FOXO1 is a master regulator of memory programming in CAR T cells.

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2-6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.

Generation of a new therapeutic D-peptide that induces the differentiation of acute myeloid leukemia cells through A TLR-2 signaling pathway.

Acute myeloid leukemia (AML) is caused by clonal disorders of hematopoietic stem cells. Differentiation therapy is emerging as an important treatment modality for leukemia, given its less toxicity and wider applicable population, but the arsenal of differentiation-inducing agents is still very limited. In this study, we adapted a competitive peptide phage display platform to search for candidate peptides that could functionally induce human leukemia cell differentiation. A monoclonal phage (P6) and the corresponding peptide (pep-P6) were identified. Both L- and D-chirality of pep-P6 showed potent efficiency in inducing AML cell line differentiation, driving their morphologic maturation and upregulating the expression of macrophage markers and cytokines, including CD11b, CD14, IL-6, IL-1ß, and TNF-α. In the THP-1 xenograft animal model, administration of D-pep-P6 was effective in inhibiting disease progression. Importantly, exposure to D-pep-P6 induced the differentiation of primary human leukemia cells isolated AML patients in a similar manner to the AML cell lines. Further mechanism study suggested that D-pep-P6 induced human leukemia cell differentiation by directly activating a TLR-2 signaling pathway. These findings identify a novel D-peptide that may promote leukemia differentiation therapy.

FOXO1 is a master regulator of CAR T memory programming.

Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3-7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.

Dopamine Signaling Promotes Tissue-Resident Memory Differentiation of CD8+ T Cells and Antitumor Immunity.

Tissue-resident memory CD8+ T (TRM) cells have been associated with robust protective antitumor immune responses and improved prognosis of patients with cancer. Therefore, therapeutic strategies that modulate either the production or activity of TRM cells could be effective for treating cancer. Using a high-throughput drug screen, we showed that the neurotransmitter dopamine drives differentiation of CD8+ T cells into CD103+ TRM cells. In murine syngeneic tumor xenograft models and clinical human colon cancer samples, DRD5 served as the major functional dopamine receptor on CD8+ T cells and positively correlated with TRM cell density. DRD5 deficiency led to a failure of CD8+ T cells to accumulate in tissues, resulting in impaired TRM cell formation, reduced effector function, and uncontrolled disease progression. Moreover, dopamine treatment promoted the antitumor activity of CD8+ T cells and suppressed colorectal cancer growth in immunocompentent mouse models, and ex vivo preconditioning with dopamine enhanced the in vivo efficacy of chimeric antigen receptor (CAR)-T cells. Finally, in a patient with colorectal cancer cohort, dopamine expression was positively associated with patient survival and CD8+ T-cell infiltration. These findings suggest that dopaminergic immunoregulation plays an important role in the differentiation of CD8+ cells into CD103+ TRM cells and thereby modulates TRM-elicited antitumor immunity in colorectal cancer. SIGNIFICANCE: Identification of an immunostimulatory function of dopamine signaling by promoting tissue-resident memory T-cell differentiation and sustaining T-cell effector functions reveals potential therapeutic strategies and prognostic biomarkers for colorectal cancer.

Broad-spectrum diffractive network via ensemble learning.

We propose a broad-spectrum diffractive deep neural network (BS-D2NN) framework, which incorporates multiwavelength channels of input lightfields and performs a parallel phase-only modulation using a layered passive mask architecture. A complementary multichannel base learner cluster is formed in a homogeneous ensemble framework based on the diffractive dispersion during lightwave modulation. In addition, both the optical sum operation and the hybrid (optical-electronic) maxout operation are performed for motivating the BS-D2NN to learn and construct a mapping between input lightfields and truth labels under heterochromatic ambient lighting. The BS-D2NN can be trained using deep learning algorithms to perform a kind of wavelength-insensitive high-accuracy object classification.

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) sensitize melanoma cells to MEK inhibition and inhibit metastasis and relapse by inducing degradation of AXL.

Melanoma is highly heterogeneous with diverse genomic alterations and partial therapeutic responses. The emergence of drug-resistant tumor cell clones accompanied by a high AXL expression level is one of the major challenges for anti-tumor clinical care. Recent studies have demonstrated that high AXL expression in melanoma cells mediated drug resistance, epithelial-mesenchymal transition (EMT), and elevated survival of cancer stem cells (CSCs). Given that we have identified several non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin potently induce the degradation of AXL, we questioned whether NSAIDs could counteract the AXL-mediated neoplastic phenotypes. In this study, we found that NSAIDs downregulate PKA activity via the PGE2 /EP2/cAMP/PKA signaling pathway and interrupt the PKA-dependent interaction between CDC37 and HSP90, resulting in an incorrect AXL protein folding and finally AXL degradation through the ubiquitination-proteasome system (UPS) pathway. Furthermore, NSAIDs not only sensitized the MEK inhibitor treatment but also reduced EMT and relapse mediated by AXL in tumor tissue. Our findings suggest that the combination of inhibitors and NSAIDs, especially aspirin, could be a simple but efficient modality to treat melanoma in which AXL is a key factor for drug resistance, metastasis, and relapse.

