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Historically poor clinical results of tumor vaccines have been attributed to weakly immunogenic antigen targets, limited specificity, and vaccine platforms that fail to induce high-quality polyfunctional T cells, central to mediating cellular immunity. We show here that the combination of antigen selection, construct design, and a robust vaccine platform based on the Synthetically Modified Alpha Replicon RNA Technology (SMARRT), a self-replicating RNA, leads to control of tumor growth in mice. Therapeutic immunization with SMARRT replicon-based vaccines expressing tumor-specific neoantigens or tumor-associated antigen were able to generate polyfunctional CD4 + and CD8 + T cell responses in mice. Additionally, checkpoint inhibitors, or co-administration of cytokine also expressed from the SMARRT platform, synergized to enhance responses further. Lastly, SMARRT-based immunization of non-human primates was able to elicit high-quality T cell responses, demonstrating translatability and clinical feasibility of synthetic replicon technology for therapeutic oncology vaccines., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

Lentiviral vectors (LVs) pseudotyped with the measles virus hemagglutinin (H) and fusion (F) glycoproteins have been reported to more efficiently transduce hematopoietic stem and progenitor cells (HSPCs) compared with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped LVs. However, a limit to H/F LV use is the low titer of produced vector. Here we show that measles receptor (CD46) expression on H/F transfected HEK293T vector-producing cells caused adjacent cell membrane fusion, resulting in multinucleate syncytia formation and death prior to peak vector production, leading to contaminating cell membranes that co-purified with LV. H/F LVs produced in CD46 null HEK293T cells, generated by CRISPR/Cas9-mediated knockout of CD46, produced 2-fold higher titer vector compared with LVs produced in CD46 + HEK293T cells. This resulted in approximately 2- to 3-fold higher transduction of HSPCs while significantly reducing target cell cytotoxicity caused by producer cell contaminates. Improved H/F LV entry into HSPCs and distinct entry mechanisms compared with VSV-G LV were also observed by confocal microscopy. Given that vector production is a major source of cost and variability in clinical trials of gene therapy, we propose that the use of CD46 null packaging cells may help to address these challenges.

Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34 + hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E 2 (PGE 2 ) as a positive mediator of lentiviral transduction of CD34 + cells. Supplementation with PGE 2 increased lentiviral vector (LVV) transduction of CD34 + cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34 + cells from multiple normal human donors and from patients with β-thalassemia or sickle cell disease. Notably, PGE 2 increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE 2 improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE 2 may be useful in clinical gene therapy applications using lentivirally modified HSPCs., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Skin-resident T cells have been shown to play important roles in tissue homeostasis and wound repair, but their role in UV radiation (UVR)-mediated skin injury and subsequent tissue regeneration is less clear. In this study, we demonstrate that acute UVR rapidly activates skin-resident T cells in humans and dendritic epidermal γδ T cells (DETCs) in mice through mechanisms involving the release of ATP from keratinocytes. Following UVR, extracellular ATP leads to an increase in CD69 expression, proliferation, and IL-17 production, and to changes in DETC morphology. Furthermore, we find that the purinergic receptor P2X7 and caspase-1 are necessary for UVR-induced IL-1 production in keratinocytes, which increases IL-17 secretion by DETCs. IL-17, in turn, induces epidermal TNF-related weak inducer of apoptosis and growth arrest and DNA damage-associated gene 45, two molecules linked to the DNA repair response. Finally, we demonstrate that DETCs and human skin-resident T cells limit DNA damage in keratinocytes. Taken together, our findings establish a novel role for skin-resident T cells in the UVR-associated DNA repair response and underscore the importance of skin-resident T cells to overall skin regeneration., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The epidermis, the outer layer of the skin, forms a physical and antimicrobial shield to protect the body from environmental threats. Skin injury severely compromises the epidermal barrier and requires immediate repair. Dendritic epidermal T cells (DETC) reside in the murine epidermis where they sense skin injury and serve as regulators and orchestrators of immune responses. Here, we determined that TCR stimulation and skin injury induces IL-17A production by a subset of DETC. This subset of IL-17A-producing DETC was distinct from IFN-γ producers, despite similar surface marker profiles. Functionally, blocking IL-17A or genetic deletion of IL-17A resulted in delayed wound closure in animals. Skin organ cultures from Tcrd-/-, which lack DETC, and Il17a-/- mice both exhibited wound-healing defects. Wound healing was fully restored by the addition of WT DETC, but only partially restored by IL-17A-deficient DETC, demonstrating the importance of IL-17A to wound healing. Following skin injury, DETC-derived IL-17A induced expression of multiple host-defense molecules in epidermal keratinocytes to promote healing. Together, these data provide a mechanistic link between IL-17A production by DETC, host-defense, and wound-healing responses in the skin. These findings establish a critical and unique role of IL-17A-producing DETC in epidermal barrier function and wound healing.

γδ T cells respond rapidly to keratinocyte damage, providing essential contributions to the skin wound healing process. The molecular interactions regulating their response are unknown. Here, we identify a role for interaction of plexin B2 with the CD100 receptor in epithelial repair. In vitro blocking of plexin B2 or CD100 inhibited γδ T cell activation. Furthermore, CD100 deficiency in vivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ T cell response to keratinocyte damage. Ligation of CD100 in γδ T cells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providing a mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ T cells and the neighboring cells they support., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Gammadelta T cells present in epithelial tissues provide a crucial first line of defense against environmental insults, including infection, trauma, and malignancy, yet the molecular events surrounding their activation remain poorly defined. Here we identify an epithelial gammadelta T cell-specific costimulatory molecule, junctional adhesion molecule-like protein (JAML). Binding of JAML to its ligand Coxsackie and adenovirus receptor (CAR) provides costimulation leading to cellular proliferation and cytokine and growth factor production. Inhibition of JAML costimulation leads to diminished gammadelta T cell activation and delayed wound closure akin to that seen in the absence of gammadelta T cells. Our results identify JAML as a crucial component of epithelial gammadelta T cell biology and have broader implications for CAR and JAML in tissue homeostasis and repair.