The origin of Langerhans cells (LCs), which are skin epidermis-resident macrophages, remains unclear. Current lineage tracing of LCs largely relies on the promoter-Cre-LoxP system, which often gives rise to contradictory conclusions with different promoters. Thus, reinvestigation with an improved tracing method is necessary. Here, using a laser-mediated temporal-spatial resolved cell labeling method, we demonstrated that most adult LCs originated from the ventral wall of the dorsal aorta (VDA), an equivalent to the mouse aorta, gonads, and mesonephros (AGM), where both hematopoietic stem cells (HSCs) and non-HSC progenitors are generated. Further fine-fate mapping analysis revealed that the appearance of LCs in adult zebrafish was correlated with the development of HSCs, but not T cell progenitors. Finally, we showed that the appearance of tissue-resident macrophages in the brain, liver, heart, and gut of adult zebrafish was also correlated with HSCs. Thus, the results of our study challenged the EMP-origin theory for LCs.

Human cytomegalovirus (HCMV) causes serious HCMV-related diseases in immunocompromised people. Vaccination is the most effective measure to control infection with the pathogen, yet no vaccine has been licensed till now. We performed a head-to-head comparison of the protective abilities of multiple DNA vaccines in murine model of murine cytomegalovirus (MCMV) infection.

Genetic optimization of Nucleic Acid immunogens is important for potentially improving their immune potency. A COVID-19 DNA vaccine is in phase III clinical trial which is based on a promising highly developable technology platform. Here, we show optimization in mice generating a pGX-9501 DNA vaccine encoding full-length spike protein, which results in induction of potent humoral and cellular immune responses, including neutralizing antibodies, that block hACE2-RBD binding of live CoV2 virus in vitro. Optimization resulted in improved induction of cellular immunity by pGX-9501 as demonstrated by increased IFN-γ expression in both CD8+ and CD4 + T cells and this was associated with more robust antiviral CTL responses compared to unoptimized constructs. Vaccination with pGX-9501 induced subsequent protection against virus challenge in a rigorous hACE2 transgenic mouse model. Overall, pGX-9501 is a promising optimized COVID-19 DNA vaccine candidate inducing humoral and cellular immunity contributing to the vaccine's protective effects.

Docosahexaenoic acid (DHA) supplementation is beneficial for several chronic diseases; however, its effect on immune regulation is still debated. Given the prevalence of cytomegalovirus (CMV) infection and because natural killer (NK) cells are a component of innate immunity critical for controlling CMV infection, the current study explored the effect of a DHA-enriched diet on susceptibility to murine (M) CMV infection and the NK cell effector response to MCMV infection.

Systemic immune-inflammation index (SII) has been demonstrated to be closely associated with the poor prognosis of nasopharyngeal carcinoma (NPC). However, the role of SII during treatment of NPC has not been reported. This study aimed to determine the prognostic value of SII during treatment for NPC patients.

Tuberous sclerosis complex 1 (Tsc1) is known to regulate the development and function of various cell types, and RORγt is a critical transcription factor in the immune system. However, whether Tsc1 participates in regulating RORγt-expressing cells remains unknown.

The clinical features of EBV-positive diffuse large B cell lymphoma (DLBCL) indicate a poorer prognosis than EBV-negative DLBCL. Currently, there is no efficacious drug for EBV-positive DLBCL. The cytokine interleukin-21 (IL-21) has been reported to be pro-apoptotic in DLBCL cell lines and is being explored as a new therapeutic strategy for this type of lymphomas. However, our previous studies showed that IL-21 stimulation of EBV-positive DLBCL cell lines leads to increased proliferation. Here, analysis of a rare clinical sample of EBV-positive DLBCL, in combination with a NOD/SCID mouse xenograft model, confirmed the effect of IL-21 on the proliferation of EBV-positive DLBCL cells. Using RNA-sequencing, we identified the pattern of differentially-expressed genes following IL-21 treatment and verified the expression of key genes at the protein level using western blotting. We found that IL-21 upregulates expression of the host MYC and AP-1 (composed of related Jun and Fos family proteins) and STAT3 phosphorylation, as well as expression of the viral LMP-1 protein. These proteins are known to promote the G1/S phase transition to accelerate cell cycle progression. Furthermore, in NOD/SCID mouse xenograft model experiments, we found that IL-21 treatment increases glucose uptake and angiogenesis in EBV-positive DLBCL tumours. Although more samples are needed to validate these observations, our study reconfirms the adverse effects of IL-21 on EBV-positive DLBCL, which has implications for the drug development of DLBCL.

