Background Immunotherapy is emerging as a promising cure for cancer.However,a severe problem in this area is theimmune tolerance to tumor cells and tumor-associated antigens,as evidenced by the ability of cancer to escape immunesurveillance.To overcome this problem this work examined the potential of improving the antigenicity of myeloma bymetabolic engineering of its cell surface carbohydrate antigens (i.e.,glycoengineering) and presentation of the modifiedtumor antigens by dendritic cells(DCs) to generate cytotoxic T-lymphocytes (CTLs).Methods CD138~+ myeloma cells were isolated from 11 multipe myeloma(MM)patients by the immunomagnetic beadmethod.The MM cells were treated with N-propionyl-D-mannosamine(ManNPr),a synthetic analog ofN-acetyl-D-mannosamine (ManNAc),the natural biosynthetic precursor of N-acetyl sialic acid(NeuNAc),to expressunnatural N-propionylated sialoglycans.The glycoengineered cells were then induced to apoptosis,and the apoptoticproducts were added to cultured functional DCs that could present the unnatural carbohydrate antigens to autologousT-lymphocytes.Results It was found that the resultant DCs could activate CD4~+ and CD8~+ T-lymphocytes,resulting in increasedexpression of T cell surface markers,including CD8CD28 and CD4CD29.Moreover,upon stimulation byglycoengineered MM cells,these DC-activated T-lymphocytes could release significantly higher levels of IFN-γ (P<0.05).Lactate dehydrogenase(LDH)assays further showed that the stimulated T-lymphocytes were cytotoxic toglycoengineered MM cells.Conclusions This work demonstrated that glycoengineered myeloma cells were highly antigenic and the CTLs inducedby the DCs loaded with the unnatural myeloma antigens were specifically cytotoxic to the glycoengineered myeloma.This may provide a new strategy for overcoming the problem of immune tolerance for the development of effectiveimmunotherapies for MM.Chin Med J 2007;120(19):1678-1684 Background Immunotherapy is emerging as a promising cure for cancer. Despite, a severe problem in this area is theimmune tolerance to tumor cells and tumor-associated antigens, as evidenced by the ability of cancer to escape immunesurveillance.To overcome this problem this work examined the potential of improving the antigenicity of myeloma bymetabolic engineering of its cell surface carbohydrate antigens (ie, glycoengineering) and presentation of the modified tumor antigens by dendritic cells (DCs) to generate cytotoxic T-lymphocytes (CTLs). Methods CD 138 ~ + myeloma cells were isolated from 11 multipe myeloma (MM) patients by the immunomagnetic beadmethod. MM cells were treated with N-propionyl-D-mannosamine (ManNPr), a synthetic analog of N-acetyl-D-mannosamine (ManNAc), the natural biosynthetic precursor of N -acetyl sialic acid (NeuNAc), to expressunnatural N-propionylated sialoglycans. The glycoengineered cells were then induced to apoptosis, and the apoptotic products were added to cultured fun ctional DCs that could present the unnatural carbohydrate antigens to autologous T-lymphocytes. Results It was found that the resultant DCs could activate CD4 ~ + and CD8 ~ + T-lymphocytes, resulting in increasedexpression of T cell surface markers, including CD8CD28 and CD4CD29. Moreover , when reactive byglycoengineered MM cells, these DC-activated T-lymphocytes could release significantly higher levels of IFN-γ (P <0.05) .actact dehydrogenase (LDH) assays further showed that the stimulated T-lymphocytes were cytotoxic to glycoengineered MM cells. This work demonstrates that glycoengineered myeloma cells were highly antigenic and the CTLs inducedby the DCs loaded with the unnatural myeloma antigens were specifically cytotoxic to the glycoengineered myeloma.This may provide a new strategy for overcoming the problem of immune tolerance for the development of effective immunotherapy for MM . Chin Med J 2007; 120 (19): 1678-1684