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

Frank MO, Kaufman J, Parveen S, Blachère NE, Orange DE, Darnell RB - J Transl Med (2014)

Bottom Line: Dendritic cells are currently under investigation for their ability to generate anti-cancer immune responses.This difference could not be accounted for by dendritic cell vaccine dose, cell surface phenotype or dendritic cell function in vitro.Enhanced immunogenicity of adherence dendritic cell vaccinations may be due to the presence of lymphocytes during dendritic cell culture.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuro-oncology, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA. frankm@rockefeller.edu.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Fig2: 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.

Mentions: To test whether Adherence or Selected DCs express different levels of markers of maturation, DCs were prepared using both methods from the same donor’s PBMC. Adherence and Selected DCs were assessed for the surface markers CD14, HLA-DR, CD83, CD86, CD40 and CCR7, but no differences were found (Figure 2A). We next evaluated the in vitro function of the two types of DCs. First, we assessed their ability to phagocytose apoptotic cells, which could affect antigen presentation. Selected or Adherence immature iDCs were cultured with PKH26 stained apoptotic lymphocytes. Cells positive for both HLA-DR staining and PKH26 dye indicated DC phagocytosis of apoptotic cells. 66.4% of Selected and 67% of Adherence iDCs phagocytosed apoptotic cells, indicating no difference in their phagocytic activity (Figure 2B). This process was calcium dependent and inhibited in the presence of EDTA, indicating that apoptotic cells were phagocytosed and not positive for both markers simply by adhering to one another or other non-specific mechanisms.Figure 2


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

Frank MO, Kaufman J, Parveen S, Blachère NE, Orange DE, Darnell RB - J Transl Med (2014)

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.
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Related In: Results  -  Collection

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Fig2: 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.
Mentions: To test whether Adherence or Selected DCs express different levels of markers of maturation, DCs were prepared using both methods from the same donor’s PBMC. Adherence and Selected DCs were assessed for the surface markers CD14, HLA-DR, CD83, CD86, CD40 and CCR7, but no differences were found (Figure 2A). We next evaluated the in vitro function of the two types of DCs. First, we assessed their ability to phagocytose apoptotic cells, which could affect antigen presentation. Selected or Adherence immature iDCs were cultured with PKH26 stained apoptotic lymphocytes. Cells positive for both HLA-DR staining and PKH26 dye indicated DC phagocytosis of apoptotic cells. 66.4% of Selected and 67% of Adherence iDCs phagocytosed apoptotic cells, indicating no difference in their phagocytic activity (Figure 2B). This process was calcium dependent and inhibited in the presence of EDTA, indicating that apoptotic cells were phagocytosed and not positive for both markers simply by adhering to one another or other non-specific mechanisms.Figure 2

Bottom Line: Dendritic cells are currently under investigation for their ability to generate anti-cancer immune responses.This difference could not be accounted for by dendritic cell vaccine dose, cell surface phenotype or dendritic cell function in vitro.Enhanced immunogenicity of adherence dendritic cell vaccinations may be due to the presence of lymphocytes during dendritic cell culture.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Neuro-oncology, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA. frankm@rockefeller.edu.

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.

No MeSH data available.


Related in: MedlinePlus