Cross presentation of antigen by dendritic cells: mechanisms and implications for immunotherapy.

Dendritic cells (DCs) possess the specialized potential to present exogenously derived antigen to cytotoxic T lymphocytes to elicit an immune response. This process, termed cross presentation, is crucial in the generation of immune response to viruses, tumors and in autoimmune disease. The ability of DCs to cross-present exogenous antigen to cytotoxic T lymphocytes makes them an attractive target for exploitation in immunotherapy. In recent years, significant advances have been made in understanding the mechanism of cross-presentation and the DC subsets involved. The recent discovery of the human cross presenting DC has given this field a new lease of life. In this report, the authors provide an overview of cross-presentation of antigen by DCs, focusing on the current understanding of the molecular mechanisms of the process. The authors also discuss the DC subsets involved in cross presentation and its role in health and disease

Induced pluripotent stem cells: challenges and opportunities for cancer immunotherapy.

Despite recent advances in cancer treatment over the past 30 years, therapeutic options remain limited and do not always offer a cure for malignancy. Given that tumor-associated antigens (TAA) are, by definition, self-proteins, the need to productively engage autoreactive T cells remains at the heart of strategies for cancer immunotherapy. These have traditionally focused on the administration of autologous monocyte-derived dendritic cells (moDC) pulsed with TAA, or the ex vivo expansion and adoptive transfer of tumor-infiltrating lymphocytes (TIL) as a source of TAA-specific cytotoxic T cells (CTL). Although such approaches have shown some efficacy, success has been limited by the poor capacity of moDC to cross present exogenous TAA to the CD8(+) T-cell repertoire and the potential for exhaustion of CTL expanded ex vivo. Recent advances in induced pluripotency offer opportunities to generate patient-specific stem cell lines with the potential to differentiate in vitro into cell types whose properties may help address these issues. Here, we review recent success in the differentiation of NK cells from human induced pluripotent stem (iPS) cells as well as minor subsets of dendritic cells (DCs) with therapeutic potential, including CD141(+)XCR1(+) DC, capable of cross presenting TAA to naïve CD8(+) T cells. Furthermore, we review recent progress in the use of TIL as the starting material for the derivation of iPSC lines, thereby capturing their antigen specificity in a self-renewing stem cell line, from which potentially unlimited numbers of naïve TAA-specific T cells may be differentiated, free of the risks of exhaustion

Directed differentiation of human induced pluripotent stem cells into dendritic cells displaying tolerogenic properties and resembling the CD141+ subset

The advent of induced pluripotent stem cells (iPSCs) has begun to revolutionize cell therapy by providing a convenient source of rare cell types not normally available from patients in sufficient numbers for therapeutic purposes. In particular, the development of protocols for the differentiation of populations of leukocytes as diverse as naïve T cells, macrophages and Natural Killer (NK) cells provides opportunities for their scale-up and quality control prior to administration. One population of leukocytes whose therapeutic potential has yet to be explored is the subset of conventional dendritic cells (DCs) defined by their surface expression of CD141. While these cells stimulate cytotoxic T cells in response to inflammation through the cross-presentation of viral and tumor-associated antigens in an MHC class I-restricted manner, under steady-state conditions, CD141+ DCs resident in interstitial tissues are focused on the maintenance of homeostasis through the induction of tolerance to local antigens. Here we describe protocols for the directed differentiation of human iPSCs into a mixed population of CD11c+ DCs through the spontaneous formation of embryoid bodies and exposure to a cocktail of growth factors, the scheduled withdrawal of which serves to guide the process of differentiation. Furthermore, we describe the enrichment of DCs expressing CD141 through depletion of CD1c+ cells, thereby obtaining a population of ‘untouched’ DCs unaffected by cross-linking of surface CD141. The resulting cells display characteristic phagocytic and endocytic capacity and acquire an immunostimulatory phenotype following exposure to inflammatory cytokines and TLR agonists. Nevertheless, under steady-state conditions, these cells share some of the tolerogenic properties of tissue-resident CD141+ DCs, which may be further reinforced by exposure to a range of pharmacological agents including IL-10, rapamycin, dexamethasone and 1,25-dihydoxyvitamin D3. Our protocols therefore provide access to a novel source of DCs analogous to the CD141+ subset under steady-state conditions in vivo and may, therefore, find utility in the treatment of a range of disease states requiring the establishment of immunological tolerance

iPS cells reprogrammed from primary dendritic cells provide an abundant source of immunostimulatory dendritic cells for use in immunotherapy

