AIM Lycium barbarum L.is a renowned Yin strengthening agent in traditional Chinese medicine.Lycium barbarum L.polysaccharide-protein complex is well-known for its immunoregulatory and antitumor effects.LBPF4-OL is the glycan part of Lycium barbarum L.polysaccharide-protein complex fraction 4 (LBPF4).The purpose of this study was to identify the immune target cell and binding site of LBPF4-OL METHODS Miltenyi MicroBeads were used to purify the lymphocyte.Lymphocyte proliferation was analyzed with [3H] TdR incorporation method.Activation of T and B cells was analyzed by flow cytometry (FCM).Cytokine concentration was determined by multiplex bead analysis or ELISA.TLR4 gene mutation mice C3H/HeJ, antibody block and biolayer interferometry (BLI) were used to verify the binding sites.RESULTS Lymphocyte proliferation assay indicated that LBPF4-OL could not induce T and B lymphocytes proliferation spontaneously, but B ceil proliferation could take place in the presence of activated macrophages or LPS.FCM analysis found that LBPF4-OL could not activate T and B cells when co-cultured with splenocyte for 24 h in vitro.But it significantly prompted CD86 and MHC-Ⅱ molecules expression on macrophage in vivo.ELISA assay showed that LBPF4-OL greatly increased the levels of TNF-α and IL-1 β on macrophage supernatant.Multiplex bead analysis showed that LBPF4-OL induced IL-6, IL-8, IL-10 and TNF-α production in a concentration dependent manner, but had no effect on IL-2 and IL-13 production.The possible binding sits of CD19 on B lymphocyte, TLR4, TLR2 and CR3 on macrophages were studied, and TLR4 as the binding site of LBPF4-OL was further verified in C3H/HeJ mice.Results showed that LBPF4-OL obviously induced the proliferation of splenocyte and the secretion of TNF-α and IL-1 β in macrophage from wildtype mice C3H/HeN.But the proliferation of splenocyte from mutation mice C3H/HeJ reduced significantly.BLI results showed that LBPF4-OL could bind directly with TLR4 in vitro.CONCLUSION Both B lymohocytes and macrophage are target ceils of LBPF4-OL, and TLR4 is one of the binding sites on macrophage.