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

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.

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.

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.

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.

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.

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.

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.

Figure 2

(A–C) T cell recall assays in culture after DC immunization. Pre- and postimmunization specimens were thawed and cocultured with MP-pulsed DCs (unpulsed DCs as controls) for 7 d. After 7-d culture, MP-specific T cells were quantified by MHC tetramers (A), ELISPOT (B), and CTL assay (C). (A) MHC tetramer assay. Data are expressed as percent CD8⁺ T cells binding A*0201–MP tetramer. (B) ELISPOT assay. On day 7, cells were transferred to an ELISPOT plate and restimulated (Restim) overnight with specific antigen (10 μg/ml MP) or left without restimulation (as controls), and antigen-specific IFN-γ–producing cells were quantified using an ELISPOT. SEM for all measurements is <30%. (C) CTL assay. MP-specific lysis was measured using MP-pulsed T2 targets. Data shown are after subtracting lysis with control unpulsed T2 targets and from cells after expansion using unpulsed DCs.

Figure 2

(A–C) T cell recall assays in culture after DC immunization. Pre- and postimmunization specimens were thawed and cocultured with MP-pulsed DCs (unpulsed DCs as controls) for 7 d. After 7-d culture, MP-specific T cells were quantified by MHC tetramers (A), ELISPOT (B), and CTL assay (C). (A) MHC tetramer assay. Data are expressed as percent CD8⁺ T cells binding A*0201–MP tetramer. (B) ELISPOT assay. On day 7, cells were transferred to an ELISPOT plate and restimulated (Restim) overnight with specific antigen (10 μg/ml MP) or left without restimulation (as controls), and antigen-specific IFN-γ–producing cells were quantified using an ELISPOT. SEM for all measurements is <30%. (C) CTL assay. MP-specific lysis was measured using MP-pulsed T2 targets. Data shown are after subtracting lysis with control unpulsed T2 targets and from cells after expansion using unpulsed DCs.

Figure 2

(A–C) T cell recall assays in culture after DC immunization. Pre- and postimmunization specimens were thawed and cocultured with MP-pulsed DCs (unpulsed DCs as controls) for 7 d. After 7-d culture, MP-specific T cells were quantified by MHC tetramers (A), ELISPOT (B), and CTL assay (C). (A) MHC tetramer assay. Data are expressed as percent CD8⁺ T cells binding A*0201–MP tetramer. (B) ELISPOT assay. On day 7, cells were transferred to an ELISPOT plate and restimulated (Restim) overnight with specific antigen (10 μg/ml MP) or left without restimulation (as controls), and antigen-specific IFN-γ–producing cells were quantified using an ELISPOT. SEM for all measurements is <30%. (C) CTL assay. MP-specific lysis was measured using MP-pulsed T2 targets. Data shown are after subtracting lysis with control unpulsed T2 targets and from cells after expansion using unpulsed DCs.

Figure 3

Priming of KLH-specific T cells in vivo. (A) Antigen-dependent proliferation. Pre- and postimmunization PBMCs were thawed together and cultured in the absence or presence of KLH (10 μg/ml). Data shown are KLH-specific proliferation after subtracting [³H]TdR incorporation in control wells. SEM for all measurements is <30%. (B) KLH-specific IFN-γ– and IL-4–producing cells from before and after DC immunization were quantified in freshly isolated uncultured PBMCs using an ELISPOT assay. KLH-specific SFCs were calculated after subtracting data from control wells without antigen.

Figure 3

Priming of KLH-specific T cells in vivo. (A) Antigen-dependent proliferation. Pre- and postimmunization PBMCs were thawed together and cultured in the absence or presence of KLH (10 μg/ml). Data shown are KLH-specific proliferation after subtracting [³H]TdR incorporation in control wells. SEM for all measurements is <30%. (B) KLH-specific IFN-γ– and IL-4–producing cells from before and after DC immunization were quantified in freshly isolated uncultured PBMCs using an ELISPOT assay. KLH-specific SFCs were calculated after subtracting data from control wells without antigen.