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Background: We studied humoral and cellular responses against SARS-CoV-2 longitudinally in a homogeneous population of healthy young/middle-aged men of South Asian ethnicity with mild COVID-19. Met...

Significance Cure of autoimmune type 1 diabetes (T1D) requires both reversal of autoimmunity and regeneration or resupply of insulin-producing β cells. We have observed that combination therapy with induction of haploidentical mixed chimerism and administration of gastrin and epidermal growth factor (EGF) cures firmly established T1D. The predominant source of β cell regeneration in mice comes from reactivation of dysfunctional insulinlo β cells and transdifferentiation of α cells. These studies have provided insights into β cell regeneration mechanisms in firmly established autoimmune T1D, in particular, reactivation of the insulinlo β cells after reversal of autoimmunity by induction of haploidentical mixed chimerism. These studies also provide a preclinical scientific basis for the feasibility of cure of long-standing T1D in humans., Type 1 diabetes (T1D) results from the autoimmune destruction of β cells, so cure of firmly established T1D requires both reversal of autoimmunity and restoration of β cells. It is known that β cell regeneration in nonautoimmune diabetic mice can come from differentiation of progenitors and/or transdifferentiation of α cells. However, the source of β 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 β 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 β 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) β 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 β cells and transdifferentiation of α cells can provide sufficient new functional β cells to reach euglycemia in firmly established T1D.

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 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., Monoamine oxidase A (MAO-A) is an outer mitochondrial membrane-bound enzyme best known for its function in the brain, but also linked to cancer progression. Here, the authors show that MAO-A is expressed in tumor associated macrophages, promoting their immunosuppressive properties, and that MAO inhibition suppresses tumor growth in preclinical models.

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.

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.

Natural killer T (NKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer. This has made NKT cells attractive agents for cell-based cancer therapies. Various approaches have been applied in clinical trials to target NKT cells, and the recent advances in chimeric antigen receptor (CAR) have also shown great potential in increasing the efficacy of NKT cells. In this chapter, we first introduce the biology and importance of NKT cells in antitumor immunity. Then, we summarize the preclinical data and clinical trials utilizing NKT cell-based immunotherapies. Finally, we discuss the challenges and future work that could be done to unleash the full potential of NKT cells for cancer immunotherapy.

Background: We studied humoral and cellular responses against SARS-CoV-2 longitudinally in a homogeneous population of healthy young/middle-aged men of South Asian ethnicity with mild COVID-19. Methods: In total, we recruited 994 men (median age: 34 years) post-COVID-19 diagnosis. Repeated cross-sectional surveys were conducted between May 2020 and January 2021 at six time points – day 28 (n = 327), day 80 (n = 202), day 105 (n = 294), day 140 (n = 172), day 180 (n = 758), and day 280 (n = 311). Three commercial assays were used to detect anti-nucleoprotein (NP) and neutralizing antibodies. T cell response specific for Spike, Membrane and NP SARS-CoV-2 proteins was tested in 85 patients at day 105, 180, and 280. Results: All serological tests displayed different kinetics of progressive antibody reduction while the frequency of T cells specific for different structural SARS-CoV-2 proteins was stable over time. Both showed a marked heterogeneity of magnitude among the studied cohort. Comparatively, cellular responses lasted longer than humoral responses and were still detectable nine months after infection in the individuals who lost antibody detection. Correlation between T cell frequencies and all antibodies was lost over time. Conclusion: Humoral and cellular immunity against SARS-CoV-2 is induced with differing kinetics of persistence in those with mild disease. The magnitude of T cells and antibodies is highly heterogeneous in a homogeneous study population. These observations have implications for COVID-19 surveillance, vaccination strategies, and post-pandemic planning.

Targeting tumour-associated macrophages (TAMs) is a promising strategy to modify the immunosuppressive tumour 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 observed MAO-A induction in mouse and human TAMs. MAO-A-deficient mice exhibited decreased TAM immunosuppressive functions corresponding with enhanced antitumour immunity. MAOI treatment induced TAM reprogramming and suppressed tumour growth in preclinical mouse syngeneic and human xenograft tumour models. Combining MAOI and anti-PD-1 treatments resulted in synergistic tumour suppression. Clinical data correlation studies associated high intratumoural MAOA expression with poor patient survival in a broad range of cancers. We further demonstrated 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.

