mRNA transfection of DC in the immature or mature state: comparable in vitro priming of Th and cytotoxic T lymphocytes against DC electroporated with tumor cell line-derived mRNA

BACKGROUND: The use of mRNA in vaccine studies has generally been through loading or transfection of immature DC followed by a maturation step. A recent study has suggested that this strategy may result in inferior priming of cytotoxic T lymphocytes (CTL). Furthermore the study did not address any possible effects on the priming of CD4(+) T-cell responses. METHODS: We compared mRNA transfection of mature DC with that of immature DC, using as a read-out their capacity to prime autologous T cells during one cycle of in vitro stimulation. In this model system we used mRNA from the tumor cell line Jurkat E6. DC transfected at either the immature stage (day 5) or mature stage (day 7) displayed a similar phenotype. RESULTS: Interestingly, no major differences in their ability to prime CD4 and CD8 T-cell responses were observed. As in vitro priming to some extent may reflect the capacity of these DC to prime T cells in vivo after vaccination, these studies support the use of mRNA-transfected mature DC in clinical protocols. DISCUSSION: Transfection of DC at the end of the maturation process represents a logistical improvement in the GMP production of mRNA-transfected DC for clinical protocols.     

RNA-containing adenovirus/polyethylenimine transfer complexes effectively transduce dendritic cells and induce antigen-specific T cell responses

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells in initiating primary immune responses. Given the unique properties of DCs, gene-modified DCs represent a particularly attractive approach for immunotherapy of diseases such as cancer. METHODS: Gene-modified DCs were obtained by a receptor-mediated gene delivery system using adenovirus (Ad) particles as ligand and RNA or DNA condensed by polyethylenimine (PEI). In vitro transcribed polyadenylated or non-polyadenylated RNA was used. RNA-transduced DCs were generated expressing chicken ovalbumin (OVA) or chimeric constructs thereof, and compared with DNA-transduced DCs. RESULTS: Ad/PEI transfection complexes efficiently delivered RNA into DCs. Such RNA-transduced DCs induced OVA-specific T cell responses more effectively than DNA-transduced DCs. Furthermore, DCs transduced with polyadenylated RNA were more potent in stimulating CD4(+) and CD8(+) T cell responses than DCs transduced with non-polyadenylated RNA and this was particularly important for CD4(+) T cell responses. CONCLUSIONS: Ad/PEI/RNA transfection is an efficient means for generating RNA-transduced DCs and for stimulating antigen-specific T cell responses. Polyadenylation of RNA enhances CD8(+) T cell responses and is essential for CD4(+) T cell responses.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.     

Mucosally delivered dendritic cells activate T cells independently of IL-12 and endogenous APCs

In vitro manipulated dendritic cells (DC) have increasingly been used as a promising vaccine formulation against cancer and infectious disease. However, improved understanding of the immune mechanisms is needed for the development of safe and efficacious mucosal DC immunization. We have developed a murine model of respiratory mucosal immunization by using a genetically manipulated DC vaccine. Within 24 h of intranasal delivery, the majority of vaccine DCs migrated to the lung mucosa and draining lymph nodes and elicited a significant level of T cells capable of IFN-gamma secretion and CTL in the airway lumen as well as substantial T cell responses in the spleen. And such T cell responses were associated with enhanced protection against respiratory mucosal intracellular bacterial challenge. In comparison, parenteral i.m. DC immunization did not elicit marked airway luminal T cell responses and immune protection regardless of strong systemic T cell activation. Although repeated mucosal DC delivery boosted Ag-specific T cells in the airway lumen, added benefits to CD8 T cell activation and immune protection were not observed. By using MHC-deficient vaccine DCs, we further demonstrated that mucosal DC immunization-mediated CD8 and CD4 T cell activation does not require endogenous DCs. By using IL-12-deficient vaccine DCs, we also observed that IL-12(-/-) DCs failed to migrate to the lymph nodes but remained capable of T cell activation. Our observations indicate that mucosal delivery of vaccine DCs represents an effective approach to enhance mucosal T cell immunity, which may operate independent of vaccine IL-12 and endogenous DCs.     

IL-1 beta enhances CD40 ligand-mediated cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC activation.

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-gamma and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, IL-1beta, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34(+)-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1beta is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia coli or CD40L, whereas neither CD34(+)-derived DC nor peripheral blood DC produce IL-1beta. Finally, DC activated with CD40L and IL-1beta induce higher levels of IFN-gamma secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1beta is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC. The synergy between CD40L and IL-1beta highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses.     

