Integrated single-cell transcriptome and T cell receptor profiling reveals defects of T cell exhaustion in pulmonary tuberculosis.

Tuberculosis-affected lungs with chronic inflammation harbor abundant immunosuppressive immune cells but the nature of such inflammation is unclear. Dysfunction in T cell exhaustion, while implicated in chronic inflammatory diseases, remains unexplored in tuberculosis. Given that immunotherapy targeting exhaustion checkpoints exacerbates tuberculosis, we speculate that T cell exhaustion is dysfunctional in tuberculosis. Using integrated single-cell RNA sequencing and T cell receptor profiling we reported defects in exhaustion responses within inflamed tuberculosis-affected lungs. Tuberculosis lungs demonstrated significantly reduced levels of exhausted CD8+ T cells and exhibited diminished expression of exhaustion-related transcripts among clonally expanded CD4+ and CD8+ T cells. Additionally, clonal expansion of CD4+ and CD8+ T cells bearing T cell receptors specific for CMV was observed. Expanded CD8+ T cells expressed the cytolytic marker GZMK. Hence, inflamed tuberculosis-affected lungs displayed dysfunction in T cell exhaustion. Our findings likely hold implications for understanding the reactivation of tuberculosis observed in patients undergoing immunotherapy targeting the exhaustion checkpoint.

5HT2A modulation attenuates pancreatic cancer induced pain mouse model by inhibiting HDAC.

PURPOSE: Patients have been severely suffered from cancer associated pain, and pancreatic cancer is the most severe form of cancer associated with pain. There are very few options available to manage it. The present report evaluated the effect of 5HT2A on pancreatic cancer associated pain. METHODS: Pancreatic cancer was induced by injecting SW 1,990 cells (~3×106 in a 20 µL suspension) into the pancreas and formed a 2-3-mm vesicle using an inoculator fitted with a 26-gauge needle in BALB/c-nu mice. Survival rate and body weight of the mice were observed. Pain behaviour testing was performed at the end of each week (third and fourth week) after surgery. Inflammatory mediators and HDAC 2 proteins were determined in the spinal tissue using quantitative real-time polymerase chain reaction. RESULTS: There was improvement in the survival rate and body weight in 5HT2A antagonist treated group than pancreatic cancer group of mice. Moreover, 5HT2A antagonist ameliorated the alteration in pain behaviour of pancreatic cancer mice. mRNA expression of HDAC2 and level of inflammatory cytokines were reduced in the spinal tissue of 5HT 2A antagonist treated group than pancreatic cancer group of mice. CONCLUSIONS: Data revealed that 5HT2A antagonist ameliorates pain associated with pancreatic cancer mice by HDAC inhibition and inflammatory cytokines. The result of investigation supports that modulation of 5HT2A receptor could be used clinically to protects neuropathic pain in pancreatic cancer.

An intranasal combination vaccine induces systemic and mucosal immunity against COVID-19 and influenza.

Despite prolonged surveillance and interventions, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses continue to pose a severe global health burden. Thus, we developed a chimpanzee adenovirus-based combination vaccine, AdC68-HATRBD, with dual specificity against SARS-CoV-2 and influenza virus. When used as a standalone vaccine, intranasal immunization with AdC68-HATRBD induced comprehensive and potent immune responses consisting of immunoglobin (Ig) G, mucosal IgA, neutralizing antibodies, and memory T cells, which protected the mice from BA.5.2 and pandemic H1N1 infections. When used as a heterologous booster, AdC68-HATRBD markedly improved the protective immune response of the licensed SARS-CoV-2 or influenza vaccine. Therefore, whether administered intranasally as a standalone or booster vaccine, this combination vaccine is a valuable strategy to enhance the overall vaccine efficacy by inducing robust systemic and mucosal immune responses, thereby conferring dual lines of immunological defenses for these two viruses.

Construction of an infectious clone for enterovirus A89 and mutagenesis analysis of viral infection and cell binding.

