Off-the-shelf third-party HSC-engineered iNKT cells for ameliorating GvHD while preserving GvL effect in the treatment of blood cancers.

Allo-HSCT is a curative therapy for hematologic malignancies owing to GvL effect mediated by alloreactive T cells; however, the same T cells also mediate GvHD, a severe side effect limiting the widespread application of allo-HSCT in clinics. Invariant natural killer T (iNKT) cells can ameliorate GvHD while preserving GvL effect, but the clinical application of these cells is restricted by their scarcity. Here, we report the successful generation of third-party HSC-engineered human iNKT (3rdHSC-iNKT) cells using a method combining HSC gene engineering and in vitro HSC differentiation. The 3rdHSC-iNKT cells closely resembled the CD4-CD8-/+ subsets of endogenous human iNKT cells in phenotype and functionality. These cells displayed potent anti-GvHD functions by eliminating antigen-presenting myeloid cells in vitro and in xenograft models without negatively impacting tumor eradication by allogeneic T cells in preclinical models of lymphoma and leukemia, supporting 3rdHSC-iNKT cells as a promising off-the-shelf cell therapy candidate for GvHD prophylaxis.

Development of allogeneic HSC-engineered iNKT cells for off-the-shelf cancer immunotherapy.

Cell-based immunotherapy has become the new-generation cancer medicine, and "off-the-shelf" cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. Here, by combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we generate human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development.

Methods for Studying Mouse and Human Invariant Natural Killer T Cells.

Invariant natural killer T (iNKT) cells are a unique subset of T lymphocytes that recognize lipid antigens presented by nonpolymorphic major histocompatibility complex (MHC) I-like molecule CD1d. iNKT cells play essential roles in regulating immune responses against cancer, viral infection, autoimmune disease, and allergy. However, the study and application of iNKT cells have been hampered by their very small numbers (0.01-1% in mouse and human blood). Here, we describe protocols to (1) generate mouse iNKT cells from mouse mononuclear cells or from mouse hematopoietic stem cells engineered with iNKT T cell receptor (TCR) gene (denoted as mMNC-iNKT cells or mHSC-iNKT cells, respectively), (2) generate human iNKT cells from human peripheral blood mononuclear cells or from human HSC cells engineered with iNKT TCR gene (denoted as hPBMC-iNKT cells or hHSC-iNKT cells, respectively), and (3) characterize mouse and human iNKT cells in vitro and in vivo.

Targeting monoamine oxidase A-regulated tumor-associated macrophage polarization for cancer immunotherapy.

Targeting tumor-associated macrophages (TAMs) is a promising strategy to modify the immunosuppressive tumor microenvironment and improve cancer immunotherapy. Monoamine oxidase A (MAO-A) is an enzyme best known for its function in the brain; small molecule MAO inhibitors (MAOIs) are clinically used for treating neurological disorders. Here we observe MAO-A induction in mouse and human TAMs. MAO-A-deficient mice exhibit decreased TAM immunosuppressive functions corresponding with enhanced antitumor immunity. MAOI treatment induces TAM reprogramming and suppresses tumor growth in preclinical mouse syngeneic and human xenograft tumor models. Combining MAOI and anti-PD-1 treatments results in synergistic tumor suppression. Clinical data correlation studies associate high intratumoral MAOA expression with poor patient survival in a broad range of cancers. We further demonstrate that MAO-A promotes TAM immunosuppressive polarization via upregulating oxidative stress. Together, these data identify MAO-A as a critical regulator of TAMs and support repurposing MAOIs for TAM reprogramming to improve cancer immunotherapy.

Targeting monoamine oxidase A for T cell-based cancer immunotherapy.

Monoamine oxidase A (MAO-A) is an enzyme best known for its function in the brain, where it breaks down neurotransmitters and thereby influences mood and behavior. Small-molecule MAO inhibitors (MAOIs) have been developed and are clinically used for treating depression and other neurological disorders. However, the involvement of MAO-A in antitumor immunity has not been reported. Here, we observed induction of the Maoa gene in tumor-infiltrating immune cells. Maoa knockout mice exhibited enhanced antitumor T cell immunity and suppressed tumor growth. MAOI treatment significantly suppressed tumor growth in preclinical mouse syngeneic and human xenograft tumor models in a T cell-dependent manner. Combining MAOI and anti-PD-1 treatments generated synergistic tumor suppression effects. Clinical data correlation studies associated intratumoral MAOA expression with T cell dysfunction and decreased patient survival in a broad range of cancers. We further demonstrated that MAO-A restrains antitumor T cell immunity through controlling intratumoral T cell autocrine serotonin signaling. Together, these data identify MAO-A as an immune checkpoint and support repurposing MAOI antidepressants for cancer immunotherapy.

Reversal of autoimmunity by mixed chimerism enables reactivation of ß cells and transdifferentiation of α cells in diabetic NOD mice.

Type 1 diabetes (T1D) results from the autoimmune destruction of ß cells, so cure of firmly established T1D requires both reversal of autoimmunity and restoration of ß cells. It is known that ß cell regeneration in nonautoimmune diabetic mice can come from differentiation of progenitors and/or transdifferentiation of α cells. However, the source of ß cell regeneration in autoimmune nonobese diabetic (NOD) mice remains unclear. Here, we show that, after reversal of autoimmunity by induction of haploidentical mixed chimerism, administration of gastrin plus epidermal growth factor augments ß cell regeneration and normalizes blood glucose in the firmly established diabetic NOD mice. Using transgenic NOD mice with inducible lineage-tracing markers for insulin-producing ß cells, Sox9+ ductal progenitors, Nestin+ mesenchymal stem cells, and glucagon-producing α cells, we have found that both reactivation of dysfunctional low-level insulin expression (insulinlo) ß cells and neogenesis contribute to the regeneration, with the latter predominantly coming from transdifferentiation of α cells. These results indicate that, after reversal of autoimmunity, reactivation of ß cells and transdifferentiation of α cells can provide sufficient new functional ß cells to reach euglycemia in firmly established T1D.

miR-194 is a marker of hepatic epithelial cells and suppresses metastasis of liver cancer cells in mice.

UNLABELLED: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by interacting with the 3' untranslated region (3'-UTR) of multiple mRNAs. Recent studies have linked miRNAs to the development of cancer metastasis. In this study, we show that miR-194 is specifically expressed in the human gastrointestinal tract and kidney. Moreover, miR-194 is highly expressed in hepatic epithelial cells, but not in Kupffer cells or hepatic stellate cells, two types of mesenchymal cells in the liver. miR-194 expression was decreased in hepatocytes cultured in vitro, which had undergone a dedifferentiation process. Furthermore, expression of miR-194 was low in liver mesenchymal-like cancer cell lines. The overexpression of miR-194 in liver mesenchymal-like cancer cells reduced the expression of the mesenchymal cell marker N-cadherin and suppressed invasion and migration of the mesenchymal-like cancer cells both in vitro and in vivo. We further demonstrated that miR-194 targeted the 3'-UTRs of several genes that were involved in epithelial-mesenchymal transition and cancer metastasis. CONCLUSION: These results support a role of miR-194, which is specifically expressed in liver parenchymal cells, in preventing liver cancer cell metastasis.