Fusions of dendritic cells with breast carcinoma stimulate the expansion of regulatory T cells while concomitant exposure to IL-12, CpG oligodeoxynucleotides, and anti-CD3/CD28 promotes the expansion of activated tumor reactive cells

Vaccination of patients with dendritic cell (DC)/breast carcinoma fusions stimulated antitumor immune responses in a majority of patients with metastatic disease but only a subset demonstrate evidence of tumor regression. To define the factors that limit vaccine efficacy, we examined the biological characteristics of DC/breast carcinoma fusions as APCs and the nature of the vaccine-mediated T cell response. We demonstrate that fusion of DCs with breast carcinoma cells up-regulates expression of costimulatory and maturation markers and results in high levels of expression of IL-12 consistent with their role as activated APCs. Fusion cells also express the chemokine receptor CCR7, consistent with their ability to migrate to the draining lymph node. However, DC/breast cancer fusions stimulate a mixed T cell response characterized by the expansion of both activated and regulatory T cell populations, the latter of which is characterized by expression of CTLA-4, FOXP3, IL-10, and the suppression of T cell responses. Our results demonstrate that IL-12, IL-18, and TLR 9 agonist CpG oligodeoxynucleotides reduce the level of fusion-mediated regulatory T cell expansion. Our results also demonstrate that sequential stimulation with DC/breast carcinoma fusions and anti-CD3/CD28 results in the marked expansion of activated tumor-specific T cells. These findings suggest that DC/breast carcinoma fusions are effective APCs, but stimulate inhibitory T cells that limit vaccine efficacy. In contrast, exposure to TLR agonists, stimulatory cytokines, and anti-CD3/CD28 enhances vaccine efficacy by limiting the regulatory T cell response and promoting expansion of activated effector cells.     

Inhibition of myeloid dendritic cell accessory cell function and induction of T cell anergy by alcohol correlates with decreased IL-12 production

Alcohol consumption inhibits accessory cell function and Ag-specific T cell responses. Myeloid dendritic cells (DCs) coordinate innate immune responses and T cell activation. In this report, we found that in vivo moderate alcohol intake (0.8 g/kg of body weight) in normal volunteers inhibited DC allostimulatory capacity. Furthermore, in vitro alcohol treatment during DC differentiation significantly reduced allostimulatory activity in a MLR using naive CD4(+) T cells, and inhibited tetanus toxoid Ag presentation by DCs. Alcohol-treated DCs showed reduced IL-12, increased IL-10 production, and a decrease in expression of the costimulatory molecules CD80 and CD86. Addition of exogenous IL-12 and IL-2, but not neutralization of IL-10, during MLR ameliorated the reduced allostimulatory capacity of alcohol-treated DCs. Naive CD4(+) T cells primed with alcohol-treated DCs showed decreased IFN-gamma production that was restored by exogenous IL-12, indicating inhibition of Th1 responses. Furthermore, CD4(+) T cells primed with alcohol-treated DCs were hyporesponsive to subsequent stimulation with the same donor-derived normal DCs, suggesting the ability of alcohol-treated DCs to induce T cell anergy. LPS-induced maturation of alcohol-treated immature DCs partially restored the reduced allostimulatory activity, whereas alcohol given only during DC maturation failed to inhibit DC functions, suggesting that alcohol primarily impairs DC differentiation rather than maturation. NFkappaB activation, a marker of DC maturation was not affected by alcohol. Taken together, alcohol both in vitro and in vivo can impair generation of Th1 immune responses via inhibition of DC differentiation and accessory cell function through mechanisms that involve decreased IL-12 induction.     

On the role of APC-activation for in vitro versus in vivo T cell priming

Professional antigen-presenting cells take up antigens for processing and presentation in association with MHC class I and II molecules. When APCs receive the right stimuli, they undergo a maturation process and migrate to secondary lymphoid organs to trigger T cell activation. In this study, we compared side-by-side in vivo and in vitro activation of T cells. Transgenic CD8(+) T cells specific for the p33 epitope, derived from the lymphocytic choriomeningitis virus glycoprotein, were labeled with CFSE and injected into syngeneic mice or alternatively, co-cultured in vitro with APCs. The p33 epitope was delivered as free peptide or genetically fused to virus-like particles. Whereas proliferation of specific T cells was comparable in both systems, the production of IFN-gamma and the expression of CD25 showed important differences. Induction of effector function and expression of activation markers were strongly enhanced in vitro by both the free peptide and VLPs. Surprisingly, addition of CpG-containing immune-stimulating DNA for activation of APCs dramatically increased effector T cell differentiation in vitro, whereas no enhancement could be observed in vitro. Thus, activation of professional APCs was mandatory for induction of effector CD8(+) T cell responses in vivo, while this step was largely dispensable in vitro.     

