Consequences of persistent antigen presentation following administration of HIV-1-derived lentiviral vectors

Lentiviral vectors (LVs) are promising tools for in vivo gene delivery, either to correct genetic defects or for vaccination. Intravenous administration of LVs results in stable transduction and expression of the transgene in antigen presenting cells (APCs) from the spleen. Therefore, it was decided to investigate the reasons for and the consequences of sustained antigen expression in these cells after systemic in vivo administration of LVs. Intravenous injection of a LV encoding green fluorescent protein (GFP) resulted in transduction of lymphocytes, macrophages and all subsets of dendritic cells (DCs) in the spleen, detected 5 days later. In the case of macrophages and DCs, the percentage of transduced cells increased between 5 and 30 days after injection. The transduction of dividing precursors contributes to the persistence of the transgene-expressing DCs, as shown by BrdU incorporation. Expression of ovalbumin (OVA) resulted in a reduced number of transgeneexpressing cells after 30 days. However, the remaining transduced cells stimulated proliferation and activation of OVA-specific CD8+ T cells up to 3 months after LV administration, in spite of a reduction in the activation status of transduced DCs over time. Mice also maintained cytolytic activity against OVA-pulsed targets following a single immunisation. In conclusion, this thesis shows that LVs can transduce DCs and macrophages, leading to persistent antigen expression. These modified APCs are functional and capable of activating T cells. Therefore, LVs can be used as tools for persistent genetic modification of APCs, opening the opportunity for their use in long-term immunomodulation

α-PD-1 therapy elevates Treg/Th balance and increases tumor cell pSmad3 that are both targeted by α-TGFβ antibody to promote durable rejection and immunity in squamous cell carcinomas

Abstract Background Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFβ signaling. Methods To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFβ or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. Results We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFβ monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFβ, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFβ antibody administration attenuates these effects. We show that α-TGFβ acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFβ acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery. Conclusions We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFβ-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFβ combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFβ/α-PD-1 combination therapy (NCT02947165).https://deepblue.lib.umich.edu/bitstream/2027.42/148212/1/40425_2018_Article_493.pd

Crowd guilds: Worker-led reputation and feedback on crowdsourcing platforms

Crowd workers are distributed and decentralized. While decentralization is designed to utilize independent judgment to promote high-quality results, it paradoxically undercuts behaviors and institutions that are critical to high-quality work. Reputation is one central example: crowdsourcing systems depend on reputation scores from decentralized workers and requesters, but these scores are notoriously inflated and uninformative. In this paper, we draw inspiration from historical worker guilds (e.g., in the silk trade) to design and implement crowd guilds: centralized groups of crowd workers who collectively certify each other’s quality through double-blind peer assessment. A two-week field experiment compared crowd guilds to a traditional decentralized crowd work model. Crowd guilds produced reputation signals more strongly correlated with ground-truth worker quality than signals available on current crowd working platforms, and more accurate than in the traditional model

α-PD-1 therapy elevates Treg/Th balance and increases tumor cell pSmad3 that are both targeted by α-TGFβ antibody to promote durable rejection and immunity in squamous cell carcinomas

BACKGROUND: Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFβ signaling. METHODS: To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFβ or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. RESULTS: We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFβ monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFβ, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFβ antibody administration attenuates these effects. We show that α-TGFβ acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFβ acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery. CONCLUSIONS: We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFβ-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFβ combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFβ/α-PD-1 combination therapy (NCT02947165)

Consequences of persistent antigen presentation following administration of HIV-1-derived lentiviral vectors.

Lentiviral vectors (LVs) are promising tools for in vivo gene delivery, either to correct genetic defects or for vaccination. Intravenous administration of LVs results in stable transduction and expression of the transgene in antigen presenting cells (APCs) from the spleen. Therefore, it was decided to investigate the reasons for and the consequences of sustained antigen expression in these cells after systemic in vivo administration of LVs. Intravenous injection of a LV encoding green fluorescent protein (GFP) resulted in transduction of lymphocytes, macrophages and all subsets of dendritic cells (DCs) in the spleen, detected 5 days later. In the case of macrophages and DCs, the percentage of transduced cells increased between 5 and 30 days after injection. The transduction of dividing precursors contributes to the persistence of the transgene-expressing DCs, as shown by BrdU incorporation. Expression of ovalbumin (OVA) resulted in a reduced number of transgeneexpressing cells after 30 days. However, the remaining transduced cells stimulated proliferation and activation of OVA-specific CD8+ T cells up to 3 months after LV administration, in spite of a reduction in the activation status of transduced DCs over time. Mice also maintained cytolytic activity against OVA-pulsed targets following a single immunisation. In conclusion, this thesis shows that LVs can transduce DCs and macrophages, leading to persistent antigen expression. These modified APCs are functional and capable of activating T cells. Therefore, LVs can be used as tools for persistent genetic modification of APCs, opening the opportunity for their use in long-term immunomodulation.