To assess the long-term consequences of coronavirus disease (COVID-19) among health care workers (HCWs) in China (hereafter surviving HCWs).

Background: Pre- and post-treatment plasma Epstein-Barr virus (EBV) DNA are important biomarkers for the prognosis of nasopharyngeal carcinoma (NPC). This study was performed to determine the prognostic potential of integrating EBV DNA levels in plasma measured pre-treatment (pre-EBV) and 3 months post-treatment (3 m-EBV). Materials and methods: A total of 543 incident non-metastatic NPC patients treated with intensity-modulated radiotherapy, with or without chemotherapy, were reviewed. Patients were divided into four subgroups based on pre-EBV and 3 m-EBV status. The data for pre-EBV and 3 m-EBV samples were integrated, and the predictability of the survival of patients with NPC was analyzed. Results: There were significant differences in the 5-year progression-free survival, distant metastasis-free survival, locoregional relapse-free survival, and overall survival among the four patient subgroups (P<0.001). Patients who tested negative for both pre-EBV and 3 m-EBV had the best prognosis, followed by patients who tested positive for pre-EBV and negative for 3 m-EBV, and those who tested negative for pre-EBV and positive for 3 m-EBV; however, patients who tested positive for both pre-EBV and 3 m-EBV had the poorest chances of survival. Multivariate analyses demonstrated that integration of pre-EBV and 3 m-EBV data was an independent predictor of NPC progression in patients. Receiver operating characteristic curve analysis further confirmed that the combination of pre-EBV and 3 m-EBV had a greater prognostic value than pre-EBV or 3 m-EBV alone. Conclusions: Integrating pre-EBV and 3 m-EBV data could provide more accurate risk stratification and better prognostic prediction in NPC.

Purkinje cell (PC) synapses onto cerebellar nuclei (CbN) neurons convey signals from the cerebellar cortex to the rest of the brain. PCs are inhibitory neurons that spontaneously fire at high rates, and many uniform sized PC inputs are thought to converge onto each CbN neuron to suppress or eliminate firing. Leading theories maintain that PCs encode information using either a rate code, or by synchrony and precise timing. Individual PCs are thought to have limited influence on CbN neuron firing. Here, we find that single PC to CbN synapses are highly variable in size, and using dynamic clamp and modelling we reveal that this has important implications for PC-CbN transmission. Individual PC inputs regulate both the rate and timing of CbN firing. Large PC inputs strongly influence CbN firing rates and transiently eliminate CbN firing for several milliseconds. Remarkably, the refractory period of PCs leads to a brief elevation of CbN firing prior to suppression. Thus, PC-CbN synapses are suited to concurrently convey rate codes, and generate precisely-timed responses in CbN neurons. Variable input sizes also elevate the baseline firing rates of CbN neurons by increasing the variability of the inhibitory conductance. Although this reduces the relative influence of PC synchrony on the firing rate of CbN neurons, synchrony can still have important consequences, because synchronizing even two large inputs can significantly increase CbN neuron firing. These findings may be generalized to other brain regions with highly variable sized synapses.