Cell types differentiated from induced pluripotent stem cells (iPSCs) are frequently arrested in their development program, more closely resembling a fetal rather than an adult phenotype, potentially limiting their utility for downstream clinical applications. The fetal phenotype of iPSC‐derived dendritic cells (ipDCs) is evidenced by their low expression of MHC class II and costimulatory molecules, impaired secretion of IL‐12, and poor responsiveness to conventional maturation stimuli, undermining their use for applications such as immune‐oncology. Given that iPSCs display an epigenetic memory of the cell type from which they were originally derived, we investigated the feasibility of reprogramming adult DCs to pluripotency to determine the impact on the phenotype and function of ipDCs differentiated from them. Using murine bone marrow‐derived DCs (bmDCs) as proof of principle, we show here that immature DCs are tractable candidates for reprogramming using non‐integrating Sendai virus for the delivery of Oct4, Sox2, Klf4, and c‐Myc transcription factors. Reprogramming efficiency of DCs was lower than mouse embryonic fibroblasts (MEFs) and highly dependent on their maturation status. Although control iPSCs derived from conventional MEFs yielded DCs that displayed a predictable fetal phenotype and impaired immunostimulatory capacity in vitro and in vivo, DCs differentiated from DC‐derived iPSCs exhibited a surface phenotype, immunostimulatory capacity, and responsiveness to maturation stimuli indistinguishable from the source DCs, a phenotype that was retained for 15 passages of the parent iPSCs. Our results suggest that the epigenetic memory of iPSCs may be productively exploited for the generation of potently immunogenic DCs for immunotherapeutic applications

iPS cells reprogrammed from primary dendritic cells provide an abundant source of immunostimulatory dendritic cells for use in immunotherapy

Cell types differentiated from induced pluripotent stem cells (iPSCs) are frequently arrested in their development program, more closely resembling a fetal rather than an adult phenotype, potentially limiting their utility for downstream clinical applications. The fetal phenotype of iPSC‐derived dendritic cells (ipDCs) is evidenced by their low expression of MHC class II and costimulatory molecules, impaired secretion of IL‐12, and poor responsiveness to conventional maturation stimuli, undermining their use for applications such as immune‐oncology. Given that iPSCs display an epigenetic memory of the cell type from which they were originally derived, we investigated the feasibility of reprogramming adult DCs to pluripotency to determine the impact on the phenotype and function of ipDCs differentiated from them. Using murine bone marrow‐derived DCs (bmDCs) as proof of principle, we show here that immature DCs are tractable candidates for reprogramming using non‐integrating Sendai virus for the delivery of Oct4, Sox2, Klf4, and c‐Myc transcription factors. Reprogramming efficiency of DCs was lower than mouse embryonic fibroblasts (MEFs) and highly dependent on their maturation status. Although control iPSCs derived from conventional MEFs yielded DCs that displayed a predictable fetal phenotype and impaired immunostimulatory capacity in vitro and in vivo, DCs differentiated from DC‐derived iPSCs exhibited a surface phenotype, immunostimulatory capacity, and responsiveness to maturation stimuli indistinguishable from the source DCs, a phenotype that was retained for 15 passages of the parent iPSCs. Our results suggest that the epigenetic memory of iPSCs may be productively exploited for the generation of potently immunogenic DCs for immunotherapeutic applications

A Highly Optimized Protocol for Reprogramming Cancer Cells to Pluripotency Using Nonviral Plasmid Vectors

In spite of considerable interest in the field, reprogramming induced pluripotent stem cells (iPSCs) directly from cancer cells has encountered considerable challenges, including the extremely low reprogramming efficiency and instability of cancer-derived iPSCs (C-iPSCs). In this study, we aimed to identify the main obstacles that limit cancer cell reprogramming. Through a detailed multidimensional kinetic optimization, a highly optimized protocol is established for reprogramming C-iPSCs using nonviral plasmid vectors. We demonstrated how the initial cancer cell density seeded could be the most critical factor ultimately affecting C-iPSCs reprogramming. We have consistently achieved an unprecedented high C-iPSC reprogramming efficiency, establishing stable colonies with typical iPSC morphology, up to 50% of which express the iPSC phenotypic (Oct3/4, Sox2, Nanog) and enzymatic (alkaline phosphatase) markers. Furthermore, established C-iPSC lines were shown to be capable of forming teratomas in vivo, containing cell types and tissues from each of the embryonic germ layers, fully consistent with their acquisition of pluripotency. This protocol was tested and confirmed in two completely unrelated human lung adenocarcinoma (A549) and mouse melanoma (B16f10) cancer cell lines and thus offers a potentially valuable method for generating effectively virus-free C-iPSCs for future applications

Upregulation of heat shock proteins and the promotion of damage-associated molecular pattern signals in a colorectal cancer model by modulated electrohyperthermia

In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHT-provoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168- and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals. Significant cell death response upon mEHT treatment was accompanied by the early upregulation (4-h post-treatment) of heat shock protein (Hsp70 and Hsp90) mRNA levels. In situ, the treatment resulted in spatiotemporal occurrence of a DAMP protein signal sequence featured by the significant cytoplasmic to cell membrane translocation of calreticulin at 4 h, Hsp70 between 14 and 24 h and Hsp90 between 24- and 216-h post-treatment. The release of high-mobility group box1 protein (HMGB1) from tumor cell nuclei from 24-h post-treatment and its clearance from tumor cells by 48 h was also detected. Our results suggest that mEHT treatment can induce a DAMP-related signal sequence in colorectal cancer xenografts that may be relevant for promoting immunological cell death response, which need to be further tested in immune-competent animals