Day-4 Myeloid Dendritic Cells Pulsed with Whole Tumor Lysate Are Highly Immunogenic and Elicit Potent Anti-Tumor Responses

“Day-7” myeloid DCs are commonly used in the clinic. However, there is a strong need to develop DCs faster that have the same potent immunostimulatory capacity as “Day-7” myeloid DCs and at the same time minimizing time, labor and cost of DC preparations. Although “2 days” DCs can elicit peptide-specific responses, they have not been demonstrated to engulf, process and present complex whole tumor lysates, which could be more convenient and personalized source of tumor antigens than defined peptides. In this preclinical study, we evaluated the T-cell stimulatory capacity of Day-2, Day-4, and Day-7 cultured monocyte-derived DCs loaded with SKOV3 cell whole lysate prepared by freeze-thaw or by UVB-irradiation followed by freeze-thaw, and matured with lipopolysaccharide (LPS) and interferon (IFN)-gamma. DCs were evaluated for antigen uptake, and following maturation with LPS and IFN-gamma, DCs were assessed for expression of CD80, CD40, CD86, ICAM-1 and CCR7, production of IL-12p70 and IP-10, and induction of tumor-specific T-cell responses. Day-4 and Day-7 DCs exhibited similar phagocytic abilities, which were superior to Day-2 DCs. Mature Day-7 DCs expressed the highest CD40 and ICAM-1, but mature Day-4 DCs produced the most IL-12p70 and IP-10. Importantly, Day-4 and Day-7 DCs derived from ovarian cancer patients stimulated equally strongly tumor-specific T-cell responses. This is the first study demonstrating the highly immunogenic and strong T-cell stimulatory properties of Day-4 myeloid DCs, and provided important preclinical data for rapid development of potent whole tumor lysate-loaded DC vaccines that are applicable to many tumor types

Day-4 DCs, but not Day-2 DCs, were as phagocytic as Day-7 DCs in actively engulfing ovarian tumor lysate.

<p>(A) Day-2, Day-4, and Day-7 DCs were cocultured with UVB lysate (UVBL) or freeze-thaw lysate (FTL) of PKH26-labeled SKOV3 cells at 37°C for 4 h or 24 h to determine active phagocytosis of tumor lysates. Representative dot plot results from 1 out of 6 donors are shown here. DCs that had engulfed PKH26-labeled tumor lysate appear as the HLA-DR⁺ PKH26⁺ double-positive population, and are expressed as the percentage of the total number of HLA-DR⁺ DCs as indicated in the upper-right quadrant of the dot plots. (B) DCs were cocultured with lysate at 4°C for 4 h or 24 h as controls to determine passive transfer of the PKH26 dye to DCs. Representative dot plot results from 1 out of 6 donors are shown here. HLA-DR⁺ PKH26⁺ double-positive DCs are expressed as the percentage of the total number of HLA-DR⁺ DCs as indicated in the upper-right quadrant of the dot plots. (C) Summary results of the percentages of Day-2, Day-4, and Day-7 DCs that have engulfed PKH26-labeled UVBL or FTL after 4 h or 24 h, at both 4°C and 37°C. Data displayed are the means ± standard errors of six independent experiments. There are no significant differences among Day-2, Day-4 and Day-7 DCs for the precentage of DCs taking up UVBL (ANOVA <i>P</i> value = 0.13) or FTL (ANOVA <i>P</i> value = 0.59) at 4 h. However, there is a significant difference for % of DCs taking up UVBL at 24 h (ANOVA <i>P</i> value = 0.02). By <i>post hoc</i> paired testing, % of Day-2 DCs taking up UVBL was significantly lower than either Day-7 or Day-4 DCs (*<i>P</i> values = 0.05 and 0.02, respectively). Highly significant differences were also observed for the uptake of FTL at 24 h (ANOVA**<i>P</i> value<0.001). The % of Day-2 DCs taking up FTL was significantly lower than either Day-7 or Day-4 DCs (**<i>P</i> values<0.001 for each). The differences between Day-4 and Day-7 DCs were insignificant for uptake of both UVBL (<i>P</i> value = 0.90) and FTL (<i>P</i> value = 0.92).</p

Day-4 and Day-7 DCs, but not Day-2 DCs acquired phenotypic features of differentiated DCs.

<p>Elutriated monocytes derived from normal healthy donors were cultured for 2, 4, or 7 days in serum-free AIM-V media supplemented with recombinant GM-CSF and IL-4, and analyzed by flow cytometry at the end of the culture period. (A) Representative histograms from 1 out of 6 donors were shown here. The MFIs indicated in the histograms were derived by subtracting the MFI of the test sample from the isotype control. Day-2 DCs expressed slightly higher CD14, CD68, and CD33 markers than Day-4 and Day-7 DCs, while Day-4 DCs were similar to Day-7 DCs in immunophenotype. (B) The average MFIs of the different DC maturation markers of 6 normal healthy donors were shown here. ** <i>P</i> value = <0.001; highly significant when comparing the MFI of Day-4 or Day-7 DCs to the MFI of Day-2 DCs by Student's unpaired <i>t</i> test.</p

Day-4 DCs derived from EOC patients, stimulated potent and specific autologous ovarian tumor-reactive T cell responses <i>in vitro</i>.

