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Tables S1-S2

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.

Figures S1-S6

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.

The SNARE hypothesis proposes that synaptic vesicles dock at presynaptic membranes via interactions among the vesicular, integral membrane proteins VAMP (vesicle-associated membrane protein) and synaptotagmin and the target membrane proteins SNAP25 (synaptosome-associated protein with an Mr of 25 kDa) and syntaxin-1. Non-neuronal cells express isoforms of these proteins, believed to mediate secretory vesicle docking and/or fusion. Secretion in neuronal and non-neuronal systems differs in time course, Ca2+ dependence, and regulatory input. It is not known whether the non-neuronal protein isoforms form complexes akin to those of their neuronal counterparts. In this study, we defined the binding characteristics of three SNARE proteins: SNAP23, VAMP-2, and syntaxin-4. Binary, saturable interactions among all three partners (VAMP-2-syntaxin-4, VAMP-2-SNAP23, and SNAP23-syntaxin-4) were measured in vitro. Unlike its neuronal counterpart, SNAP23 did not potentiate VAMP-2 binding to its putative t-SNARE partner, syntaxin-4. The susceptibility of SNARE proteins to phosphorylation by exogenous kinases and their impact on binary interactions were explored. Syntaxin-4 was efficiently phosphorylated by casein kinase II (CKII) and cAMP-dependent protein kinase (PKA) (incorporating 0.8 and 3.9 mol of phosphate/mol of syntaxin-4, respectively), while syntaxin-1 was only strongly phosphorylated by CKII. Each of the syntaxin isoforms was weakly phosphorylated by protein kinase C (PKC) (0.05 mol of phosphate/mol of syntaxin-4). Importantly, PKA but not casein kinase II phosphorylation of syntaxin-4 disrupted its binding to SNAP23. We hypothesize that PKA may modulate syntaxin-4-dependent SNARE complex formation to regulate exocytosis in non-neuronal cells.

Several findings have recently questioned the long held hypothesis that cytokines belonging to the Th2 pathway are protective in T-cell-mediated autoimmunity. Among them, there is our previous report that pancreatic expression of IL-4 activated islet antigen-specific BDC2.5 T cells and rendered them able to trigger insulin-dependent diabetes mellitus in ins-IL-4/BDC2.5 mice (Mueller et al., Immunity, 7, 1997). Here we analyze the mechanisms underlying IL-4-mediated activation of the self-reactive BDC2.5 T cells. IL-4 is mainly known as the Th2-driving cytokine. However, IL-4 is also critical for DC maturation and upregulation of antigen uptake and presentation by macrophages. In our model, we found that pancreatic expression of IL-4 activated self-reactive BDC2.5 T cells by increasing islet antigen presentation by macrophages and dendritic cells. IL-4 could have triggered self-antigen presentation within the pancreatic islets both by driving maturation of DC from a tolerizing to a priming state and by increasing self-antigen uptake by macrophages.

In the presence of interferon-gamma (IFN-gamma), pancreatic ductal epithelial cells grow continuously, and islets undergo neogenesis. To determine whether these new islets are derived from conventional precursors, we tested whether IFN-gamma can complement the loss of transcription factors known to regulate pancreatic development. We analyzed the effect of a transgene on lethality in mice lacking the transcription factors Pax4, Pax6, or Pdx-1, by intercrossing such mice with transgenic mice whose pancreatic cells make IFN-gamma (ins-IFN-gamma mice). However, IFN-gamma expression did not rescue these mice from the lethal mutations, because no homozygous knockout mice carrying the IFN-gamma transgene survived, despite the survival of all other hemizygous gene combinations. This outcome demonstrates that the pathway for IFN-gamma regeneration requires the participation of Pax4, Pax6, and Pdx-1. We conclude that the striking islet regeneration observed in the ins-IFN-gamma NOD strain is regulated by the same transcription factors that control initial pancreatic development.

The function of natural killer T (NKT) cells in the immune system has yet to be determined. There is some evidence that their defect is associated with autoimmunity, but it is still unclear how they play a role in regulating the pathogenesis of T cell–mediated autoimmune diseases. It was originally proposed that NKT cells could control autoimmunity by shifting the cytokine profile of autoimmune T cells toward a protective T helper 2 cell (Th2) type. However, it is now clear that the major function of NKT cells in the immune system is not related to their interleukin (IL)-4 secretion. In fact, NKT cells mainly secrete interferon (IFN)-γ and, activated in the presence of IL-12, acquire a strong inflammatory phenotype and cytotoxic function.