Research On T/B Cells Sensitized By ALDH^high CSC DCs And Cytokines Can Target At ALDH^high CSC In Human HNSCC In Vitro

Objective: To date,the available cancer preventive vaccines function by preventing viral infection rather than cancer.The field of ‘cancer vaccines’ that are not anti-infection vaccines is directed to immunotherapy of diagnosed tumors.In our preliminary work,we isolate the cancer stem cells by marker ALDEFLUOR+ /ALDHhigh.The DCs pulsed by ALDEFLUOR+ / ALDHhigh cancer stem cells lysate can induced significantly anti-cancer in response to ALDHhigh CSCs.In contrast,DCs pulsed with lysate of ALDHlow cells(ALDHlow-DC)resulted in limited sensitization/priming of autologous T,B lymphocytes to produce cytokines and antibodies.We want to establish the concept that the antigenicity/immunogenicity of ALDHhigh human head and neck squamous cell carcinoma(HNSCC)cancer stem cells(CSC)is distinct from that of ALDHlow non-CSCs.In HNSCC,T,B lymphocytes sensitized by DC pulsed with ALDEFLUOR+/ ALDHhigh cells lysates can render greater and specific antitumor efficacy in humoral immunity and cellular immunity.Methods:1.From peripheral blood collected from patients with HNSCC,we obtained PBMCs.CD3+T lymphocytes,CD19+B lymphocytes and CD3-/CD19-PBMCs from PBMCs by MACs.T,B lymphocytes are activated and DC are cultured from CD3-/CD19-PBMCs.2.HNSCC cancer cells are passaged.ALDH expression was detected using the ALDEFLUOR kit.Fluorescence-activated cell sorting gates were established using the inhibited control(DEAB).Equal numbers of ALDEFLUOR+/ALDHhigh CSCs and ALDEFLUOR-/ALDHlow non-CSCs were collected.The tumor cells were made into lysate by frozen and melted.DCs were pulsed by ALDHhigh and ALDHlow cancer cells lysate.3.T,B lymphocytes which were sensitized by CSC-DC/ non-CSC-DC/ PBS-DC were checked by cytokine production,CTL activity,and antibody secretion of these primed T,B cells in response to ALDHhigh CSCs/ ALDHlow non-CSCs.Results:1.The ELISA results indicate that T,B lymphocytes which were sensitized by CSC-DC can produce more IFN-γ,GM-CSF,Ig M and Ig G.2.CTL activity was assessed using the LDH release assay.T lymphocytes which were sensitized by CSC-DC can kill more CSCs tumor cells.3.For flow cytometry,we found that binding to ALDHhigh CSCs by Ab produced by B cells sensitized with ALDHhigh-DC and stimulated with ALDHhigh-CSCs is higher than the binding to the ALDHhigh CSCs by Ab produced by B cells sensitized with ALDHlow-DC and stimulated with ALDHlow cells.The expression of HLA-ABC was drastically reduced.Conclusions:1.DCs generated from the PBMC can be pulsed by ALDHhigh CSCs and ALDHlow non-CSCs.HLA-ABC.Down-regulation of HLA-ABC molecules on the ALDHhigh CSCs may represent one of the mechanisms for this sub population of cells to escape from the immune surveillance.2.DCs pulsed by ALDHhigh CSCs lysate can induce anti-HNSCC CSC cellular immunity3.DCs pulsed by ALDHhigh CSCs lysate can induce anti-HNSCC CSC humoral immunity。Binding results thus reveal enhanced binding to ALDHhigh-CSCs by antibodies produced by B cells sensitized with ALDHhigh-DC and stimulated with ALDHhigh-CSCs.