The Immune Effect Of β2-microglolulin Antibody Activating Dendritic Cells From Non-Hodgkin Lymphomas In Vitro

Objective Dendritic cells(DCs)are the most potential professional antigen presenting cells(APCs)as a promising tool and target for cancer immunotherapy.But weak tumor antigens restricted the function of Dcs to solve the problems of minimal residual diseases(MRDs)and recidivism of malignancies.However,special monoclonal antibodies could induce many malignant cells to apoptosis,and some researches had revealed that certain antibodies could mediate DCs to activate auto-T lymphocytes to improve anti-tumor effects.So,it will be a possible method to cure malignancies.β2-microglobulin(β2-MG)is the invariant chain of the major histocompatibility(MHC)classⅠmolecules on the cell surface of all nucieated cells. Freeβ2-MG is found in body fluid of patients with malignancies as a result of shedding from cell surfaces or intracellular release.Clinical researches show elevated levels ofβ2-MG correlate with a poor prognosis in lymphomas.Butβ2-MG antibody is a typicalβ2-MG monopoly antibody,which enables NHL and other human hematological malignancies to apoptosis.Meanwhile,β2-MG exists in the surface of DCs,which constitudes MHC-Ⅰmolecules,CD1a molecules and Fc receptors.The purpose of this study is whetherβ2-MG antibody can connect with the associated molecules on the surface of DCs to induce the activation of cellular immunity for eliminating NHL cells.Methods Bone marrow mononuclear cells(BMMNCs)derived from the new-diagnosed NHL patients.DCs were cultured in vitro by the conventional methods as GM-CSF,IL-4 and TNF-a.25ug/mlβ2-MG antibody,25ug/mlβ2-MG and 25ug/ml connectedβ2-MG/β2-MG antibody were added to DCs on day 5 respectively as group A,group B and group C.At some times,25ug/ml IgG cultured with DCs as control group A and unpulsed DCs designed as blank control group.DCs morphous were observed under light microscope.The immune phenotypes of DCs were analysed by flow cytometry.The level of IFN-γin cultural supernatants was detected by ELISA and the anti-tumor effects were measured by MTT.Results The levels of immune phenotypes onβ2-MG-loaded DCs surface including CD1a,CD80,CD83,CD86 and HLA-DR,weren't more upregulated than those of unpulsed DCs analysed by flow cytometry neither the day 5 after 3 hours of addingβ2-MG nor the sequential day 8 after 1 day of adding TNF-a(P＞0.05).Also,the secreting level of IFN-γwasn't up to unpulsed DCs obviously whenβ2-MG-loaded DCs stimulated auto-T lymphocytes(296.37±40.04vs384.42±23.86pg/ml,P＞0.05). However,β2-MG antibody could promote DCs mature.From cell morphous,the cultural cells prossessed the typical DC-like morphology on day 8.Moreover,the mature phenotypes levels of DCs including CD80,CD83,CD86 and HLA-DR were more noticeable to rise than those stimulated byβ2-MG(P＜0.05).With the incubation of theβ2-MG antibody-loaded DCs and auto-T lymphocytes,the level of IFN-γin the cultural supernatants was upper than that of unpulsed DCs orβ2-MG-loaded DCs(628.99±30.59 vs 384.42±23.86 or 296.37±40.04,P＜0.05)and the findings of cytotoxic activities to Raji cells(NHL cells)were increased obviously(P＜0.05).In addition,β2-MG/β2-MG antibody stimulated DCs were capable to induce CTL effects and kill Raji cells.The cytotoxic activity had no statistical differences between theβ2-MG/β2-MG antibody combination andβ2-MG antibody alone.Conclusion As a tumor associated antigen,β2-MG had a weaker affinity to DCs, which could not evidently induce DCs mature and activate the CD4~+Th1 cells of NHL patients.In contrast,β2-MG antibody could mediate DCs to activate CD4~+Th1 cells and CTL for eliminating NHL cells.In addition,the effect ofβ2-MG/β2-MG antibody combinated DCs illustrated freeβ2-MG didn't prevent the activation of DCs mediated byβ2-MG antibody.So,the study provided a novel model to reinforce the application of DC vaccines in NHL treatment.