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The TLR3 agonist poly(I:C) targets CD8+ T cells and augments their antigen-specific responses upon their adoptive transfer into naive recipient mice.

ABSTRACT: We have recently reported that the toll-like receptor 3 (TLR3) agonist poly(I:C) induces adjuvant effects to post vaccination CD8+ T cells responses through rapid induction of innate mediators, including NK cells, macrophages, dendritic cells (DCs), and inflammatory cytokines. However, whether this TLR3 agonist directly targets CD8+ T cells needs to be carefully investigated. In this study, we found that optimal post vaccination CD8+ T cell responses to ex vivo DC-based vaccination requires triggering of TLR3 signaling pathway in DCs in vitro as well as in the recipient host, indicating a role for other cell types. Real-time PCR analysis revealed that TLRs (TLR1-TLR13) are expressed in purified (>99% pure) CD4+ and CD8+ T cells from C57BL/6 and BALB/c mice, where the magnitude of the expression was strain and cell type dependent. In vitro, treatment of these purified T cells with poly(I:C) modulated the expression of TLRs including TLR3. Furthermore, non-specific and antigen-specific stimulation of CD8+ T cells by phorbol myristate acetate and MHC class I peptide-pulsed splenocytes, respectively, modulated TLR expression in purified CD4+ and CD8+ T cells. Importantly, brief conditioning of purified naïve TCR transgenic OT-1 (CD8+) T cells in vitro with poly(I:C) induced activation of these cells in absence of antigen stimulation. Interestingly, when these in vitro poly(I:C)-conditioned OT-1 cells were adoptively transferred into naïve recipient followed by peptide vaccination, they showed superior expansion and activation to their naïve counterparts. These results suggest that CD8+ T cells can be activated by triggering their TLR3. Furthermore, the data support the notion of direct involvement of TLRs in adaptive immune responses.

SUBMITTER: Salem ML

PROVIDER: S-EPMC3072892 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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The TLR3 agonist poly(I:C) targets CD8+ T cells and augments their antigen-specific responses upon their adoptive transfer into naïve recipient mice.

Salem Mohamed L ML Diaz-Montero C Marcela CM El-Naggar Sabry A SA Chen Yian Y Moussa Omar O Cole David J DJ

Vaccine 20081121 4

We have recently reported that the toll-like receptor 3 (TLR3) agonist poly(I:C) induces adjuvant effects to post vaccination CD8+ T cells responses through rapid induction of innate mediators, including NK cells, macrophages, dendritic cells (DCs), and inflammatory cytokines. However, whether this TLR3 agonist directly targets CD8+ T cells needs to be carefully investigated. In this study, we found that optimal post vaccination CD8+ T cell responses to ex vivo DC-based vaccination requires trig ...[more]

PMID: 19027047

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Project description:Macrophages orchestrate inflammation and control the promotion or inhibition of tumors and metastasis. Ferumoxytol (FMT), a clinically approved iron oxide nanoparticle, possesses anti-tumor therapeutic potential by inducing pro-inflammatory macrophage polarization. Toll-like receptor 3 (TLR3) activation also potently enhances the anti-tumor response of immune cells. Herein, the anti-tumor potential of macrophages harnessed by FMT combined with the TLR3 agonist, poly (I:C) (PIC), and FP-NPs (nanoparticles composed of amino-modified FMT (FMT-NH2) surface functionalized with PIC) was explored. Methods: Proliferation of B16F10 cells co-cultured with macrophages was measured using immunofluorescence or flow cytometry (FCM). Phagocytosis was analyzed using FCM and fluorescence imaging. FP-NPs were prepared through electrostatic interactions and their properties were characterized using dynamic light scattering, transmission electron microscopy, and gel retardation assay. Anti-tumor and anti-metastasis effects were evaluated in B16F10 tumor-bearing mice, and tumor-infiltrating immunocytes were detected by immunofluorescence staining and FCM. Results: FMT, PIC, or the combination of both hardly impaired B16F10 cell viability. However, FMT combined with PIC synergistically inhibited their proliferation by shifting macrophages to a tumoricidal phenotype with upregulated TNF-? and iNOS, increased NO secretion and augmented phagocytosis induced by NOX2-derived ROS in vitro. Combined treatment with FMT/PIC and FMT-NH2/PIC respectively resulted in primary melanoma regression and alleviated pulmonary metastasis with elevated pro-inflammatory macrophage infiltration and upregulation of pro-inflammatory genes in vivo. In comparison, FP-NPs with properties of internalization by macrophages and accumulation in the lung produced a more pronounced anti-metastatic effect accompanied with decreased myeloid-derived suppressor cells, and tumor-associated macrophages shifted to M1 phenotype. In vitro mechanistic studies revealed that FP-NPs nanoparticles barely affected B16F10 cell viability, but specifically retarded their growth by steering macrophages to M1 phenotype through NF-?B signaling. Conclusion: FMT synergized with the TLR3 agonist PIC either in combination or as a nano-composition to induce macrophage activation for primary and metastatic melanoma regression, and the nano-composition of FP-NPs exhibited a more superior anti-metastatic efficacy.

