Dendritic cells (DCs) are antigen-presenting cells with a unique ability to induce primary immune responses. DCs capture and transfer information from the outside world to the cells of the adaptive immune system. DCs are not only critical for the induction of primary immune responses, but may also be important for the induction of immunological tolerance, as well as for the regulation of the type of T cell-mediated immune response. Although our understanding of DC biology is still in its infancy, we are now beginning to use DC-based immunotherapy protocols to elicit immunity against cancer and infectious diseases.

Immunobiology of Dendritic Cells

Recognition of pathogens is mediated by a set of germline-encoded receptors that are referred to as pattern-recognition receptors (PRRs). These receptors recognize conserved molecular patterns (pathogen-associated molecular patterns), which are shared by large groups of microorganisms. Toll-like receptors (TLRs) function as the PRRs in mammals and play an essential role in the recognition of microbial components. The TLRs may also recognize endogenous ligands induced during the inflammatory response. Similar cytoplasmic domains allow TLRs to use the same signaling molecules used by the interleukin 1 receptors (IL-1Rs): these include MyD88, IL-1R--associated protein kinase and tumor necrosis factor receptor--activated factor 6. However, evidence is accumulating that the signaling pathways associated with each TLR are not identical and may, therefore, result in different biological responses.

Toll-like receptors: critical proteins linking innate and acquired immunity.

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.

https://science.sciencemag.org/content/sci/303/5663/1526.full.pdf

Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8

The clinical relevance of T cells in the control of a diverse set of human cancers is now beyond doubt. However, the nature of the antigens that allow the immune system to distinguish cancer cells from noncancer cells has long remained obscure. Recent technological innovations have made it possible to dissect the immune response to patient-specific neoantigens that arise as a consequence of tumor-specific mutations, and emerging data suggest that recognition of such neoantigens is a major factor in the activity of clinical immunotherapies. These observations indicate that neoantigen load may form a biomarker in cancer immunotherapy and provide an incentive for the development of novel therapeutic approaches that selectively enhance T cell reactivity against this class of antigens.

http://science.sciencemag.org/content/sci/348/6230/69.full.pdf

Neoantigens in cancer immunotherapy

MicroRNAs (miRNAs) are an abundant class of ∼22-nucleotide regulatory RNAs found in plants and animals. Some miRNAs of plants, Caenorhabditis elegans, and Drosophila play important gene-regulatory roles during development by pairing to target mRNAs to specify posttranscriptional repression of these messages. We identify three miRNAs that are specifically expressed in hematopoietic cells and show that their expression is dynamically regulated during early hematopoiesis and lineage commitment. One of these miRNAs, miR-181, was preferentially expressed in the B-lymphoid cells of mouse bone marrow, and its ectopic expression in hematopoietic stem/progenitor cells led to an increased fraction of B-lineage cells in both tissue-culture differentiation assays and adult mice. Our results indicate that microRNAs are components of the molecular circuitry that controls mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development.

/pdf/micrornas-modulate-hematopoietic-lineage-differentiation-2chw0g0ujk.pdf

MicroRNAs Modulate Hematopoietic Lineage Differentiation

In this study, we investigated whether elimination of CD4+/CD25+ Tregs using the recombinant IL-2 diphtheria toxin conjugate DAB(389)IL-2 (also known as denileukin diftitox and ONTAK) is capable of enhancing the immunostimulatory efficacy of tumor RNA-transfected DC vaccines. We show that DAB(389)IL-2 is capable of selectively eliminating CD25-expressing Tregs from the PBMCs of cancer patients without inducing toxicity on other cellular subsets with intermediate or low expression of CD25. DAB(389)IL-2-mediated Treg depletion resulted in enhanced stimulation of proliferative and cytotoxic T cell responses in vitro but only when DAB(389)IL-2 was omitted during T cell priming. DAB(389)IL-2 significantly reduced the number of Tregs present in the peripheral blood of metastatic renal cell carcinoma (RCC) patients and abrogated Treg-mediated immunosuppressive activity in vivo. Moreover, DAB(389)IL-2-mediated elimination of Tregs followed by vaccination with RNA-transfected DCs significantly improved the stimulation of tumor-specific T cell responses in RCC patients when compared with vaccination alone. Our findings may have implications in the design of immune-based strategies that may incorporate the Treg depletion strategy to achieve potent antitumor immunity with therapeutic impact.