Most cancer immunotherapy approaches aim to stimulate cytotoxic CD8+ T lymphocytes to reject tumor cells. Due to the tumor-mediated suppressive micro-environment, of which the major contributor is regulatory T cells (Tregs), promising preclinical approaches were disappointing in clinical settings. Our recent study demonstrated that transient interruption of Tregs could induce CD8+ T cell responses to reject tumors in an animal model. The long-term tumor protective effect has yet not to be investigated. In this study, mice with Treg depletion rejected tumors and were rechallenged to study anti-tumor memory immune responses. The effects of major immune cell subsets on tumor protection were explored. Finally, we demonstrate that transient depletion of Tregs during primary tumor challenge can result in long-lasting protection against the tumor rechallenge. Skin-resident memory T cells (sTRM) were major factors in rejecting rechallenged tumors even when peripheral T cells were deficient. These findings highlight a promising strategy for empowering tissue-resident memory T cells for cancer prevention and immunotherapy in humans by interrupting Tregs.

Natural killer (NK) cells play important roles in controlling virus-infected and malignant cells. The identification of new molecules that can activate NK cells may effectively improve the antiviral and antitumour activities of these cells. In this study, by using a commercially available metabolism-related compound library, we initially screened the capacity of compounds to activate NK cells by determining the ratio of interferon-gamma (IFN-γ)+ NK cells by flow cytometry after the incubation of peripheral blood mononuclear cells (PBMCs) with IL-12 or IL-15 for 18 h. Our data showed that eight compounds (nafamostat mesylate (NM), loganin, fluvastatin sodium, atorvastatin calcium, lovastatin, simvastatin, rosuvastatin calcium, and pitavastatin calcium) and three compounds (NM, elesclomol, and simvastatin) increased the proportions of NK cells and CD3+ T cells that expressed IFN-γ among PBMCs cultured with IL-12 and IL-15, respectively. When incubated with enriched NK cells (purity ≥ 80.0%), only NM enhanced NK cell IFN-γ production in the presence of IL-12 or IL-15. When incubated with purified NK cells (purity ≥ 99.0%), NM promoted NK cell IFN-γ secretion in the presence or absence of IL-18. However, NM showed no effect on NK cell cytotoxicity. Collectively, our study identifies NM as a selective stimulator of IFN-γ production by NK cells, providing a new strategy for the prevention and treatment of infection or cancer in select populations.

ABSTRACTThe COVID-19 disease caused by infection with SARS-CoV-2 and its variants is devastating to the global public health and economy. To date, over a hundred COVID-19 vaccines are known to be under development, and the few that have been approved to fight the disease are using the spike protein as the primary target antigen. Although virus-neutralizing epitopes are mainly located within the RBD of the spike protein, the presence of T cell epitopes, particularly the CTL epitopes that are likely to be needed for killing infected cells, has received comparatively little attention. This study predicted several potential T cell epitopes with web-based analytic tools and narrowed them down from several potential MHC-I and MHC-II epitopes by ELIspot and cytolytic assays to a conserved MHC-I epitope. The epitope is highly conserved in current viral variants and compatible with a presentation by most HLA alleles worldwide. In conclusion, we identified a CTL epitope suitable for evaluating the CD8+ T cell-mediated cellular response and potentially for addition into future COVID-19 vaccine candidates to maximize CTL responses against SARS-CoV-2.

Nitric oxide (NO) is a short-lived signaling molecule that plays a pivotal role in cardiovascular system. Organic nitrates represent a class of NO-donating drugs for treating coronary artery diseases, acting through the vasodilation of systemic vasculature that often leads to adverse effects. Herein, we design a nitrate-functionalized patch, wherein the nitrate pharmacological functional groups are covalently bound to biodegradable polymers, thus transforming small-molecule drugs into therapeutic biomaterials. When implanted onto the myocardium, the patch releases NO locally through a stepwise biotransformation, and NO generation is remarkably enhanced in infarcted myocardium because of the ischemic microenvironment, which gives rise to mitochondrial-targeted cardioprotection as well as enhanced cardiac repair. The therapeutic efficacy is further confirmed in a clinically relevant porcine model of myocardial infarction. All these results support the translational potential of this functional patch for treating ischemic heart disease by therapeutic mechanisms different from conventional organic nitrate drugs.

