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Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells.

Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N - J. Exp. Med. (2001)

Bottom Line: Injection of mature DCs rapidly enhances antigen-specific CD4+ and CD8+ T cell immunity in humans.Here we describe the immune response to a single injection of immature DCs pulsed with influenza matrix peptide (MP) and keyhole limpet hemocyanin (KLH) in two healthy subjects.In contrast to prior findings using mature DCs, injection of immature DCs in both subjects led to the specific inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells and the appearance of MP-specific interleukin 10-producing cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021, USA. dhodapm@rockvax.rockefeller.edu

ABSTRACT
Immunostimulatory properties of dendritic cells (DCs) are linked to their maturation state. Injection of mature DCs rapidly enhances antigen-specific CD4+ and CD8+ T cell immunity in humans. Here we describe the immune response to a single injection of immature DCs pulsed with influenza matrix peptide (MP) and keyhole limpet hemocyanin (KLH) in two healthy subjects. In contrast to prior findings using mature DCs, injection of immature DCs in both subjects led to the specific inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells and the appearance of MP-specific interleukin 10-producing cells. When pre- and postimmunization T cells were boosted in culture, there were greater numbers of MP-specific major histocompatibility complex tetramer-binding cells after immunization, but these had reduced interferon production and lacked killer activity. These data demonstrate the feasibility of antigen-specific inhibition of effector T cell function in vivo in humans and urge caution with the use of immature DCs when trying to enhance tumor or microbial immunity.

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Immune responses in uncultured T cells. (A and B) MP-, gag-, and influenza (Flu)-specific IFN-γ–producing cells from before and after DC immunization were quantified in freshly isolated uncultured PBMCs using an ELISPOT assay. Data for influenza specific cells is per 105 cells. SEM for all measurements is <20%. (A) Im1; (B) Im2. (C and D). Pre- and postimmunization samples were thawed together and assayed for antigen-specific T cells secreting IFN-γ, IL-4, and IL-10 using a 16-h ELISPOT assay. Antigens were HLA A2.1–restricted peptides from influenza MP, HIV-gag (gag), and CMV pp65 (CMV). Positive controls for the assays included SEA for IFN-γ and IL-10 and PHA for IL-4 (not shown). SEM for all measurements is <20%. (C) Im1; (D) Im2. (E) Use of peptide-pulsed DCs as APCs in the ELISPOT. Pre- and postimmunization specimens were examined using peptide-pulsed mature DCs as APCs (PBMC/DC ratio 30:1) in the ELISPOT. SEM for all measurements is <20%. (F) Quantification of MP-specific T cells using MHC tetramers in uncultured cells. Pre-/postimmunization specimens were stained with A*0201–MP tetramers at 37°C and analyzed by flow cytometry. Data shown are gated for CD8+ T cells and expressed as percent CD8+ T cells binding A*0201–MP tetramer.
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Figure 1: Immune responses in uncultured T cells. (A and B) MP-, gag-, and influenza (Flu)-specific IFN-γ–producing cells from before and after DC immunization were quantified in freshly isolated uncultured PBMCs using an ELISPOT assay. Data for influenza specific cells is per 105 cells. SEM for all measurements is <20%. (A) Im1; (B) Im2. (C and D). Pre- and postimmunization samples were thawed together and assayed for antigen-specific T cells secreting IFN-γ, IL-4, and IL-10 using a 16-h ELISPOT assay. Antigens were HLA A2.1–restricted peptides from influenza MP, HIV-gag (gag), and CMV pp65 (CMV). Positive controls for the assays included SEA for IFN-γ and IL-10 and PHA for IL-4 (not shown). SEM for all measurements is <20%. (C) Im1; (D) Im2. (E) Use of peptide-pulsed DCs as APCs in the ELISPOT. Pre- and postimmunization specimens were examined using peptide-pulsed mature DCs as APCs (PBMC/DC ratio 30:1) in the ELISPOT. SEM for all measurements is <20%. (F) Quantification of MP-specific T cells using MHC tetramers in uncultured cells. Pre-/postimmunization specimens were stained with A*0201–MP tetramers at 37°C and analyzed by flow cytometry. Data shown are gated for CD8+ T cells and expressed as percent CD8+ T cells binding A*0201–MP tetramer.

Mentions: Two subjects (Im1 and Im2) received a single subcutaneous injection of 2 × 106 immature DCs pulsed with the influenza peptide, MP, and KLH (Table ). All DC injections in this study were well tolerated without any clinical toxicity and serologic/clinical evidence of autoimmunity. Before immunization, MP-specific IFN-γ–producing T cells were detectable in both subjects as expected, because most adults have been exposed to the influenza virus. However, after DC immunization, there was a decline in MP-specific IFN-γ–producing cells (Fig. 1a and Fig. b). The number of MP-specific effectors reached a nadir 7–30 d after immunization and improved thereafter. In contrast, there was little decline in total influenza effector T cell function, indicating that the decrease in MP effector function was specific for the immunizing peptide. Similar data were obtained when cryopreserved cells were assayed together (Fig. 1c and Fig. d), although the absolute reactivity was higher with fresh cells, as reported previously 4. As a control, no decline in antigen-specific IFN-γ–producing cells to HLA A*0201–restricted CMV peptide was observed. The loss of MP-specific IFN-γ–producing cells persisted, even when peptide-pulsed mature DCs were used as APCs in the ELISPOT (Fig. 1 E). In fact, the use of DCs increased the preimmunization measurements, so that the decrease in effectors after immature DC injection was even more striking.


Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells.

Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N - J. Exp. Med. (2001)

Immune responses in uncultured T cells. (A and B) MP-, gag-, and influenza (Flu)-specific IFN-γ–producing cells from before and after DC immunization were quantified in freshly isolated uncultured PBMCs using an ELISPOT assay. Data for influenza specific cells is per 105 cells. SEM for all measurements is <20%. (A) Im1; (B) Im2. (C and D). Pre- and postimmunization samples were thawed together and assayed for antigen-specific T cells secreting IFN-γ, IL-4, and IL-10 using a 16-h ELISPOT assay. Antigens were HLA A2.1–restricted peptides from influenza MP, HIV-gag (gag), and CMV pp65 (CMV). Positive controls for the assays included SEA for IFN-γ and IL-10 and PHA for IL-4 (not shown). SEM for all measurements is <20%. (C) Im1; (D) Im2. (E) Use of peptide-pulsed DCs as APCs in the ELISPOT. Pre- and postimmunization specimens were examined using peptide-pulsed mature DCs as APCs (PBMC/DC ratio 30:1) in the ELISPOT. SEM for all measurements is <20%. (F) Quantification of MP-specific T cells using MHC tetramers in uncultured cells. Pre-/postimmunization specimens were stained with A*0201–MP tetramers at 37°C and analyzed by flow cytometry. Data shown are gated for CD8+ T cells and expressed as percent CD8+ T cells binding A*0201–MP tetramer.
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Related In: Results  -  Collection

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Figure 1: Immune responses in uncultured T cells. (A and B) MP-, gag-, and influenza (Flu)-specific IFN-γ–producing cells from before and after DC immunization were quantified in freshly isolated uncultured PBMCs using an ELISPOT assay. Data for influenza specific cells is per 105 cells. SEM for all measurements is <20%. (A) Im1; (B) Im2. (C and D). Pre- and postimmunization samples were thawed together and assayed for antigen-specific T cells secreting IFN-γ, IL-4, and IL-10 using a 16-h ELISPOT assay. Antigens were HLA A2.1–restricted peptides from influenza MP, HIV-gag (gag), and CMV pp65 (CMV). Positive controls for the assays included SEA for IFN-γ and IL-10 and PHA for IL-4 (not shown). SEM for all measurements is <20%. (C) Im1; (D) Im2. (E) Use of peptide-pulsed DCs as APCs in the ELISPOT. Pre- and postimmunization specimens were examined using peptide-pulsed mature DCs as APCs (PBMC/DC ratio 30:1) in the ELISPOT. SEM for all measurements is <20%. (F) Quantification of MP-specific T cells using MHC tetramers in uncultured cells. Pre-/postimmunization specimens were stained with A*0201–MP tetramers at 37°C and analyzed by flow cytometry. Data shown are gated for CD8+ T cells and expressed as percent CD8+ T cells binding A*0201–MP tetramer.
Mentions: Two subjects (Im1 and Im2) received a single subcutaneous injection of 2 × 106 immature DCs pulsed with the influenza peptide, MP, and KLH (Table ). All DC injections in this study were well tolerated without any clinical toxicity and serologic/clinical evidence of autoimmunity. Before immunization, MP-specific IFN-γ–producing T cells were detectable in both subjects as expected, because most adults have been exposed to the influenza virus. However, after DC immunization, there was a decline in MP-specific IFN-γ–producing cells (Fig. 1a and Fig. b). The number of MP-specific effectors reached a nadir 7–30 d after immunization and improved thereafter. In contrast, there was little decline in total influenza effector T cell function, indicating that the decrease in MP effector function was specific for the immunizing peptide. Similar data were obtained when cryopreserved cells were assayed together (Fig. 1c and Fig. d), although the absolute reactivity was higher with fresh cells, as reported previously 4. As a control, no decline in antigen-specific IFN-γ–producing cells to HLA A*0201–restricted CMV peptide was observed. The loss of MP-specific IFN-γ–producing cells persisted, even when peptide-pulsed mature DCs were used as APCs in the ELISPOT (Fig. 1 E). In fact, the use of DCs increased the preimmunization measurements, so that the decrease in effectors after immature DC injection was even more striking.

Bottom Line: Injection of mature DCs rapidly enhances antigen-specific CD4+ and CD8+ T cell immunity in humans.Here we describe the immune response to a single injection of immature DCs pulsed with influenza matrix peptide (MP) and keyhole limpet hemocyanin (KLH) in two healthy subjects.In contrast to prior findings using mature DCs, injection of immature DCs in both subjects led to the specific inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells and the appearance of MP-specific interleukin 10-producing cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021, USA. dhodapm@rockvax.rockefeller.edu

ABSTRACT
Immunostimulatory properties of dendritic cells (DCs) are linked to their maturation state. Injection of mature DCs rapidly enhances antigen-specific CD4+ and CD8+ T cell immunity in humans. Here we describe the immune response to a single injection of immature DCs pulsed with influenza matrix peptide (MP) and keyhole limpet hemocyanin (KLH) in two healthy subjects. In contrast to prior findings using mature DCs, injection of immature DCs in both subjects led to the specific inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells and the appearance of MP-specific interleukin 10-producing cells. When pre- and postimmunization T cells were boosted in culture, there were greater numbers of MP-specific major histocompatibility complex tetramer-binding cells after immunization, but these had reduced interferon production and lacked killer activity. These data demonstrate the feasibility of antigen-specific inhibition of effector T cell function in vivo in humans and urge caution with the use of immature DCs when trying to enhance tumor or microbial immunity.

Show MeSH
Related in: MedlinePlus