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. 2021 Sep 22;6(18):e149301. doi: 10.1172/jci.insight.149301

Figure 3. Nox2-deficient Tregs express higher levels of CCR4 and CCR8 than WT Tregs driving migration into heart allografts.

Figure 3

Tregs were purified from spleens and lymph nodes of Nox2-deficient Tregs (Nox2–/–) and WT mice and assessed for (A) mRNA levels of chemokine receptors (n = 12); (B and C) CCR4 and CCR8 protein levels by flow cytometry (n = 6); (D) chemotaxis in vitro toward CCL1 and CCL22; and (E and F) actin polymerization stimulated by CCL1 and CCL22. Some cells were incubated with Ly294002 (5 μM). Full minus one (FMO) antibody was used as a negative control. Graphs and images represent 1 of 3 independent experiments. Scale bar: 32 μm. (G, H, and K) WT and Nox2–/– Tregs were stained with different color cell tracers and tested for infiltration (after adoptive transfer) into CB6F1 hearts transplanted in C57BL/6 recipients (n = 4) (G and H) or adherence on cardiac ECs (K). (I and J) In vitro activation and binding of ICAM-1 in Tregs. Histograms and mean data represent 1 of 2 independent experiments. Data are shown as mean ± SEM. *P < 0.05 for indicated comparisons, Mann-Whitney 2-tailed t test in C, H, and K; Kruskal-Wallis followed by Dunn’s post test in D and E; and 2-way ANOVA followed by Bonferroni’s post test in J.