Escherichia coli expressing recombinant antigen and listeriolysin O stimulate class I-restricted CD8+ T cells following uptake by human APC

Vaccination against cancer or intracellular pathogens requires stimulation of class I-restricted CD8(+) T cells. It is therefore important to develop Ag delivery vectors that will promote cross-presentation by APCs and stimulate appropriate inflammatory responses. Toward this goal, we tested the potential of Escherichia coli as an Ag delivery vector in in vitro human culture. Bacteria expressing enhanced green fluorescent protein were internalized efficiently by dendritic cells, as shown by flow cytometry and fluorescence microscopy. Phenotypic changes in DC were observed, including up-regulation of costimulatory molecules and IL-12p40 production. We tested whether bacteria expressing recombinant Ags could stimulate human T cells using the influenza matrix protein as a model Ag. Specific responses against an immunodominant epitope were seen using IFN-gamma ELISPOT assays when the matrix protein was coexpressed with listeriolysin O, but not when expressed alone. THP-1 macrophages were also capable of stimulating T cells after uptake of bacteria, but showed slower kinetics and lower overall levels of T cell stimulation than dendritic cells. Increased phagocytosis of bacteria induced by differentiation of THP-1 increased their ability to stimulate T cells, as did opsonization. Presentation was blocked by proteasome inhibitors, but not by lysosomal protease inhibitors leupeptin and E64. These results demonstrate that recombinant E. coli can be engineered to direct Ags to the cytosol of human phagocytic APCs, and suggest possible vaccine strategies for generating CD8(+) T cell responses against pathogens or tumors.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.     

Myeloid and plasmacytoid dendritic cells are susceptible to recombinant adenovirus vectors and stimulate polyfunctional memory T cell responses

Although replication-incompetent recombinant adenovirus (rAd) type 5 is a potent vaccine vector for stimulating T and B cell responses, high seroprevalence of adenovirus type 5 (Ad5) within human populations may limit its clinical utility. Therefore, alternative adenovirus serotypes have been studied as vaccine vectors. In this study, we characterized the ability of rAd5 and rAd35 to infect and induce maturation of human CD11c(+) myeloid dendritic cells (MDCs) and CD123(+) plasmacytoid dendritic cells (PDCs), and their ability to stimulate Ag-specific T cells. Both MDCs and PDCs were found to express the primary receptor for Ad35 (CD46) but not Ad5 (coxsackie-adenovirus receptor; CAR). Both dendritic cell (DC) subsets were also more susceptible to rAd35 than to rAd5. MDCs were more susceptible to both rAd35 and rAd5 than were PDCs. Whereas rAd35 used CD46 for entry into DCs, entry of rAd5 may be through a CAR-independent pathway. Exposure to rAd35 but not rAd5 induced high levels of IFN-alpha in PDCs and phenotypic differentiation in both DC subsets. MDCs and PDCs exposed to either rAd5 or rAd35 encoding for CMV pp65 were able to present pp65 and activate CMV-specific memory CD8(+) and CD4(+) T cells in a dose-dependent manner, but MDCs stimulated the highest frequencies of pp65-specific T cells. Responding T cells expressed multiple functions including degranulation (CD107a surface mobilization) and production of IFN-gamma, IL-2, TNF-alpha, and MIP-1beta. Thus, the ability of rAd35 to naturally target important DC subsets, induce their maturation, and appropriately present Ag to T cells may herald greater in vivo immunogenicity than has been observed with rAd5.     

Partial activation of neonatal CD11c+ dendritic cells and induction of adult-like CD8+ cytotoxic T cell responses by synthetic microspheres

Neonatal cytotoxic T cell responses have only been elicited to date with immunogens or delivery systems inducing potent direct APC activation. To define the minimal activation requirements for the induction of neonatal CD8(+) cytotoxic responses, we used synthetic microspheres (MS) coated with a single CD8(+) T cell peptide from lymphocytic choriomeningitis virus (LCMV) or HIV-1. Unexpectedly, a single injection of peptide-conjugated MS without added adjuvant induced CD4-dependent Ag-specific neonatal murine cytotoxic responses with adult-like CTL precursor frequency, avidity for Ag, and frequency of IFN-gamma-secreting CD8(+) splenocytes. Neonatal CD8(+) T cell responses to MS-LCMV were elicited within 2 wk of a single immunization and, upon challenge, provided similar protection from viral replication as adult CTLs, demonstrating their in vivo competence. As previously reported, peptide-coated MS elicited no detectable activation of adult CD11c(+) dendritic cells (DC). In contrast, CTL responses were associated with a partial activation of neonatal CD11c(+) DC, reflected by the up-regulation of CD80 and CD86 expression but no concurrent changes in MHC class II or CD40 expression. However, this partial activation of neonatal DC was not sufficient to circumvent the requirement for CD4(+) T cell help. The effective induction of neonatal CD8(+) T cell responses by this minimal Ag delivery system demonstrates that neonatal CD11c(+) DC may mature sufficiently to stimulate naive CD8(+) neonatal T cells, even in the absence of strong maturation signals.     