<p>Patients' T cells were cocultured with autologous Day-2, Day-4 or Day-7 mature DCs previously pulsed with SKOV3 UVBL or FTL for 2 weeks and then interrogated for reactivity against tumor antigens. The IFN-γ responses of patients' T cells previously stimulated with UVBL-pulsed DCs (closed squares or black bars), FTL-pulsed DCs (closed circles or white bars) or unpulsed mature DCs (closed triangles or grey bars) are shown here, (A) For patients 1 and 2 who were HLA-A2⁻, the T cells were tested in the presence of autologous DCs pulsed with SKOV3 FTL, autologous unpulsed DCs, or media (T cells only). (B) For patient 3, who was HLA-A2⁺, the T cells were also tested against live HLA-A2⁺ SKOV3 cells, HLA-A2⁺ T2 pulsed with HER-2/neu 369 or 689 peptides, autologous DCs pulsed with HLA-A2⁻ SKOV3 FTL, autologous unpulsed DCs, or media (T cells only). The results are the means of the number of IFN-γ spots per 10⁶ T cells ± standard error. The asterisks indicate those columns differing significantly (<i>P</i> value = 0.001; Student's paired <i>t</i> test) from the media (i.e. T cells only) controls.</p

Day-4 DCs produce the highest levels of IL-12p70 and IP-10.

<p>Day-2, Day-4, and Day-7 DCs were first pulsed with either UVBL or FTL for 16 h, and then stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) to determine (A) IL-12p70 and, (B) IP-10 production. Supernatants from the DC-tumor lysate cocultures were collected and evaluated by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028732#s2" target="_blank">Materials and Methods</a>. The results are from 4 different normal healthy donors and are expressed as mean (pg/ml) ± standard errors.</p

Day-2, Day-4 and Day-7 DCs pulsed with UVBL or FTL mature normally following LPS and IFN-γ stimulation.

<p>Following lysate loading, DCs were stimulated with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) for 16 h. DCs were identified by HLA-DR, CD11c and one of the markers shown. Unpulsed immature DCs (iDCs) and mature (mDCs) were set up in parallel and harvested at the same time as the other tumor lysate-loaded mature DCs for analysis. (A–C) Upregulation of maturation markers CD80, CD40, CD86, ICAM-1, and CCR7 is observed on mature Day-2, Day-4 and Day-7 DCs. The open histograms represent the isotype control, while shaded histograms represent the DC markers. The solid line marks the MFI of the different markers on iDCs for each condition. Representative histogram results from 1 out of 6 donors are shown here. (D) Fold-increase in expression levels of maturation and adhesion markers on mDCs, UVBL-loaded DCs (UVBL-DC) or FTL-loaded DCs (FTL-DC) over iDC was determined by expressing the MFIs as a ratio of the DCs to unpulsed iDCs. Highly significant differences were detected in CD86 (**<i>P</i> value = 0.002), ICAM-1 (**<i>P</i> value = <0.001), and CCR7 (**<i>P</i> value = 0.002, <0.001 and 0.008, respectively; ANOVA followed by <i>post-hoc</i> testing) on unpulsed matured Day-4 DCs compared to unpulsed matured Day-7 DCs. Similar highly significant differences were detected in CD86 and CCR7 (**<i>P</i> value = 0.001, and 0.01, respectively; ANOVA followed by <i>post-hoc</i> testing) on Day-4 UVBL-DCs compared to Day-7 UVBL-DCs. CD86 and ICAM-1 (**<i>P</i> value = 0.001, and 0.01, respectively; ANOVA followed by <i>post-hoc</i> testing) were also significantly different on Day-4 FTL-DCs compared to Day-7 FTL-DCs. (E) The overall immunophenotypes of DCs loaded with UVBL or FTL were similar to unpulsed mature DCs, however Day-4 DCs loaded with UVBL consistently expressed slightly higher levels of most markers including CD80, CD86 and CCR7 relative to any other lysate-DC preparations.</p

Day-4 DCs derived from normal healthy HLA-A2⁺ donors pulsed with UVBL or FTL and matured with LPS and IFN-γ, stimulate potent and specific autologous ovarian tumor-reactive T cell responses <i>in vitro</i>.

<p>Day-2, Day-4, and Day-7 DCs were pulsed with lysate for 16 h and matured with LPS (60 EU/ml) and IFN-γ (2000 IU/ml) for a further 16 h before being used for priming T cells. After 2 weeks of <i>in vitro</i> stimulation, T cells were harvested and evaluated for their ability to recognize ovarian tumor-associated antigens. The IFN-γ responses of T cells previously stimulated with UVBL-pulsed DCs (grey bars), FTL-pulsed DCs (black bars) or unpulsed mature [no antigen] DCs (white bars) are shown here. Data were obtained from 3 different individuals and displayed as the means ± standard errors. Day-2 DCs result in no significant T cell priming, while Day-4 and Day-7 DCs elicit T cells that are able to produce significant amounts of IFN-γ in response to live HLA-A2⁺ SKOV3 cells or to HLA-A2⁺ T2 cells pulsed with HER-2/neu_369–377 (H369) or HER-2/neu_689–697 (H689). Using Kruskal-Wallis test by <i>post-hoc</i> paired testing, Day-2 DCs are significantly lower than either Day-7 or Day-4 DCs (*<i>P</i> values = 0.05 for all tests), and no differences are detected when comparing Day-4 and Day-7 DCs (<i>P</i> values ≥ 0.28). FTL-pulsed DCs perform better than UVBL-pulsed DCs in priming ovarian-specific IFN-γ T cell responses.</p