Dendritic cell vaccines containing lymphocytes produce improved immunogenicity in patients with cancer

Mayu O Frank, Julia Kaufman, Salina Parveen, Nathalie E Blachère, Dana E Orange, Robert B Darnell, Mayu O Frank, Julia Kaufman, Salina Parveen, Nathalie E Blachère, Dana E Orange, Robert B Darnell

Abstract

Background: Dendritic cells are currently under investigation for their ability to generate anti-cancer immune responses. No consensus has been reached as to the optimal method of dendritic cell vaccine preparation and is a barrier to success in the field.

Methods: Over a course of three separate dendritic cell vaccine studies to treat cancer, we tested two different methods for preparing dendritic cells from peripheral blood mononuclear cells: adherence and antibody-selected CD14+ cells.

Results: Surprisingly, we found that patients who received dendritic cell vaccines generated by the adherence method mounted increased T cell proliferation in response to vaccination. This difference could not be accounted for by dendritic cell vaccine dose, cell surface phenotype or dendritic cell function in vitro. One notable difference between the two vaccine preparation methods was that the dendritic cell vaccine cultures generated by the adherence method contained up to 10% lymphocytes, and these lymphocytes were proliferating and producing IFNγ in response to antigen in vitro at the time of administration.

Conclusions: Enhanced immunogenicity of adherence dendritic cell vaccinations may be due to the presence of lymphocytes during dendritic cell culture.

Trial registration: Clinicaltrials.gov identifiers: NCT00289341, NCT00345293, and NCT00893945.

Figures

Figure 1
Figure 1
Adherence DCs elicit increased proliferation of lymphocytes post-vaccination. (A)3H thymidine proliferation responses to prostate cancer cells lines, PC3, LNCaP or no antigen of lymphocytes from patients vaccinated with either Selected or Adherence DCs pulsed with PC3. CD14+ monocytes were used as APCs. Results are presented as average pre-vaccination counts per minute (CPM) subtracted from average post-vaccination CPM for each antigen group. The results are the average of 3 to 6 replicate wells. Black line indicates median. * indicates p < 0.05. NS = not statistically significant. (B) Pooled 3H thymidine proliferation responses to KLH or no antigen of lymphocytes from patients vaccinated with either Selected or Adherence DCs from 3 clinical trials. ● indicates response of patient in DC/LNCaP study, ▲ indicates response of patient in DC/PC3 study, and ■ indicates response of patient in DC pulsed with autologous brain tumor study.
Figure 2
Figure 2
In vitro assays of phenotype and function do not predict differences in immunogenicity. (A) Cell surface marker staining. Selected or Adherence DCs made from the same donor were stained with CD14, CD83, HLA-DR, CD86, CD40 and CCR7 antibody. This is representative of 3 repeated experiments. (B) Phagocytosis assay. HLA-DR stained Selected or Adherence iDCs were cultured with PKH26 stained apoptotic lymphocytes with or without EDTA for 24 hours. Cells are gated on HLA-DR. Cells staining positive for both HLA-DR and PKH26 indicate phagocytosis of apoptotic cells by DC groups. The data shown is representative of 3 repeated experiments. (C) Allo-MLR. Selected and Adherence DCs were made from cells from 3 donors. Average CPMs are shown for syngeneic and allogeneic responses at the DC:T cell ratio of 1:30. Solid black line represents the mean. NS = not statistically significant. (D) Lymphocyte proliferation. Adherence or Selected DCs co-cultured with apoptotic 3T3 cells (DC/ctrl) or with influenza-infected 3T3 cells (DC/flu), were cultured with syngeneic CD14- cells. The cultures were assessed for proliferation by 3H thymidine incorporation and the data shown is representative of 3 repeated experiments. NS = not statistically significant. (E) IFNγ ELISPOT. Purified syngeneic CD8 or CD4 T cells were plated in an ELISPOT with either non-infected DCs (DC) or influenza-infected (DCF) made by the 2 methods. The data shown is an average of triplicate wells and representative of 3 repeated experiments. NS = not statistically significant.
Figure 3
Figure 3
Lymphocytes in Adherence DC vaccine preparations are activated and proliferating. (A) Representative flow cytometry profile of forward scatter versus side scatter of Adherence and Selected DC vaccine preparations. CD4, CD8, and CD19 antibody staining of cells in the lymphocytes gate in the Adherence DC plot is shown by histogram. (B) DCs made with the Adherence method were stained with antibodies to lymphocyte markers, CD4, CD8 or CD19 and activation markers CD40, CD83, CD69 and HLA-DR and analyzed by flow cytometry. The results shown are representative of 3 different donors.
Figure 4
Figure 4
DC vaccines prepared by Adherence support lymphocyte proliferation and cytokine production. (A) DCs made by the 2 methods were assessed for proliferation by 3H thymidine incorporation at 48, 72, and 96 hours beyond day 8, (the day DCs would have been administered as vaccine). Proliferation responses for Adherence DCs are shown in red and Selected DCs are shown in purple. The results are averages of triplicate wells. * indicates p < 0.05. (B) Supernatants of Adherence and Selected DC cultures were examined for pro-inflammatory cytokines on Day 6 and Day 8. Data for IL-2 is shown for 3 donors. Black line represents the mean. * indicates p < 0.05. NS = not statistically significant.
Figure 5
Figure 5
Proliferation and cytokine production in DC vaccine prepared by adherence method is antigen specific. (A) Adherence DCs were co-cultured with apoptotic 3T3 (DC/ctrl) or with 3T3 infected with influenza (DC/flu) and assessed for proliferation by 3H thymidine incorporation at 24, 48 and 96 hours beyond day 8 (the day DCs would have been administered as a vaccine). Results are of triplicate wells. * indicates p < 0.05. (B) Adherence DC/ctrl and DC/flu were stained with CD8 antibody, influenza-M1 specific dextramer and IFNγ antibody. The original PBMC population was used as the comparison group. The upper panels shows CD8+ cells that are gated on M1+ staining. The lower panels show M1+ cells that are gated on IFNγ + cells.

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