Enterovirus A89 (EV-A89) is an unconventional strain belonging to the Enterovirus A species. Limited research has been conducted on EV-A89, leaving its biological and pathogenic properties unclear. Developing reverse genetic tools for EV-A89 would help to unravel its infection mechanisms and aid in the development of vaccines and anti-viral drugs. In this study, an infectious clone for EV-A89 was successfully constructed and recombinant enterovirus A89 (rEV-A89) was generated. The rEV-A89 exhibited similar characteristics such as growth curve, plaque morphology, and dsRNA expression with parental strain. Four amino acid substitutions were identified in the EV-A89 capsid, which were found to enhance viral infection. Mechanistic studies revealed that these substitutions increased the virus's cell-binding ability. Establishing reverse genetic tools for EV-A89 will significantly contribute to understanding viral infection and developing anti-viral strategies.IMPORTANCEEnterovirus A species contain many human pathogens and have been classified into conventional cluster and unconventional cluster. Most of the research focuses on various conventional members, while understanding of the life cycle and infection characteristics of unconventional viruses is still very limited. In our study, we constructed the infectious cDNA clone and single-round infectious particles for the unconventional EV-A89, allowing us to investigate the biological properties of recombinant viruses. Moreover, we identified key amino acids residues that facilitate EV-A89 infection and elucidate their roles in enhancing viral binding to host cells. The establishment of the reverse genetics system will greatly facilitate future study on the life cycle of EV-A89 and contribute to the development of prophylactic vaccines and anti-viral drugs.

Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research.

The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications, enabling the development of effective therapeutic and preventive strategies. In this study, our initial attempts to introduce a NanoLuc luciferase (NLuc) reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies. We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus. Therefore, we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71 (EV-A71), coxsackievirus A10 (CVA10), coxsackievirus A7 (CVA7), coxsackievirus A16 (CVA16), namely EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT, and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency. Notably, the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses. A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics. Moreover, these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages. In addition, the HiBiT-tagged viruses were successfully tested in antiviral drug assays, and the sensitivity of the viruses to drugs was not affected by the HiBiT tag. Ultimately, our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal, convenient, and invaluable method for advancing research in the realm of enterovirus virology. Furthermore, HiBiT-tagged enteroviruses exhibit great potential for diverse applications, including the development of antivirals and the elucidation of viral infection mechanisms.

PB2 residue 473 contributes to the mammalian virulence of H7N9 avian influenza virus by modulating viral polymerase activity via ANP32A.

Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity.IMPORTANCEThe H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes multi-organ damage, which includes hepatic dysfunction, as observed in over 50% of COVID-19 patients. Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (ACE2) is the primary receptor for SARS-CoV-2 entry into host cells, and studies have shown the presence of intracellular virus particles in human hepatocytes that express ACE2, but at extremely low levels. Consequently, we asked if hepatocytes might express receptors other than ACE2 capable of promoting the entry of SARS-CoV-2 into cells. To address this question, we performed a genome-wide CRISPR-Cas9 activation library screening and found that Asialoglycoprotein receptor 1 (ASGR1) promoted SARS-CoV-2 pseudovirus infection of HeLa cells. In Huh-7 cells, simultaneous knockout of ACE2 and ASGR1 prevented SARS-CoV-2 pseudovirus infection. In the immortalized THLE-2 hepatocyte cell line and primary hepatic parenchymal cells, both of which barely expressed ACE2, SARS-CoV-2 pseudovirus could successfully establish an infection. However, after treatment with ASGR1 antibody or siRNA targeting ASGR1, the infection rate significantly dropped, suggesting that SARS-CoV-2 pseudovirus infects hepatic parenchymal cells mainly through an ASGR1-dependent mechanism. We confirmed that ASGR1 could interact with Spike protein, which depends on receptor binding domain (RBD) and N-terminal domain (NTD). Finally, we also used Immunohistochemistry and electron microscopy to verify that SARS-CoV-2 could infect primary hepatic parenchymal cells. After inhibiting ASGR1 in primary hepatic parenchymal cells by siRNA, the infection efficiency of the live virus decreased significantly. Collectively, these findings indicate that ASGR1 is a candidate receptor for SARS-CoV-2 that promotes infection of hepatic parenchymal cells.

