Aim: to observe whether the donor's marrow (BM) cells treated by GM-CSF and TGF-β in vitro were beneficial in inducing specific hypoergia to donor's lymphocyte in recipient.
Methods: donor's marrow cell were cultured with GM-CSF and TGF-β in vitro and its maturities were tested by treating with LPS. BALB/c mice were divided into three groups and received treatment as following: (1) BM pretreatment group: donor's BM cell mentioned above were injected into caudal vein with 10(5); cell each mouse; (2) donor's splenocyte group: C57BL/6 mouse's splenocyte were injected through caudal vein with 10(5); cell each mouse; (3) Negative control group: PBS with the same volume were injected through caudal vein. The treatment were performed twice with one week internal in each group. One week after the second treatment, all of the mice were challenged by splenocyte of C57BL/6 mouse in abdominal cavity with 10(5); cell each mouse. Three days later, the allo-reactivities were tested: the proliferation of recipient mouse lymphocyte to donor's lymphocyte were test by single mix lymphocyte reaction, the level of serum IFN-γ and IL-10 were tested with ELISA methods. CD4(+);CD25(high); Treg cell and expression of killer cell lectin-like receptor G1 (KLRG1) on NK cell were tested with flow cytometric technique and the NK cytotoxicity were measured by LDH release method.
Results: the donor's marrow cell treated by GM-CSF and TGF-β in vitro could resist the maturation promotion by LPS. Compared with donor's lymphocyte pretreatment, pre-treatment with BM cell induced decreased IL-10, increased CD4(+);CD25(high); Treg cell in spleen, decreased proliferation to donor's lymphocyte in vitro. Additionally, NK cell cytotoxicity was also decreased.
Conclusion: the donor's marrow cell treated by GM-CSF and TGF-βcan induce the donor specific tolerance in some degree, and modulation of NK cell maybe participate in inducing immunological tolerance.