Adult acute lymphoblastic leukemia (ALL) is a highly aggressive cancer with poor clinical prognosis and high relapse rate. Whereas allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for highly differentiated hematologic malignancies, allo-HSCT provides only limited benefits for treating ALL with relapses frequent. Additionally, the development of graft-versus-host-disease (GVHD) remains a major concern for allo-HSCT recipients. Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that express both a semi-invariant T cell receptor and natural killer cell markers. They recognize lipid antigens presented by non-polymorphic CD1d and have been reported to directly target CD1d+ solid tumors. Many B-ALL, including the P210 B-ALL cell line, are also CD1d+. Clinically, increased iNKT cells have simultaneously been correlated with reduced risk of developing acute GVHD and lower rates of malignant relapse. Because iNKT cells comprise only a tiny fraction of total T cells, we established a protocol to insert an iNKT TCR into murine HSC, thereby generating retrogenic mice with 5-10x expansion of iNKT cells in spleen and liver. Although engineered T cell therapy, including CD19-CART therapy, has proven to be effective in inducing remission in many B-ALL patients, when used as a monotherapy, relapse remains a major obstacle. We therefore tested whether a combination of allo-HSCT and iNKT cell therapy could robustly prevent GVHD while preserving graft-versus-leukemia effects (GVL). To test whether the addition of iNKT cells into allo-graft was capable of preventing acute GVHD (aGVHD), lethally irradiated BALB/C were given B6 bone marrow (2.5 x 106) and whole splenocytes (2.5 x 106, 5 x 106, or 10 x 106) from either B6 or retrogenic B6-iNKT mice. Mice that received 5 x 106 B6 splenocytes developed lethal aGVHD and became moribund by Day 10. Half of the mice that received 2.5 x 106 B6 splenocytes develop lethal aGVHD and died by Day 20. In contrast, only half of the mice that received 5 x 106 or 10 x 106 B6-iNKT splenocytes developed lethal aGVHD by Day 20, and all recipients of 2.5 x 106 B6 splenocytes remained alive by Day 20, suggesting the addition of iNKT cells into allograft may reduce the severity of aGVHD. To test whether adding iNKT cells to allograft could enhance GVL without causing GVHD, BALB/C mice with pre-established P210 B-ALL leukemia were given allo-HSCT with or without iNKT cell addition. Initially, our data showed freshly isolated iNKT cells were rapidly cleared following transplantation. We therefore included lethally irradiated P210 loaded with α-Galactosylceramide (α-Galcer) as adjuvant, and were able to see robust expansion of iNKT cells in vitro and in vivo (>20% vs Disclosures No relevant conflicts of interest to declare.

Direct lineage conversion is a promising approach to generate therapeutically important cell types for disease modeling and tissue repair. However, the survival and function of lineage-reprogrammed cells in vivo over the long term has not been examined. Here, using an improved method for in vivo conversion of adult mouse pancreatic acinar cells toward beta cells, we show that induced beta cells persist for up to 13 months (the length of the experiment), form pancreatic islet-like structures and support normoglycemia in diabetic mice. Detailed molecular analyses of induced beta cells over 7 months reveal that global DNA methylation changes occur within 10 d, whereas the transcriptional network evolves over 2 months to resemble that of endogenous beta cells and remains stable thereafter. Progressive gain of beta-cell function occurs over 7 months, as measured by glucose-regulated insulin release and suppression of hyperglycemia. These studies demonstrate that lineage-reprogrammed cells persist for >1 year and undergo epigenetic, transcriptional, anatomical and functional development toward a beta-cell phenotype.

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. (HEPATOLOGY 2010;.)

The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and a sensor and detoxifier of both xenobiotics and endobiotics. Recent studies also show that CAR participates in metabolism of glucose and lipid, and has an important role in fatty liver disease and diabetes. In this study, we investigate the roles of CAR in chronic and acute alcohol-induced liver injuries. The results showed that absence of CAR in rodents led to significantly increased susceptibility to chronic alcohol-induced liver injury, which was accompanied with elevated hepatocyte apoptosis and fat accumulation. However, pre-activation of CAR by a CAR agonist, TCPOBOP, strongly enhanced the hepatic toxicity by both chronic and acute alcohol infusion in wild-type, but not in CAR(-/-) mice. Gene expression analyses indicated that CAR pre-activation and alcohol infusion synergistically decreased the expression of enzymes that metabolize the alcohol in liver. These results support a role of CAR in modulating alcoholic liver injury and imply a risk of synergistic liver toxicity induced by alcohol and CAR activation.

Nat. Biotechnol. 32, 1223–1230 (2014); published online 17 November 2014; corrected after print 14 May 2015 In the version of this article initially published, Yingying Zhang's name was spelled Yinying Zhang. The error has been corrected in the HTML and PDF versions of the article.