Fig. 4.

Macrophages infiltrating the grafts early during granulocyte colony‐stimulating factor (G‐CSF)/rapamycin treatment induce T regulatory type 1 (Tr1)‐cell expansion in vitro. Graft‐infiltrating cells harvested earlier were pooled by group [G‐CSF/rapamycin‐treated (closed circles) and untreated (open circles) mice] and used for co‐culture experiments. A total of 100 000 cells were cultured with CD4⁺ cells, isolated by negative immunomagnetic selection from naive splenocytes of CD45.1 C57BL/6 mice, at a 1 : 1 ratio in 96‐well round‐bottomed plates in complete medium for 5 or 7 days. Upon collection, Tr1 cells were detected by flow cytometry: LAG3⁺CD4⁺CD25⁻CD45.1⁺ and represented over time.