Safety and immunogenicity of a quadrivalent influenza vaccine in adults aged 60 years or above: a phase III randomized controlled clinical study.

To control seasonal influenza epidemics in elders, a quadrivalent, inactivated, split-virion influenza vaccine (IIV4) comprising A and B lineages is produced for young individuals and adults aged ≥60 years. In this phase III, randomized, double-blind, active-controlled trial, we compared safety and immunogenicity of IIV4 with a licensed quadrivalent inactivated vaccine (IIV4-HL) produced by Hualan Biological Engineering during the 2019 influenza season. Participants were randomly assigned to receive IIV4 (n = 959) or IIV4-HL (n = 959). Compared to IIV4-HL, geometric mean titers (GMT) of hemagglutination inhibition (HAI) titers and seroconversion rate (SCR) of IIV4 demonstrated better antibody responses in A lineages (H1N1 and H3N2) (P < .01) and equivalent antibody responses in B lineages (B/Yamagata and B/Victoria) (P > .01) in both age groups. After immunization, IIV4 provided a satisfactory SCR and seroprotection rate (SPR) in elders. No discernible variation in immunogenicity was observed between the two age cohorts. In both age groups, IIV4 and IIV4-HL recipients experienced similar levels of solicited and unsolicited adverse events (AEs), and the incidence of AEs was low in both vaccine groups. Most AEs were of mild-to-moderate severity and no grade 3 AEs in IIV4 group, but AEs in adults aged 60-65 were little higher than in adults over 65 years in IIV4 and IIV4-HL groups (IIV4: 14.66% vs. 10.36%; IIV4-HL:14.67% vs. 11.43%). Totally, IIV4 was generally well tolerated and induced high antibody titers against all four influenza strains in elderly, making it a compelling alternative for the elderly aged ≥60 years.Trial registration: Clinical Trials.gov: 2015L00649-2.

Brd4 Regulates the Homeostasis of CD8+ T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation.

CD8+ T cells are major components of adaptive immunity and confer robust protective cellular immunity, which requires adequate T-cell numbers, targeted migration, and efficient T-cell proliferation. Altered CD8+ T-cell homeostasis and impaired proliferation result in dysfunctional immune response to infection or tumorigenesis. However, intrinsic factors controlling CD8+ T-cell homeostasis and immunity remain largely elusive. Here, we demonstrate the prominent role of Brd4 on CD8+ T cell homeostasis and immune response. By upregulating Myc and GLUT1 expression, Brd4 facilitates glucose uptake and energy production in mitochondria, subsequently supporting naïve CD8+ T-cell survival. Besides, Brd4 promotes the trafficking of naïve CD8+ T cells partially through maintaining the expression of homing receptors (CD62L and LFA-1). Furthermore, Brd4 is required for CD8+ T cell response to antigen stimulation, as Brd4 deficiency leads to a severe defect in clonal expansion and terminal differentiation by decreasing glycolysis. Importantly, as JQ1, a pan-BRD inhibitor, severely dampens CD8+ T-cell immune response, its usage as an anti-tumor agent or latency-reversing agent for human immunodeficiency virus type I (HIV-1) should be more cautious. Collectively, our study identifies a previously-unexpected role of Brd4 in the metabolic regulation of CD8+ T cell-mediated immune surveillance and also provides a potential immunomodulation target.

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2-infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.

Amino acids and RagD potentiate mTORC1 activation in CD8+ T cells to confer antitumor immunity.

BACKGROUND: In the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8+ tumor-infiltrating lymphocytes (TILs) is unclear. METHOD: In this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8+ TILs. RESULTS: We discovered that the Rag complex, particularly RagD, was crucial for CD8+ T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8+ TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8+ T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8+ T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo. CONCLUSION: Our results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway.

Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses.

Various vaccine strategies have been proposed in response to the global COVID-19 pandemic, each with unique strategies for eliciting immune responses. Here, we developed nanoparticle vaccines by covalently conjugating the self-assembled 24-mer ferritin to the receptor binding domain (RBD) and/or heptad repeat (HR) subunits of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. Compared to monomer vaccines, nanoparticle vaccines elicited more robust neutralizing antibodies and cellular immune responses. RBD and RBD-HR nanoparticle vaccinated hACE2 transgenic mice vaccinated with RBD and/or RBD-HR nanoparticles exhibited reduced viral load in the lungs after SARS-CoV-2 challenge. RBD-HR nanoparticle vaccines also promoted neutralizing antibodies and cellular immune responses against other coronaviruses. The nanoparticle vaccination of rhesus macaques induced neutralizing antibodies, and T and B cell responses prior to boost immunization; these responses persisted for more than three months. RBD- and HR-based nanoparticles thus present a promising vaccination approach against SARS-CoV-2 and other coronaviruses.

UHRF1 Controls Thymocyte Fate Decisions through the Epigenetic Regulation of EGR1 Expression.