Project description:Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage.

Project description:BackgroundToll-like receptor-3 (TLR-3) is a critical component of innate immune system against dsRNA viruses and is expressed in the central nervous system. However, it remains unknown whether TLR3 may serve as a therapeutic target in human neuroblastoma (NB).MethodsTLR3 expression in human NB samples was examined by immunohistochemical analysis. Quantitative RT-PCR and western blot was used to determine TLR3 expression in three human NB cell lines. The effect of TLR3 agonist, polyinosinic-polycytidylic acid (poly(I:C)), on the growth of human NB cells was evaluated by WST-1 cell proliferation assay, flow cytometry analysis, and immunoblot analysis. Blockade of TLR3 signaling was achieved using TLR3 neutralizing antibody, small interference RNA, and 2-aminopurine (2-AP), an inhibitor of protein kinase R (PKR), an interferon-induced, double-stranded RNA-activated protein kinase.ResultsIn immunohistochemical studies, TLR3 mainly expressed in the cytoplasm of ganglion cells and in some neuroblastic cells, but not in the stromal cells in human NB tissues. Among three human NB cell lines analyzed, TLR3 was significantly up-regulated in SK-N-AS cells at mRNA and protein level compared with other two low TLR3- expressing NB cells. Treatment with poly(I:C) elicited significant growth inhibition and apoptosis only in high TLR3-expressing SK-N-AS cells, but not in low TLR3-expressing SK-N-FI and SK-N-DZ cells. Moreover, poly(I:C) treatment significantly stimulated the activities of PKR, interferon regulatory factor 3 (IRF-3) and caspase-3 in SK-N-AS cells. Application of TLR3 neutralizing antibody or small interference RNA (siRNA) reduced the poly(I:C)-induced inhibition of cell proliferation and apoptosis in SK-N-AS cells. On the contrary, ectopic TLR3 expression enhanced the sensitivity of low TLR3-expressing NB cells to poly(I:C). Finally, application of 2-AP attenuated the poly(I:C)-induced IRF-3 and caspase-3 activation in SK-N-AS cells.ConclusionThe present study demonstrates that TLR3 is expressed in a subset of NB cells. Besides, TLR3/PKR/IRF-3/capase-3 pathway is implicated in the selective cytotoxicity of TLR3 agonist towards high TLR3-expressing NB cells.

Project description:Objective: Toll-like receptor-3 agonist Poly-ICLC has been known to activate immune cells and induce HIV replication in pre-clinical experiments. In this study we investigated if Poly-ICLC could be used for disrupting HIV latency while simultaneously enhancing innate immune responses. Design: This was a randomized, placebo-controlled, double-blinded trial in aviremic, cART-treated HIV-infected subjects. Participants (n = 15) were randomized 3:1 to receive two consecutive daily doses of Poly-ICLC (1.4 mg subcutaneously) vs. placebo. Subjects were observed for adverse events, immune activation, and viral replication. Methods: Besides primary outcomes of safety and tolerability, several longitudinal immune parameters were evaluated including immune cell phenotype and function via flowcytometry, ELISA, and transcriptional profiling. PCR assays for plasma HIV-1 RNA, CD4+ T cell-associated HIV-1 RNA, and proviral DNA were performed to measure HIV reservoirs and latency. Results: Poly-ICLC was overall safe and well-tolerated. Poly-ICLC-related adverse events were Grade 1/2, with the exception of one Grade 3 neutropenia which was short-lived. Mild Injection site reactions were observed in nearly all participants in the Poly-ICLC arm. Transcriptional analyses revealed upregulation of innate immune pathways in PBMCs following Poly-ICLC treatment, including strong interferon signaling accompanied by transient increases in circulating IP-10 (CXCL10) levels. These responses generally peaked by 24-48 h after the first injection and returned to baseline by day 8. CD4+ T cell number and phenotype were unchanged, plasma viral control was maintained and no significant effect on HIV reservoirs was observed. Conclusions: These finding suggest that Poly-ICLC could be safely used for inducing transient innate immune responses in treated HIV+ subjects indicating promise as an adjuvant for HIV therapeutic vaccines. Trial Registration: www.ClinicalTrials.gov, identifier: NCT02071095.

Dataset Information

The TLR3 agonist poly(I:C) targets CD8+ T cells and augments their antigen-specific responses upon their adoptive transfer into naive recipient mice.

Publications

The TLR3 agonist poly(I:C) targets CD8+ T cells and augments their antigen-specific responses upon their adoptive transfer into naïve recipient mice.

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