Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells

Rodrı́guez, Ana; Régnault, Armelle; Kleijmeer, Monique J.; Ricciardi‐Castagnoli, Paola; Amigorena, Sebastián

doi:10.1038/14058

Cited by 497 publications

(390 citation statements)

References 33 publications

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“…It is noteworthy that proper expression and presentation of the encoded antigen requires the escape of RNA from the macropinosomes. Even though endosomal compartments appear to be leaky or possess channels or transporters facilitating the egression and cross presentation of antigens from endosomes [26][27][28][29][30] the penetration mechanisms of RNA through the endocytic membrane remain to be investigated.…”

Section: Discussionmentioning

confidence: 99%

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

et al. 2011

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Even though it is known for more than one decade that antigen-encoding RNA can deliver antigenic information to induce antigen-specific immunity against cancer, the nature and mechanism of RNA uptake have remained enigmatic. In this study, we investigated the pharmacokinetics of naked RNA administered into the lymph node. We observed that RNA is rapidly and selectively uptaken by lymph node dendritic cells (DCs). Furthermore, in vitro and in vivo studies revealed that the efficient internalization of RNA by human and murine DCs is primarily driven by macropinocytosis. Selective inhibition of macropinocytosis by compounds or as a consequence of DC maturation abrogated RNA internalization and delivery of encoded antigens. Our findings imply that bioavailability of recombinant RNA vaccines in vivo highly depends on the density and the maturation stage of DCs at the administration site and are of importance for the design of RNA-based clinical immunotherapy protocols. Gene Therapy (2011) Keywords: vaccination; dendritic cells; RNA; macropinocytosis INTRODUCTION Antigen-encoding RNA has emerged as an attractive new vaccine format. Major advantages of RNA are efficient delivery of the complete antigenic information, transient expression and lack of genome integration, as well as cost-efficient and easy production in large amounts and high purity. 1 Moreover, through activation of the immune system via TLR3, TLR7 and TLR8, 2,3 RNA supports the induction of immune responses against the encoded protein.Of several in vitro and in vivo strategies utilizing antigen-encoding RNA as a vaccine format, 4 two are in clinical stage. One is based on adoptive transfer of autologous dendritic cells (DCs) transfected ex vivo with antigen-encoding RNA. [5][6][7] The other employs direct injection of naked RNA. 8,9 Feasibility and safety of vaccination protocols based on intradermal administration of naked RNA have been shown in preclinical and clinical settings and partial protection from tumor challenge has been achieved in mice. 10,11 However, at present, neither in animals nor in men, systemic immune responses let alone therapeutic effects were unequivocally demonstrated. It is assumed that rapid degradation by ubiquitous RNAses constraining pharmacologically efficient dosing of unprotected RNA contributes to the still suboptimal clinical effects. Knowledge about the nature and pharmacokinetics of RNA uptake in vitro and in vivo as well as the cell-type specificity is still scarce. Recently, we discovered that the administration route of naked RNA has a crucial impact on its efficacy as a vaccine. By injecting RNA directly into lymph nodes of mice, we achieved for the first time strong systemic antigen-specific Th1 type immunity and cancer cure in preclinical animal models. 12

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Section: Discussionmentioning

confidence: 99%

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

et al. 2011

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“…This process is markedly enhanced when the antigens are internalized by phago- Spontaneous release in the absence of effector cells was <10% of maximum release by detergent. All data points represent the average of two independent experiments, which were performed with 3-5 mice per group cytosis (Shen et al, 1997), macropinocytosis (Norbury et al, 1997), or receptor-mediated endocytosis (Rodriguez et al, 1999;Machy et al, 2000). In some cases, peptides derived from the exogenous antigens appear to be generated in the endocytic compartments and then bind to MHC class I molecules on the cell surface (Zitvogel et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens

et al. 2001

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Summary Dendritic cells (DCs) loaded with tumour antigens have been successfully used to induce protective tumour immunity in murine models and human trials. However, it is still unclear which DC administration route elicits a superior therapeutic effect. Herein, we investigated the vaccine efficiency of DC2.4 cells, a murine dendritic cell line, pulsed with ovalbumin (OVA) in the murine E.G7-OVA tumour model after immunization via various routes. After a single vaccination using 1 × 10 6 OVA-pulsed DC2.4 cells, tumour was completely rejected in the intradermally (i.d.; three of four mice), subcutaneously (s.c.; three of four mice), and intraperitoneally (i.p.; one of four mice) immunized groups. Double vaccinations enhanced the anti-tumour effect in all groups except the intravenous (i.v.) group, which failed to achieve complete rejection. The anti-tumour efficacy of each immunization route was correlated with the OVA-specific cytotoxic T lymphocyte (CTL) activity evaluated on day 7 post-vaccination. Furthermore, the accumulation of DC2.4 cells in the regional lymph nodes was detected only in the i.d.-and s.c.-injected groups. These results demonstrate that the administration route of antigen-loaded DCs affects the migration of DCs to lymphoid tissues and the magnitude of antigen-specific CTL response. Furthermore, the immunization route affects vaccine efficiency.

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“…This is reminiscent of the way exogenous antigens internalized by receptor-mediated endocytosis are presented by MHC class I molecules 60 in macrophages 61,62 and in dendritic cells. 63 This type of antigen processing/ presentation requires that the endocytosed antigen be released from the endosome/lysosome compartment into the cytosol for processing by the proteasome, implying the existence of a mechanism for facilitated transmembrane transport from the lumen of endosomes into the cytosol. 64 By analogy, in B lymphocytes the internalized DNA also diffuses out of the late endosomes free to reach the nucleus and undergo transcription.…”

Section: Transgenesis Of Human B Lymphocytes G Filaci Et Almentioning

confidence: 99%

Spontaneous transgenesis of human B lymphocytes

et al. 2003

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DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional immunoglobulin (Ig) heavy chain gene under the control of a B-cell-specific promoter. Using polymerase chain reaction (PCR), reverse transcriptase-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, Epstein-Barr virus-transformed cell lines, and peripheral blood B lymphocytes of the mature naïve phenotype (IgM þ /IgD þ /CD27 À ). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells processed and presented to major histocompatibility complex (MHC)-restricted T lymphocytes a peptide expressed in the transgenic product. This is the first demonstration that primary B lymphocytes possess a program for the spontaneous internalization of DNA, which in turn imparts the cell with new immunological functions. As spontaneous transgenesis is obtained using a nonviral vector, does not require prior cell activation, and is not associated with chromosomal integration, the findings reported here open new possibilities for genetic manipulations of mature naïve B lymphocytes for therapy and vaccination.

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Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells

Cited by 497 publications

References 33 publications

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens

Spontaneous transgenesis of human B lymphocytes

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