Controlling T‑Cell Activation with Synthetic Dendritic Cells Using the Multivalency Effect

Artificial antigen-presenting cells (aAPCs) have recently gained a lot of attention. They efficiently activate T cells and serve as powerful replacements for dendritic cells in cancer immunotherapy. Focusing on a specific class of polymer-based aAPCs, so-called synthetic dendritic cells (sDCs), we have investigated the importance of multivalent binding on T-cell activation. Using antibody-functionalized sDCs, we have tested the influence of polymer length and antibody density. Increasing the multivalent character of the antibody-functionalized polymer lowered the effective concentration required for T-cell activation. This was evidenced for both early and late stages of activation. The most important effect observed was the significantly prolonged activation of the stimulated T cells, indicating that multivalent sDCs sustain T-cell signaling. Our results highlight the importance of multivalency for the design of aAPCs and will ultimately allow for better mimics of natural dendritic cells that can be used as vaccines in cancer treatment

Phenotypic characteristics of CD56⁺ IL-15 DCs.

<p>(A) CD14⁺ monocytes were cultured for 24–36 hr in the presence of GM-CSF and IL-15 (IL-15 DCs) and analyzed by flow cytometry for expression of CD11c/CD56 (left). The percentage between parentheses indicates the mean (± SEM) percentage of CD56⁺ cells among the total IL-15 DC population (<i>n</i> = 17). These CD56⁺ cells were then immunomagnetically separated, cultured for another 16–20 hr in the presence of DC maturation cocktail and analyzed for co-expression of CD11c/BDCA-1 (middle) and CD56/CD7 (right). Quadrant gates were set using corresponding isotype controls. (B) Matured CD56⁺ IL-15 DCs were further analyzed by flow cytometry for expression of the indicated NK cell-associated (CD56, CD7, CD16, CD69, NKG2D, NKp46), NKDC-associated (CD11c, B220, NKR-P1A) and DC-related surface antigens. Histogram overlays show expression of the indicated markers (solid line histograms) compared to their respective isotype controls (filled grey histograms). All plots are representative of at least 4 independent experiments.</p

Expression of lytic molecules by IL-15 DCs.

<p>(A) Matured CD56⁺ IL-15 DCs were analyzed by flow cytometry for cell surface expression of TNF-α, FasL and TRAIL (solid line histograms). Filled grey histograms represent isotype controls. Data are from one experiment representative of three. (B) Both CD56⁺ (solid line) and CD56⁻ (dashed line) IL-15 DCs were stained for intracellular expression of TRAIL, granzyme B and perforin. Filled grey histograms represent isotype controls. One representative experiment out of 6 (for TRAIL) and 9 (for granzyme B and perforin) is shown. (C) Supernatants from overnight washout cultures of CD56⁺ (▪) and CD56⁻ (□) IL-15 DCs were analyzed for granzyme B release by ELISA. Bars represent mean (± SEM) granzyme B concentations (pg/mL) from 7 experiments.*, <i>P</i><0.05.</p

Lysis of K562 cells but not of a WT1-specific CTL clone by IL-15 DCs.

<p>PKH67-labeled target cells were mixed at varying E:T ratios with mature CD56⁺ and CD56⁻ IL-15 DCs or, where indicated, with conventionally generated IL-4 DCs and then subjected to PI/Annexin-V staining after overnight incubation. Target cell viability was defined as the percentage of PI⁻/Annexin-V⁻ cells within the PKH67⁺CD11c⁻ gate. (A) Viability profiles of gated K562 tumor cells cultured alone (control) or with either CD56⁻ or CD56⁺ IL-15 DCs at an E:T ratio of 50:1. One representative experiment out of five is shown. Percentages of viable K562 cells are displayed in the lower left quadrants and expressed as mean (± SEM) of 5 independent experiments. (B) Graph depicting the specific lysis of K562 tumor cells by CD56⁺ IL-15 DCs (solid black line, ▪; <i>n</i> = 5), CD56⁻ IL-15 DCs (solid grey line, □; <i>n</i> = 5) and IL-4 DCs (dashed grey line, ○; <i>n</i> = 3) at the indicated E:T ratios. Results are expressed as mean (± SEM) percentages of specific lysis. Asterisks refer to a statistically significant difference in cytotoxic activity at the indicated E:T ratio between CD56⁺ and CD56⁻ IL-15 DCs. (C) Bar graphs showing the viability of a WT1_126–134-specific CTL clone after overnight culture in the absence or presence of either CD56⁻ (□) or CD56⁺ (▪) IL-15 DCs at an E:T ratio of 50:1. Data are presented as mean (± SEM) percentages of viable T cells from three experiments.</p

Allostimulatory capacity of CD56⁺ and CD56⁻ IL-15 DCs in a MLR.

<p>CD56⁺ and CD56⁻ IL-15 DCs were co-cultured with allogeneic, CFSE-labeled lymphocytes at a 1∶10 ratio for 5 days. CFSE-labeled cells stimulated with IL-4 DCs (1∶10 stimulator/responder ratio) or PHA/IL-2 were used as positive controls. Histograms show the degree of CFSE dilution, indicative of T cell proliferation, among gated CD3⁺CD4⁺ T cells in the absence (filled grey histograms) and presence (solid line histograms) of the indicated stimulators. Numbers above the bracketed lines indicate the background-subtracted percentages of proliferated (i.e. CFSE^low) T cells within the CD3⁺CD4⁺ gate. Data shown are representative of three donors.</p

Differential ability of CD56⁺ and CD56⁻ IL-15 DCs to stimulate a WT1-specific CTL clone.

<p>Mature CD56⁺ and CD56⁻ IL-15 DCs were electroporated (EP) with <i>WT1</i> RNA and co-cultured with an HLA-A*0201-restricted WT1_126–134-specific CTL clone. Negative controls included: T cells cultured without DCs (T cells only), <i>WT1</i> RNA-electroporated DCs cultured without T cells (DCs only), and T cells cultured with non-antigen-loaded DCs (non/mock EP). (A) After overnight incubation, IFN-γ concentrations (pg/mL) in the culture supernatants were measured by ELISA. Bars represent mean (± SEM) IFN-γ concentrations of triplicate wells of three independent experiments (**, <i>P</i> = 0.003). (B) Antigenic responses were quantified in parallel by IFN-γ ELISpot. Illustrative single-well images from an IFN-γ ELISpot plate of one representative experiment are shown. Values in parentheses indicate the mean (± SEM) number of IFN-γ spot-forming cells (SFCs) of triplicate ELISpot wells.</p

Inhibition of CD56⁺ IL-15 DC-mediated cytotoxicity by neutralizing anti-TRAIL mAbs and concanamycin A.

<p>Matured CD56⁺ IL-15 DCs were co-cultured with PKH67-labeled K562 target cells at an E:T ratio of 50:1 in the presence of either anti-TRAIL blocking mAb (left) or the granule exocytosis inhibitor concanamycin A (right). Parallel experiments were performed using TRAIL isotype-matched control mAb and medium control devoid of concanamycin A, respectively. Lysis of target cells was determined after overnight incubation using a flow cytometry-based cytotoxicity assay, as described above. Results are expressed as mean (± SEM) percentages of specific target cell lysis. Data are from 5 (for TRAIL) and 10 (for concanamycin) independent experiments. *, <i>P</i><0.05; **, <i>P</i><0.01.</p