Thymocyte differentiation is a highly complex process that is accompanied by epigenetic changes. Ubiquitin-like containing PHD ring finger 1 (UHRF1) is a critical epigenetic modifier involved in various cellular processes. In this study, we demonstrated that it is highly expressed in T cell precursors of the thymus. Further, its deficiency results in significantly reduced thymocyte cellularity and thymus size in mice. Through systematic analysis based on single-cell RNA sequencing, we found that UHRF1 deficiency thwarts αß T cell lineage development, whereas biasing γδ T lineage differentiation dampens the progression of immature single-positive cells. UHRF1 deficiency promotes the IL-17 secreting and RORγt expression in γδ T cell, indicating a Tγδ17 phenotype. Further, the analysis of gene-regulatory networks demonstrated that UHRF1 controls the expression of early growth response 1 (EGR1). UHRF1 interacts with DNA methyltransferase 1 (DNMT1) at the CpG promoter region of Egr1 loci and affects the nearby chromatin modifications of H3K9me3 and H3K4me3. Taken together, our results demonstrate that UHRF1 is a key factor that mediates the epigenetic regulation of EGR1 and, consequently, thymocyte fate decisions.

Smart inverse design of graphene-based photonic metamaterials by an adaptive artificial neural network.

The burgeoning research of graphene and other 2D materials enables many unprecedented metamaterials and metadevices for applications on nanophotonics. The design of on-demand graphene-based metamaterials often calls for the solution of a complex inverse problem within a small sampling space, which highly depends on the rich experiences from researchers of nanophotonics. Conventional optimization algorithms could be used for this inverse design, but they converge to local optimal solutions and take significant computational costs with increased nanostructure parameters. Here, we establish a deep learning method based on an adaptive batch-normalized neural network, aiming to implement smart and rapid inverse design for graphene-based metamaterials with on-demand optical responses. This method allows a quick converging speed with high precision and low computational consumption. As typical complex proof-of-concept examples, the optical metamaterials consisting of graphene/dielectric alternating multilayers are chosen to demonstrate the validity of our design paradigm. Our method demonstrates a high prediction accuracy of over 95% after very few training epochs. A universal programming package is developed to achieve the design goals of graphene-based metamaterials with low absorption and near unity absorption, respectively. Our work may find important design applications in the field of nanoscale photonics based on graphene and other 2D materials.

Adoptive Transfer of Interleukin-21-stimulated Human CD8+ T Memory Stem Cells Efficiently Inhibits Tumor Growth.

Memory stem T (TSCM) cells, a new subset of memory T cells with self-renewal and multipotent capacities, are considered as a promising candidates for adoptive cellular therapy. However, the low proportion of human TSCM cells in total CD8 T cells limits their utility. Here, we aimed to induce human CD8 TSCM cells by stimulating naive precursors with interleukin-21 (IL-21). We found that IL-21 promoted the generation of TSCM cells, described as CD45RACD45ROCD62LCCR7CD122CD95 cells, with a higher efficiency than that observed with other common γ-chain cytokines. Upon adoptive transfer into an A375 melanoma mouse model, these lymphocytes mediated much stronger antitumor responses. Further mechanistic analysis revealed that IL-21 activated the Janus kinase signal transducer and activator of transcription 3 pathway by upregulating signal transducer and activator of transcription 3 phosphorylation and consequently promoting the expression of T-bet and suppressor of cytokine signaling 1, but decreasing the expression of eomesodermin and GATA binding protein 3. Our findings provide novel insights into the generation of human CD8 TSCM cells and reveal a novel potential clinical application of IL-21.

A Cellular MicroRNA Facilitates Regulatory T Lymphocyte Development by Targeting the FOXP3 Promoter TATA-Box Motif.

The CD4+CD25+FOXP3+ regulatory T cells (Tregs) mediate immunological self-tolerance and suppress various immune responses. FOXP3 is a key transcriptional factor for the generation and development of Tregs. Its expression is regulated by various cytokines including TGF-ß, IL-2, and IL-10. It is important to further identify the regulatory factors for Tregs. Given that many microRNAs (miRNAs) could specifically interact with the core promoter region and specifically enhance the transcription of many target genes, we searched for any possible miRNA(s) targeting the core promoter region of the FOXP3 gene. We found that miR-4281, an miRNA specifically expressed in hominids, can potently and specifically upregulate FOXP3 expression by directly interacting with the TATA-box motif in the human FOXP3 promoter. Consequently, miR-4281 significantly accelerated the differentiation of human naive cells to induced Tregs (iTregs) that possess immune suppressor functions and weaken the development of graft-versus-host disease in a humanized mouse model. Interestingly, iTregs induced by the combination of TGF-ß, IL-2, and chemically synthesized miR-4281 were more stable and functional than those induced by TGF-ß and IL-2 alone. Moreover, we found that the IL-2/STAT5 signal transduction upregulates FOXP3 expression not only through the classical pathway, but also by enhancing the expression of the miR-4281 precursor gene (SNCB) and, correspondingly, miR-4281. This study reveals a novel mechanism regulating FOXP3 expression and human iTreg development and, therefore, offers a new therapeutic target to manipulate immunosuppressive system.