/pdf/enhancement-of-vaccine-mediated-antitumor-immunity-in-cancer-3einktewdg.pdf

Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.

Immunization with defined tumor antigens is currently limited to a small number of cancers where candidates for tumor rejection antigens have been identified. In this study we investigated whether pulsing dendritic cells (DC) with tumor-derived RNA is an effective way to induce CTL and tumor immunity. DC pulsed with in vitro synthesized chicken ovalbumin (OVA) RNA were more effective than OVA peptide-pulsed DC in stimulating primary, OVA-specific CTL responses in vitro. DC pulsed with unfractionated RNA (total or polyA+) from OVA-expressing tumor cells were as effective as DC pulsed with OVA peptide at stimulating CTL responses. Induction of OVA-specific CTL was abrogated when polyA+ RNA from OVA-expressing cells was treated with an OVA-specific antisense oligodeoxynucleotide and RNase H, showing that sensitization of DC was indeed mediated by OVA RNA. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against a challenge with OVA-expressing tumor cells. In the poorly immunogenic, highly metastatic, B16/F10.9 tumor model a dramatic reduction in lung metastases was observed in mice vaccinated with DC pulsed with tumor-derived RNA (total or polyA+, but not polyA- RNA). The finding that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid was highly effective in sensitizing DC shows that amplification of the antigenic content from a small number of tumor cells is feasible, thus expanding the potential use of RNA-pulsed DC-based vaccines for patients bearing very small, possibly microscopic, tumors.

https://rupress.org/jem/article-pdf/184/2/465/1108621/465.pdf

Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.

To study the effects of localized secretion of cytokines on tumor progression, the gene for human interleukin 2 (IL-2) was introduced via retroviral vectors into CMS-5 cells, a weakly immunogenic mouse fibrosarcoma cell line of BALB/c origin. Secretion of low levels of IL-2 from the tumor cells abrogated their tumorigenicity and induced a long-lasting protective immune response against a challenge with a tumorigenic dose of parental CMS-5 cells. Co-injection of IL-2-producing CMS-5 cells with unmodified tumor cells inhibited tumor formation even when highly tumorigenic doses of CMS-5 cells were used. Cytolytic activity in mice injected with parental CMS-5 cells was transient and was greatly diminished 3 wk after injection, as commonly observed in tumor-bearing animals. However, in mice injected with IL-2-producing cells, tumor-specific cytolytic activity persisted at high levels for the duration of the observation period (at least 75 d). High levels of tumor-specific cytolytic activity could also be detected in parental CMS-5 tumor-bearing animals 18 d after inoculation with tumor cells, if IL-2-producing CMS-5 cells but not unmodified parental tumor cells were used as targets. These studies highlight the potential advantages of localized secretion of cytokines mediated via gene transfer to induce potent anti-tumor immune responses.

/pdf/interleukin-2-gene-transfer-into-tumor-cells-abrogates-rgl132nm2t.pdf

Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity.

Summary To study the effects oflocalized secretion ofcytokines on tumor progression, the gene for human interleukin 2 (IL2)was introduced via retroviral vectors intoCMS-5 cells, a weakly immunogenic mouse fibrosarcoma cell line of BALB/c origin. Secretion of low levels of 11,2 from the tumor cells abrogated their tumorigenicity and induced a long-lasting protectiveimmune response against a challenge with a tumorigenic dose of parental CMS-5 cells . Co-injection of IL2-producing CMS-5 cells with unmodified tumor cells inhibited tumor formation evenwhen highly tumorigenic doses of CMS-5 cells were used . Cytolytic activity in mice injected with parental CMS-5 cells was transient and was greatly diminished 3 wk after injection, as commonly observed in tumorbearing animals. However, in mice injected with IL-2-producing cells, tumor-specific cytolytic activity persisted at high levels for the duration of the observation period (at least 75 d) . High levels oftumor-specific cytolytic activity could also be detected in parental CMS-5 tumor-bearing animals 18 d after inoculation with tumor cells, if 11,2-producing CMS-5 cells but not unmodified parental tumor cells were used as targets . These studies highlight the potential advantages of localized secretion of cytokines mediated via gene transfer to induce potent anti-tumor immune responses.

Eli Gilboa

Papers

Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.

Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.

Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity.

Tumor Cells Abrogates Tumorigenicity and Induces Protective Immunity