Apoptotic Cells Deliver Processed Antigen to Dendritic Cells for Cross-Presentation

Antigen derived from engulfed apoptotic cells can be cross-presented by dendritic cells (DCs) for the generation of major histocompatibility class I/peptide complexes. While the early events of recognition and internalization of the dying cell have been characterized, the antigen-processing pathway or pathways remain unknown. We established a mouse model assaying for the activation of polyclonal T cells reactive to antigen derived from apoptotic cells, and demonstrated two distinct pathways for the trafficking of exogenous epitopes. In the first, exogenous antigen is dependent on the DC's expression of a functional transporter associated with antigen processing (TAP). Surprisingly, we found evidence that a second pathway exists in which transfer of processed antigen from the dying cell allows formation of major histocompatibility class I/peptide complexes in TAP-deficient DCs. In vivo data suggest that in situations of stress (e.g., viral infection), this latter pathway may be more efficient, illustrating that dying cells may preselect immunologically important antigenic determinants

Tolerance to the Neuron-Specific Paraneoplastic HuD Antigen

Experiments dating back to the 1940's have led to the hypothesis that the brain is an immunologically privileged site, shielding its antigens from immune recognition. The paraneoplastic Hu syndrome provides a powerful paradigm for addressing this hypothesis; it is believed to develop because small cell lung cancers (SCLC) express the neuron-specific Hu protein. This leads to an Hu-specific tumor immune response that can develop into an autoimmune attack against neurons, presumably when immune privilege in the brain is breached. Interestingly, all SCLC express the onconeural HuD antigen, and clinically useful tumor immune responses can be detected in up to 20% of patients, yet the paraneoplastic neurologic syndrome is extremely rare. We found that HuD-specific CD8+ T cells are normally present in the mouse T cell repertoire, but are not expanded upon immunization, although they can be detected after in vitro expansion. In contrast, HuD-specific T cells could be directly activated in HuD null mice, without the need for in vitro expansion. Taken together, these results demonstrate robust tolerance to the neuronal HuD antigen in vivo, and suggest a re-evaluation of the current concept of immune privilege in the brain

Harnessing Naturally Occurring Tumor Immunity: A Clinical Vaccine Trial in Prostate Cancer

International audienceBACKGROUND:Studies of patients with paraneoplastic neurologic disorders (PND) have revealed that apoptotic tumor serves as a potential potent trigger for the initiation of naturally occurring tumor immunity. The purpose of this study was to assess the feasibility, safety, and immunogenicity of an apoptotic tumor-autologous dendritic cell (DC) vaccine.METHODS AND FINDINGS:We have modeled PND tumor immunity in a clinical trial in which apoptotic allogeneic prostate tumor cells were used to generate an apoptotic tumor-autologous dendritic cell vaccine. Twenty-four prostate cancer patients were immunized in a Phase I, randomized, single-blind, placebo-controlled study to assess the safety and immunogenicity of this vaccine. Vaccinations were safe and well tolerated. Importantly, we also found that the vaccine was immunogenic, inducing delayed type hypersensitivity (DTH) responses and CD4+ and CD8+ T cell proliferation, with no effect on FoxP3+ regulatory T cells. A statistically significant increase in T cell proliferation responses to prostate tumor cells in vitro (p = 0.002), decrease in prostate specific antigen (PSA) slope (p = 0.016), and a two-fold increase in PSA doubling time (p = 0.003) were identified when we compared data before and after vaccination.CONCLUSIONS:An apoptotic cancer cell vaccine modeled on naturally occurring tumor immune responses in PND patients provides a safe and immunogenic tumor vaccine

DNA methylation of the POU5F1 regulatory region in rabbit first embryonic lineages

