MART-1-particular CD8+ T cells in the bulk cultures were stained and monitored by FACSAria flow cytometer

MART-1-particular CD8+ T cells in the bulk cultures were stained and monitored by FACSAria flow cytometer. Flow cytometry and allostimulation assay To study the phenotype of H1.ME-DCs, the cells were stained with antibodies against CD11c, CD40, CD83, CD86, HLA-DR and HLA-A2 (BD Biosciences) and analyzed with a FACSCalibur flow cytometer (BD Biosciences). memory or effector memory phenotype. Thus, we demonstrated that immunocompetent tumour antigen-loaded DCs can be directly generated from antigenically modified hPSCs. Using such strategy, we can completely eliminate the conventional antigen-loading step and significantly simplify the production of DC vaccine from hPSCs. Dendritic cell (DC) vaccine is becoming a new therapeutic modality for cancer1,2. This therapeutic strategy exploits the power and specificity of the immune system to fight against cancer, yet avoids the devastating and life-threatening side effects of traditional cancer therapies. DC-based immunotherapy has a much better safety profile and may provide better quality of life for cancer patients. However, it remains challenging to prepare high-quality DC vaccines in large quantity to induce clinically significant anti-cancer immunity due to the complexities in making such living cell products3,4. Hence, a simplified manufacturing process is necessary to ultimately improve both the accessibility and therapeutic efficacy of DC vaccines5. Currently, most DC vaccines are generated from patient blood cells6. A large amount of peripheral blood mononuclear cells (PBMCs) are collected from the patient via an invasive leukapheresis process. Monocytes are then isolated from PBMCs and further differentiated into DCs. These monocyte-derived DCs (moDCs) are loaded with tumour antigens and matured before injection into the patient. This production process is complicate and full of technical and logistic difficulties. The end products are costly as exemplified by Dendreons Provenge, the first ever FDA-approved DC-based vaccine for prostate cancer7. The MGCD-265 (Glesatinib) qualities of Rabbit Polyclonal to RPL30 such produced DC vaccines are highly variable due to unpredictable and uncontrollable patient-to-patient variation. With these inconsistent DC products, it is difficult to optimize those critical parameters that may further improve vaccine efficacy in clinical trials. Moreover, such patient blood cell-derived MGCD-265 (Glesatinib) DC vaccines are often limited in supply, which makes it impossible to clinically evaluate the benefit of high dosage and frequent vaccination. All the above-mentioned issues are largely associated with the use of patient blood cells for DC vaccine production. To avoid these issues, it is imperative to employ an alternative platform that is reliable, standardizable and patient blood cell-independent. Naturally, in the age of pluripotency, human pluripotent stem cells (hPSCs) may well serve such a purpose8. As we have demonstrated earlier, hPSC-derived DCs (hPSC-DCs) are capable of presenting not only peptide antigen to antigen-specific CD8+ T cells9, but also glycolipid antigen to invariant natural killer T (iNKT) cells10. These proven functional capabilities of hPSC-DCs further validate the use of hPSCs to develop DC vaccines. To produce DC vaccine, antigen-loading is a crucial step that defines the specificity of vaccine-induced anti-tumour immune response. Most commonly used antigen-loading approaches include peptide-pulsing, protein-loading, tumour lysate-loading, RNA/DNA transfection and viral transduction11. These conventional approaches require not only the production of various clinical-grade tumour antigen payloads, but also the unavoidable and sometimes detrimental cell manipulations to deliver the antigen payloads into DCs. Furthermore, in large-scale manufacturing, the antigen-loading step needs to be repeated for every batch of DC vaccine, which poses a great challenge to yield consistent products. Although these conventional approaches are also applicable to hPSC-DCs9,10, a simpler antigen-loading solution is highly desirable for MGCD-265 (Glesatinib) making DC vaccine from hPSCs. To this end, we stably modified MGCD-265 (Glesatinib) the hPSCs with tumour antigen genes in this study and demonstrated that such antigenically modified hPSCs were able to differentiate into functional tumour antigen-presenting DCs. Using this novel antigen-loading strategy, no conventional antigen-loading step is required for generating tumor antigen-presenting DCs from hPSCs, thus the production of hPSC-DC cancer vaccine can be significantly simplified. Results Tumour antigen gene-modified hPSCs produce tumour antigen-expressing DCs To investigate whether hPSCs can be modified by tumour antigen gene and subsequently used to derive tumour antigen-expressing DCs, we generated a lentivector carrying a gene, designated as LV.MP (Fig. 1a). LV.MP was also containing a gene as reporter and a neomycin-resistance gene for drug selection (Fig. 1a). This lentivector was used to transduce an hPSC line, H1. After selection with G418, G418-resistent H1 lines were generated. One of these lines, H1.MP showed substantial GFP expression (Fig. 1b). Moreover, expression was also observed in H1.MP as demonstrated at both RNA level (Fig. 1c) and protein level (Fig. 1d). Both H1.MP line.

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