Cancer is a major public health threat, and researchers are constantly looking for new ways to develop effective treatments. One approach is the use of cancer vaccines, which work by boosting the body's immune system to fight cancer. The goal of this study was to develop an effective cancer vaccine using rendered-inactive tumor cells. A CMS5 fibrosarcoma tumor model in BALB/c mice and an E.G7 lymphoma tumor model in C57BL/6 mice were used to evaluate how mitomycin C-inactivated tumor cells mediated tumor protection. The results showed that immunization with inactivated CMS5 cells significantly improved tumor suppression after a challenge with live CMS5 tumor cells, but no effect was observed using the E.G7 tumor model. The results suggested that DC (dendritic cell) responses to tumor antigens are critical. The maturation and activation of DCs were effectively promoted by mitomycin C-treated CMS5 cells, as well as enhanced phagocytosis ability in vitro. The tumor-protective effects established by the vaccination of inactivated CMS5 cells were CD8+ T cell-dependent, as the antitumor responses disappeared after eliminating CD8+ T cells. It was found that the tumor-prevention efficacy was dramatically increased by combining inactivated CM55 tumor cells with anti-CD25 antibodies to temporarily deplete Treg cells (regulatory T cells). This strategy could also significantly induce the rejection against E.G7 tumors. In addition, vaccination with anti-CD25 antibodies plus inactivated CMS5 cells elicited antitumor responses against heterologous tumors. According to the findings of this study, combining the immunization of inactivated tumor cells with an anti-CD25 antibody may be an effective method for cancer prevention.

We previously reported that deletion of Foxo1, via Ncr1-iCre mice from the expression of NKp46 onward, led to enhanced natural killer (NK) cell maturation and effector function. In this model, however, the role of Foxo1 in regulating NK cell specification and early development remains exclusive. Herein, we utilized a murine model of hematopoietic-specific deletion of Foxo1 before lymphoid specification, by crossing mice carrying floxed Foxo1 alleles (Foxo1fl/fl) with Vav1-iCre mice, to revisit the role of Foxo1 on NK cell specification and early development. The data showed that hematopoietic-specific deletion of Foxo1 resulted in increased proportion and numbers of common lymphoid progenitors (CLP) (Lin-CD127+c-Kit+Sca-1+), pre-pro NK b cells (Lin-Sca-1+c-Kit-CD135-CD127+), as well as committed Lin-CD122+ cells and CD3-CD19-NKp46+ NK cells in bone marrow. Hematopoietic-specific deletion of Foxo1 also promoted NK cells proliferation in a cell-intrinsic manner, indicated by increased Ki-67 expression and more expansion of NK cell after ex vivo stimulation with IL-15. The reason for Foxo1 suppressing NK cell proliferation might be its direct transcription of the cell-cycle inhibitory genes, such as p21cip1, p27kip1, p130, Gadd45a, and Ccng2 (cyclin G2) in NK cells, supported by the evidence of decreased mRNA expression of p21cip1, p27kip1, p130, Gadd45a, and Ccng2 in Foxo1-deficient NK cells and direct binding of Foxo1 on their promoter region. Furthermore, hematopoietic-specific deletion of Foxo1 resulted in increased ratio of mature NK subsets, such as CD11b+CD27- and CD43+KLRG1+ NK cells, but decreased ratio of immature NK subsets, such as CD27+CD11b- and CD27+CD11b+ NK cells, consistent with the findings in the murine model of Ncr1-iCre mediated Foxo1 deletion. Conclusively, Foxo1 not only acts as a negative checkpoint on NK cell maturation, but also represses NK cell specification and proliferation. The relative higher expression of Foxo1 in CLP and early NK precursors may also contribute to the later NK cell proliferation and responsiveness, which warranties another separate study in the future.