International audienceThe expression of POU5F1 is controlled by cis-regulatory elements located 5' upstream from the initiation start site. This regulatory region is highly conserved among species, and especially four conserved regions (CR1 to 4) have been identified. The minimal promoter is located in the CR1 region, while the proximal and distal enhancers involved in cell specific regulation of expression are located in CR2- 3 and CR4 regions respectively. In the mouse the two enhancer regions contribute differently to gene expression depending on the developmental stage of the embryo. POU5F1 repression of expression is induced by regulatory factors binding to the 5' upstream region but also by DNA and histone methylation. DNA methylation of POU5F1 has been mostly analyzed in in vitro derived stem cells. However, promotor DNA methylation levels have been shown to differ between embryonic stem cells and the "in embryo" counterparts they are derived from. We thus decided to analyze DNA methylation of the four conserved regions of the POU5F1 upstream region in the first embryonic lineages. Therefore we took benefit from the rabbit embryo whose epiblast, hypoblast and trophoblast can be easily isolated. At that stage, POU5F1 expression is restricted to the epiblast. We evidenced an hypomethylation of the four conserved regions of POU5F1 5' upstream region in the epiblast. Interestingly even the CR4 region which is supposed to be functional in the ICM but not in the epiblast conserved a very low methylation level in this tissue. CR1 methylation was lower in day 6 embryos than in fibroblasts

Immunization with Apoptotic Cells Results in the Selective Priming of T Cells Reactive to Processed Antigen

<div><p>(A and B) C57BL/6 mice were immunized intraperitoneally with 300 HAU of influenza (A), or 5 × 10⁶ infected apoptotic 3T3 cells (B). After 14 d, splenocytes were harvested, and CD8⁺ T cells were purified. To assay for the specificity of these cells, an IFN-γ ELISPOT was performed using the following stimulators: DCs alone or DCs pulsed with either 1 μM NP_366–374 or 1 μM PA_224–233 peptide.</p> <p>(C) C57BL/6 mice were immunized intraperitoneally with 5 × 10⁶ untreated versus lactacystin-treated influenza-infected apoptotic 3T3 cells. As above, 14 d after priming, splenocytes were harvested, and CD8⁺ T cells were purified and assayed for their reactivity to NP_366–374 versus PA_224–233. In this experiment, peptide-pulsed EL4 cells were employed as the stimulators. Data are representative of two experiments. Values are averages of triplicate wells with error bars indicating standard deviation.</p></div

Processed Antigen within the Dying Cell Is Required for Efficient In Vivo Priming

<p>C57BL/6 mice were immunized intraperitoneally with 300 HAU of influenza (A), or 2 × 10⁶ infected apoptotic kidney epithelial cells derived from β₂m-deficient (B) or TAP-deficient mice (C). After 14 d, splenocytes were harvested, and CD8⁺ T cells were purified. To assay for the specificity of these cells, an IFN-γ ELISPOT was performed using the following stimulators: EL4 cells alone or EL4 cells pulsed with either 1 μM NP_366–374 or 1 μM PA_224–233 peptide. Values are averages of triplicate wells with error bars indicating standard deviation.</p

An Active Role for Apoptotic Cells in the Transfer of Antigen to DCs

<p>We propose that apoptotic cells play an active role through the transfer of processed antigen to DCs for the generation of MHC I/pep complexes. This pathway may be dominant in the presentation of infectious antigen as the virus may co-opt cellular translational machinery, resulting in high levels of viral protein, and the upregulation of stress proteins, as well as inducing apoptotic cell death. Defective ribosomal initiation products chaperoned by HSPs offer a potential source of antigen. Within the DC, HSPs derived from the internalized apoptotic cell may traffic via a retrograde transport pathway, shuttled to the <i>trans</i>-Golgi and then the ER via binding to KDEL receptors (A). Alternatively, the evidence for phagosome–ER (PHAGO-ER) fusion and/or the recycling of MHC I from the plasma membrane offers the possibility that processed antigen may interact directly with the DC's MHC I (B). As ER chaperones within the phagocytosed cell would be bound to the pool of peptides derived from newly synthesized proteins, these pathways offer the DC an accurate representation of what occurred immediately prior to death (A and B). At high concentrations of protein, we also find evidence for the DC processing the cross-presented antigen. This likely occurs via a phago–ER-to-cytosol pathway as has been previously described (C).</p