![]() Clones outlined in red and blue boxes were later screened for acceptance in allogeneic hosts. Untransfected (FS) mESCs (parental line) are shown in upper left corner. Values are shown relative to the corresponding expressions in splenocytes (thick black line on each radial graph) from B6 mice stimulated ex vivo with αCD3ε and αCD28 antibodies. c, Expression level of each inserted transgene by RT–qPCR. b, Schema showing the generation of clonal B6 mESCs that express the selected eight immunomodulatory transgenes via insertion with piggyBac (PB) and Sleeping Beauty (SB) transposon expression vectors. The underlined factors were selected to generate transgene-containing vectors. We aimed to test the long-term resistance to rejection of these engineered ESCs in fully immunocompetent hosts, which many short-term or human models of allotransplantation do not recapitulate.Ī, Candidate immunomodulatory factors and their roles in the innate and adaptive immune pathways involved in rejection of non-self. ![]() Two are secreted, with CCL21 having a heparin-binding domain for retention 33, 34 and MFGE8 binding to tissue collagen 35. Five of the eight factors are membrane bound and one is cytoplasmic. A goal in our selection criteria was to minimize effects on systemic immunity. We selected eight factors, including (1) CCL21 to disrupt dendritic cell migration 26, (2) PDL1, FASL, H2-M3 and SERPINB9 to target and/or protect against T-cell and NK-cell attack 27, 28, 29 and (3) CD47, CD200 and MFGE8 to target monocytes and macrophages 30, 31, 32. On the basis of these examples and the complexity of mammalian immunity, we collected candidate transgenes and overexpressed many of them to simultaneously interfere with antigen-presenting cells, lymphocytes and monocytes across the innate and adaptive arms of the immune system (Fig. We were encouraged by evolved immune-evading strategies in nature 19, 20, 21 and especially those where MHC expression is retained, such as the type 2 facial tumours in Tasmanian Devils 22, 23, 24, 25. We first sought to engineer PSCs that could escape immune rejection solely by overexpression of immunomodulatory transgenes and without deletion of MHC genes. Some also rely on overexpression of immunomodulatory factors 10, 11, 12, 13, 14, 15 to protect donor cells from the immune rejection caused by minor antigens 16, 17, 18. Many of these approaches involve knockout of the highly polymorphic class I and II genes in the major histocompatibility complex (MHC) to remove the major source of antigen mismatch between donor and recipients 5, 6, 7, 8, 9. In parallel to these programmes, several groups have worked to immune engineer or encapsulate pluripotent stem cells (PSCs) and PSC-derived cells to resist or escape immune rejection 4. As a response, some countries are developing iPSC banks containing cell lines that are homozygous for the most common human leucocyte antigen (HLA) alleles among a given population 2, 3. However, there will be many clinical situations where the costs and time needed to derive and differentiate autologous cells are prohibitive for instance, in patients needing immediate treatment for a given disease or injury. Alternatively, there are also ongoing and promising programmes to develop autologous cell therapies derived from induced pluripotent stem cells (iPSCs). ![]() Immunosuppressive drugs are one tool to mitigate these risks 1. Similar content being viewed by othersĪ barrier to getting cell-based therapies into the clinic is that cell products derived from a foreign donor can be immune rejected. ![]() The combination of cloaking and FailSafe technologies may allow for the generation of safe and allogeneically accepted cell lines and off-the-shelf cell products. Moreover, by using the previously reported FailSafe transgene system, which transcriptionally links a gene essential for cell division with an inducible and cell-proliferation-dependent kill switch, we generated cloaked tissues from mESCs that served as immune-privileged subcutaneous sites that protected uncloaked allogeneic and xenogeneic cells from rejection in immune-competent hosts. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses. Here we show that the overexpression of eight immunomodulatory transgenes ( Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically ‘cloak’ the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies.
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