Innate lymphoid cells (ILCs), including ILC1s, ILC2s, and ILC3s, play critical roles in regulating immunity, inflammation, and tissue homeostasis. However, limited attention is focused on the unique phenotype of ILCs in the heart tissue. In this study, we analyzed the ILC subsets in the heart by flow cytometry and found that ILC2s were the dominant population of ILCs, while a lower proportion of type 1 ILCs (including ILC1 and NK cells) and merely no ILC3s in the heart tissue of mice. Our results show that ILC2 development kinetically peaked in heart ILC2s at the age of 4 weeks after birth and later than lung ILC2s. By conducting parabiosis experiment, we show that heart ILC2s are tissue resident cells and minimally replaced by circulating cells. Notably, heart ILC2s have unique phenotypes, such as lower expression of ICOS, CD25 (IL-2Rα), and Ki-67, higher expression of Sca-1 and GATA3, and stronger ability to produce IL-4 and IL-13. In doxorubicin-induced myocardial necroptosis model of mouse heart tissue, IL-33 mRNA expression level and ILC2s were remarkably increased. In addition, IL-4 production by heart ILC2s, but not lung ILC2s, was also dramatically increased after doxorubicin treatment. Our results demonstrate that heart-resident ILC2s showed tissue-specific phenotypes and rapidly responded to heart injury. Thus, further studies are warranted to explore the potential for IL-33-elicited ILC2s response as therapeutics for attenuating heart damage.

7-Met, a derivative of soybean isoflavone, is a natural flavonoid compound that has been reported to have multiple signaling pathways regulation effects. This study investigated the therapeutic effects of 7-Met on mice with atopic dermatitis induced by fluorescein isothiocyanate (FITC), or oxazolone (OXZ). 7-Met ameliorated FITC or OXZ-induced atopic dermatitis symptoms by decreasing ear thickness, spleen index, mast cell activation, neutrophil infiltration and serum IgE levels in female BALB/c mice. In FITC-induced atopic dermatitis mice, 7-Met reduced Th1 cytokines production and regulated Th1/Th2 balance by downregulating the secretion of thymic stromal lymphopoietin (TSLP) via inactivation of the NF-κB pathway. In OXZ-induced atopic dermatitis, 7-Met functioned through the reduction of Th17 cytokine production. Our study showed that 7-Methoxyisoflavone alleviated atopic dermatitis by regulating multiple signaling pathways and downregulating chemokine production.

Purkinje cell (PC) synapses onto cerebellar nuclei (CbN) neurons allow signals from the cerebellar cortex to influence the rest of the brain. PCs are inhibitory neurons that spontaneously fire at high rates, and many PC inputs are thought to converge onto each CbN neuron to suppress its firing. It has been proposed that PCs convey information using a rate code, a synchrony and timing code, or both. The influence of PCs on CbN neuron firing was primarily examined for the combined effects of many PC inputs with comparable strengths, and the influence of individual PC inputs has not been extensively studied. Here, we find that single PC to CbN synapses are highly variable in size, and using dynamic clamp and modeling we reveal that this has important implications for PC-CbN transmission. Individual PC inputs regulate both the rate and timing of CbN firing. Large PC inputs strongly influence CbN firing rates and transiently eliminate CbN firing for several milliseconds. Remarkably, the refractory period of PCs leads to a brief elevation of CbN firing prior to suppression. Thus, individual PC-CbN synapses are suited to concurrently convey rate codes and generate precisely timed responses in CbN neurons. Either synchronous firing or synchronous pauses of PCs promote CbN neuron firing on rapid time scales for nonuniform inputs, but less effectively than for uniform inputs. This is a secondary consequence of variable input sizes elevating the baseline firing rates of CbN neurons by increasing the variability of the inhibitory conductance. These findings may generalize to other brain regions with highly variable inhibitory synapse sizes.

The understanding of the heterogeneous cellular microenvironment of colonic polyps in paediatric patients with solitary juvenile polyps (SJPs), polyposis syndrome (PJS) and Peutz-Jeghers syndrome (PJS) remains limited.