Preclinical evaluation of NF-κB-triggered dendritic cells expressing the viral oncogenic driver of Merkel cell carcinoma for therapeutic vaccination

Abstract

Background: Merkel cell carcinoma (MCC) is a rare but very aggressive skin tumor that develops after integration of a truncated form of the large T-antigen (truncLT) of the Merkel cell polyomavirus (MCV) into the host's genome. Therapeutic vaccination with dendritic cells (DCs) loaded with tumor antigens is an active form of immunotherapy, which intends to direct the immune system towards tumors which express the respective vaccination antigens.

Methods: Cytokine-matured monocyte-derived DCs of healthy donors and MCC patients were electroporated with mRNA encoding the truncLT. To permit major histocompatibility complex (MHC) class II next to class I presentation, we used an RNA construct in which the antigen was fused to a DCLamp sequence in addition to the unmodified antigen. To further improve their immunogenicity, the DCs were additionally activated by co-transfection with the constitutively active nuclear factor (NF)-κB activator caIKK. These DCs were used to stimulate autologous CD8+ T-cells or a mixture of CD4+ and CD8+ T-cells. Then the percentage of T-cells, specific for the truncLT, was quantified by interferon (IFN)γ ELISpot assays.

Results: Both the truncLT and its DCLamp-fusion were detected within the DCs by flow cytometry, albeit the latter required blocking of the proteasome. The transfection with caIKK upregulated maturation markers and induced cytokine production. After 2-3 rounds of stimulation, the T-cells from 11 out of 13 healthy donors recognized the antigen. DCs without caIKK appeared in comparison less potent in inducing such responses. When using cells derived from MCC patients, we could induce responses for 3 out of 5 patients; however, here the caIKK-transfected DCs did not display their superiority.

Conclusion: These results show that optimized DCs are able to induce MCV-antigen-specific T-cell responses. Therapeutic vaccination with such transfected DCs could direct the immune system against MCC.

Keywords: Merkel cell carcinoma; adoptive cellular immunotherapy; dendritic cells; large T-antigen; polyomavirus.

Conflict of interest statement

Conflict of interest statement: The authors declare the following potential conflict of interest: REV, GS, NS, and JD are named as inventors on a patent on caIKK-RNA-electroporated DCs (WO/2012/055551), which is held by the University of Erlangen-Nuremberg, Erlangen, Germany.

Figures

Figure 1.

Introduction of trLT constructs into cmDCs. (a) Schematic view of the antigen mRNA constructs. The truncLT (encoding amino acid 1–259) (MCV-LT 1–259) was fused C-terminally to a myc tag (Myc) for antibody detection. Additionally, the MCV-LT 1–246 was modified for MHC class II-restricted presentation by adding an N-terminal signaling peptide from Lamp1 (SIG) and C-terminally a sequence encoding human DCLamp. (b and c) cmDCs were electroporated without RNA, with trLT RNA (trLT), or with trLT-DCLamp-RNA (trLT-DCL). The indicated quantities of RNA were used per 100 µl of cell suspension. After electroporation, the proteasome inhibitor, bortezomib, was added to achieve a better detection of the electroporated proteins. Intracellular staining was performed 4 h after electroporation with an anti-myc-tag antibody. The geometric MFI and the percentage of positive cells were measured by flow cytometry. The average of 3 independent donors ± SEM is indicated (b). One representative histogram out of 3 independent donors, showing cmDCs, electroporated with 15 µg of RNA coding for the 2 different truncLT-constructs, is depicted (c). cmDC, cytokine-matured dendritic cell; MHC, major histocompatibility complex; MFI, mean fluorescence intensity; SEM, standard error of the mean; truncLT, truncated form of the large T-antigen.

Figure 2.

Electroporation of cmDCs with caIKK-RNA upregulates surface markers and induces cytokine production. cmDCs were electroporated without (no) or with 30 µg RNA/100 µl encoding caIKK (caIKK). (a) The expression kinetics of the indicated surface markers were determined 24 h (black bars), 48 h (dark grey bars), and 72 h (light grey bars) after electroporation by flow cytometry. The bars indicate the fold induction calculated towards the 24 h control condition of 9 independent donors. The error bars indicate the SEM. (b) The concentrations of IL-8, TNF, IL-6, IL-12p70, and IL-10 in the supernatants of the cells were determined 24 h after electroporation by a CBA. The bars indicate the mean values of 10 independent experiments. The error bars indicate the SEM. CBA, Cytometric Bead Array; cmDC, cytokine-matured dendritic cell; IL, interleukin; SEM, standard error of the mean; TNF, tumor necrosis factor.