Immune modulation and tolerance induction by RelB-silenced dendritic cells through RNA interference

Dendritic cells (DC), the most potent APCs, can initiate the immune response or help induce immune tolerance, depending upon their level of maturation. DC maturation is associated with activation of the NF-kappaB pathway, and the primary NF-kappaB protein involved in DC maturation is RelB, which coordinates RelA/p50-mediated DC differentiation. In this study, we show that silencing RelB using small interfering RNA results in arrest of DC maturation with reduced expression of the MHC class II, CD80, and CD86. Functionally, RelB-silenced DC inhibited MLR, and inhibitory effects on alloreactive immune responses were in an Ag-specific fashion. RelB-silenced DC also displayed strong in vivo immune regulation. An inhibited Ag-specific response was seen after immunization with keyhole limpet hemocyanin-pulsed and RelB-silenced DC, due to the expansion of T regulatory cells. Administration of donor-derived RelB-silenced DC significantly prevented allograft rejection in murine heart transplantation. This study demonstrates for the first time that transplant tolerance can be induced by means of RNA interference using in vitro-generated tolerogenic DC.     

Cutting edge: human CD4+CD25+ T cells restrain the maturation and antigen-presenting function of dendritic cells

The characteristics and functions of CD4(+)CD25(+) regulatory cells have been well defined in murine and human systems. However, the interaction between CD4(+)CD25(+) T cells and dendritic cells (DC) remains unclear. In this study, we examined the effect of human CD4(+)CD25(+) T cells on maturation and function of monocyte-derived DC. We show that regulatory T cells render the DC inefficient as APCs despite prestimulation with CD40 ligand. This effect was marginally reverted by neutralizing Abs to TGF-beta. There was an increased IL-10 secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to direct suppressor effect on CD4(+) T cells, regulatory T cells may modulate the immune response through DC.     

RNA based vaccines

The recognition that CD8(+) T-cell mediated Th1 immune responses were necessary to produce immunity to intracellular and transformed self pathogens led to intense interest in the delivery of nucleic acids, DNA, or RNA encoding candidate antigens, as vaccines. Antigen presenting cells (APC) encounter most protein and vaccine immunogens as extracellular proteins and, thus, present them on major histocompatibility complex (MHC) class II molecules leading to the activation of CD4(+) T cells. Protein antigens encoded by nucleic acids delivered to dendritic cell (DC) are produced inside the cell and, thus, can stimulate MHC class I mediated activation of CD8(+) T-cell immune responses. Unfortunately, DCs are not readily transfected with DNA (Akbari et al., 1999) resulting in the requirement for high concentrations of DNA and repeated immunizations to achieved immune responses. RNA, on the other hand, is readily taken up and expressed by DC, making it an alternative vaccine candidate. In this article, we will discuss immune responses developed, interactions between APC and RNA that activate and dictate DC activation, and preliminary studies using RNA in vivo and in vitro to develop protective immunity.     

Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with approximately 6% of naive CD4(+) T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4(+) and CD8(+) T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8(+) T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.     

Selectively impaired CD8+ but not CD4+ T cell cycle arrest during priming as a consequence of dendritic cell interaction with plasmodium-infected red cells

Individuals living in malaria-endemic areas show generally low T cell responses to malaria Ags. In this study, we show murine dendritic cell (DC) interaction with parasitized erythrocytes (pRBC) arrested their maturation, resulting in impaired ability to stimulate naive, but not recall T cell responses in vitro and in vivo. Moreover, within the naive T cell population, pRBC-treated DC were selectively deficient in priming CD8(+) but not CD4(+) T cells. Indeed, DC that had taken up pRBC were shown for the first time to efficiently prime CD4(+) T cell responses to a known protective merozoite Ag, MSP4/5. In contrast, impaired priming resulted in decreases in both proliferation and cytokine production by CD8(+) T cells. Deficient priming was observed to both a model and a Plasmodium berghei-specific CD8(+) T cell epitope. The mechanisms underlying the inability of parasite-treated DC to prime CD8(+) T cells were explored. pRBC treatment of DC from wild-type C57BL/6, but not from IL-10 knockout animals, suppressed DC-mediated T cell priming across a Transwell, suggesting active IL-10-dependent suppression. CD8(+) T cells were arrested at the G(0) stage of the cell cycle after two cell divisions post-Ag stimulation. The proliferation arrest was partially reversible by the addition of IL-2 or IL-7 to responder cultures. These results suggest that in malaria-endemic areas, priming of CD8(+) T cell responses may be more difficult to induce via vaccination than the priming of CD4(+) T cells. Moreover, pathogens may selectively target the CD8(+) T cell arm of protective immunity for immune evasion.     

Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation

Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8⁺ T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.