In situ Expression of Tumor Antigens by Messenger RNA–Electroporated Dendritic Cells in Lymph Nodes of Melanoma Patients

Abstract

Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) for cancer immunotherapy has been proved efficient and clinically safe. It obviates prior knowledge of CTL and Th epitopes in the antigen and leads to the presentation of multiple epitopes for several HLA alleles. Here we studied the migration capacity and the antigen expression of mRNA-electroporated DC (mRNA-DC) in lymph nodes after vaccination in melanoma patients. DC were electroporated with mRNA encoding gp100 or tyrosinase, labeled with indium-111 and superparamagnetic iron oxide particles, and injected intranodally in melanoma patients 24 to 48 hours before scheduled dissection of regional lymph nodes. Immunohistochemical analysis of the lymph nodes after surgery revealed that mRNA-DC migrated from the injection site into the T-cell areas of the same and subsequent lymph nodes, where they expressed the antigen encoded by the electroporated mRNA. Furthermore, vaccine-related CD8 + T-cell responses could be detected in 7 of 11 patients vaccinated with mRNA-DC. Together these data show that mature DC electroporated with mRNA encoding TAA migrate and express antigens in the lymph nodes and induce specific immune responses.

Targeting CD4+ T-Helper Cells Improves the Induction of Antitumor Responses in Dendritic Cell–Based Vaccination

“…These studies support our findings of superior immune responses in patients vaccinated with DCs loaded with tumor antigen in both MHC class I and II. Finally, the current vaccination protocol might be further improved by overcoming the disadvantages of peptide pulsing of DCs, for example, the dissociation of peptides from MHC complexes and the lack of posttranslational modification by the use of mRNA electroporation (50,51). This method has shown to result in mature and migratory DCs expressing the encoded antigens in situ (51).…”

Leukotriene C4 induces migration of human monocyte–derived dendritic cells without loss of immunostimulatory function

“…This is particularly important because DC migration through the afferent lymphatics to the T-cell area of lymph nodes has been shown to require 24 to 48 hours. 46,47 As shown in Figure 6 (right panel), DCs were matured with CD40L and LTC 4 for 2 days, cryopreserved, thawed, and then cultured for another 2 days in the absence of any cytokines or serum. After 2 days of maturation, DCs were CD14 neg and expressed high levels of the maturation markers CD40, CD83, and CD86 (Ͼ 90%).…”

CD8+ T Cell Responses against TAP-Inhibited Cells Are Readily Detected in the Human Population

“…RNA electroporation of MoDCs or B cells with a viral TAP inhibitor is feasible in clinical settings. RNA electroporation has the advantage that the nucleotides are not integrated in the host genome, like viral vectors (33,(49)(50)(51)(52)(53). In our mouse model, TAP-deficient DCs were able to prevent the outgrowth of TAPdeficient tumors (30).…”

“…For each electroporation, a maximum of 1 3 10 7 cells in 100 ml Opti-mem media (Invitrogen) was used. Cells were collected, washed, and taken up in Opti-Mem media and pulsed with a blockpulser (Bio-Rad, Hercules, CA), in a 2-mM cuvette (Bio-Rad), at 300 V, 150 mCF (33). Afterwards, cells were taken up in X-vivo medium (Lonza Group) without phenol red for 15 min at 37˚C, 5% CO 2 , and then incubated overnight in complete RPMI 1640 medium, with 8% heat inactivated FCS, 250 ng/ml LPS, 800 U/ml GM-GSF, and 500 U/ml IL-4.…”