Figure 3.

Detection of antigen-specific T-cell responses to the truncLT in healthy donors. cmDCs from healthy donors were electroporated with 30 µg RNA/100 µl cell suspension coding for caIKK alone (no antigen), or in combination with the indicated amounts of truncLT-RNA, either containing a DCLamp sequence (trLT-DCL) or not (trLT). At 4 h after electroporation these DCs were used to stimulate (stim) autologous CD8+ T-cells (a) or a 1:1 mixture out of CD4+ and CD8+ T-cells (b) for 1 week. These T-cells were restimulated twice with the same electroporated DCs, which had been cryoconserved. After the second and third round of stimulation the T-cells were examined for their reactivity towards the trLT. To provide the antigen in the readout, the T-cells were electroporated with RNA encoding the truncLT, without (trLT) or with DCLamp (trLT-DCL). As controls, the T-cells were mock-electroporated (mock) or electroporated with a control-antigen construct, also containing the DCLamp-encoding sequence (control-DCL). The electroporated T-cells were incubated overnight in an IFNγ ELISpot assay and the number of spot-forming units (sfu) per 500,000 cells, used in the assay, was determined. The data from 6–9 independent experiments are indicated; the different symbols represent different healthy donors. cmDC, cytokine-matured dendritic cell; DC, dendritic cell; IFN, interferon; truncLT, truncated form of the large T-antigen.

Figure 4.

Comparison of caIKK-optimized cmDCs with conventional cmDCs. cmDCs from healthy donors were electroporated with 15 µg/100 µl cell suspension truncLT-DCLamp (trLT-DCL) RNA alone or in combination with 30 µg of caIKK-RNA (caIKK). At 4 h after electroporation, these DCs were used to stimulate (stim) autologous CD8+ T-cells (a) or a 1:1 mixture out of CD4+ and CD8+ T-cells (b) for 1 week. These T-cells were restimulated twice with the same electroporated DCs, which had been cryoconserved. After the third round of stimulation the T-cells were examined for their reactivity towards the trLT. To provide the antigen in the readout, the T-cells were electroporated with RNA encoding the truncLT, without (trLT) or with DCLamp (trLT-DCL). As controls, the T-cells were mock-electroporated (mock) or electroporated with a control-antigen construct, also containing the DCLamp-encoding sequence (control-DCL). The electroporated T-cells were incubated overnight in an IFNγ ELISpot assay. The numbers of spots per 500,000 cells used in the assay are indicated. The data from 3 independent experiments are indicated; the different symbols represent the different experiments. cmDC, cytokine-matured dendritic cell; DC, dendritic cell; IFN, interferon; truncLT, truncated form of the large T-antigen.

Figure 5.

Responses against the trLT in patients. cmDCs were generated out of whole blood from patients diagnosed with MCC with GM-CSF and IL-4 and matured with the standard maturation cocktail. Afterwards the cells were electroporated with caIKK-RNA or trLT-DCL-RNA or with a combination of both. At 4 h after electroporation these DCs were used to stimulate autologous CD8+ T-cells (blue bars) or a 1:1 mixture out of CD4+ and CD8+ T-cells (red bars) for 1 week (depending on the material available). (a) These T-cells were stimulated for a second week with the same electroporated DCs which had been cryoconserved. Patient #1 is representative for patients #1 and #2 where no response was detected after 2 weekly stimulations. Patient #3 showed a weak response and patient #4 a strong response after 2 weekly stimulations. (b) For patient #5, enough cells were generated to perform a third stimulation (shown as open bars). For readout, the T-cells were electroporated with RNA encoding the truncLT, without (trLT) or with DCLamp (trLT-DCL). As controls, the T-cells were mock-electroporated (mock) or electroporated with a control-antigen construct, also containing the DCLamp-encoding sequence (control-DCL). The electroporated T-cells were incubated overnight in an IFNγ ELISpot assay. The numbers of spots per 500,000 cells used in the assay are indicated. Data from 4 different patients are shown; each panel represents one patient. cmDC, cytokine-matured dendritic cell; DC, dendritic cell; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; MCC, Merkel cell carcinoma; truncLT, truncated form of the large T-antigen.