestine were examined for donor CD8+ T cells or CD4+ T cells by luciferase expression. One representative of 2 independent experiments is shown (n = 3 mice/group for control, n = 5 mice for WT, and n = 5 mice for SLP75Y145FKI. p values were calculated using two-way ANOVA and Student's t test, p values are listed). See also Figures S5 and S6.