BTLA contributes to acute-on-chronic liver failure infection and mortality through CD4+ T-cell exhaustion.

B- and T-lymphocyte attenuator (BTLA) levels are increased in patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). This condition is characterized by susceptibility to infection and T-cell immune exhaustion. However, whether BTLA can induce T-cell immune exhaustion and increase the risk of infection remains unclear. Here, we report that BTLA levels are significantly increased in the circulating and intrahepatic CD4+ T cells from patients with HBV-ACLF, and are positively correlated with disease severity, prognosis, and infection complications. BTLA levels were upregulated by the IL-6 and TNF signaling pathways. Antibody crosslinking of BTLA activated the PI3K-Akt pathway to inhibit the activation, proliferation, and cytokine production of CD4+ T cells while promoting their apoptosis. In contrast, BTLA knockdown promoted their activation and proliferation. BTLA-/- ACLF mice exhibited increased cytokine secretion, and reduced mortality and bacterial burden. The administration of a neutralizing anti-BTLA antibody reduced Klebsiella pneumoniae load and mortality in mice with ACLF. These data may help elucidate HBV-ACLF pathogenesis and aid in identifying novel drug targets.

One HA stalk topping multiple heads as a novel influenza vaccine.

Classic chimeric hemagglutinin (cHA) was designed to induce immune responses against the conserved stalk domain of HA. However, it is unclear whether combining more than one HA head domain onto one stalk domain is immunogenic and further induce immune responses against influenza viruses. Here, we constructed numerous novel cHAs comprising two or three fuzed head domains from different subtypes grafted onto one stalk domain, designated as cH1-H3, cH1-H7, cH1-H3-H7, and cH1-H7-H3. The three-dimensional structures of these novel cHAs were modelled using bioinformatics simulations. Structural analysis showed that the intact neutralizing epitopes were exposed in cH1-H7 and were predicted to be immunogenic. The immunogenicity of the cHAs constructs was evaluated in mice using a chimpanzee adenoviral vector (AdC68) vaccine platform. The results demonstrated that cH1-H7 expressed by AdC68 (AdC68-cH1-H7) induced the production of high levels of binding antibodies, neutralizing antibodies, and hemagglutinin inhibition antibodies against homologous pandemic H1N1, drifted seasonal H1N1, and H7N9 virus. Moreover, vaccinated mice were fully protected from a lethal challenge with the aforementioned influenza viruses. Hence, cH1-H7 cHAs with potent immunogenicity might be a potential novel vaccine to provide protection against different subtypes of influenza virus.

A non-human primate derived anti-P-selectin glycoprotein ligand-1 antibody curtails acute pancreatitis by alleviating the inflammatory responses.

Acute pancreatitis (AP) is a devastating disease characterized by an inflammatory disorder of the pancreas. P-selectin glycoprotein ligand-1 (PSGL-1) plays a crucial role in the initial steps of the adhesive at process to inflammatory sites, blockade of PSGL-1 might confer potent anti-inflammatory effects. In this study, we generated two non-human primate derived monoclonal antibodies capable of efficiently targeting human PSGL-1, RH001-6 and RH001-22, which were screened from immunized rhesus macaques. We found that RH001-6, can effectively block the binding of P-selectin to PSGL-1, and abolish the adhesion of leukocytes to endothelial cells in vitro. In vivo, we verified that RH001-6 relieved inflammatory responses and pancreatic injury in both caerulein and l-arginine induced AP models. We also evaluated the safety profile after RH001-6 treatment in mice, and verified that RH001-6 did not cause any significant pathological damages in vivo. Taken together, we developed a novel non-human primate derived PSGL-1 blocking antibody with high-specificity, named RH001-6, which can interrupt the binding of PSGL-1 and P-selectin and attenuate inflammatory responses during AP. Therefore, RH001-6 is highly potential to be further developed into therapeutics against acute inflammatory diseases, such as AP.

Escherichia coli adhesion portion FimH polarizes M2 macrophages to M1 macrophages in tumor microenvironment via toll-like receptor 4.

Background: Macrophages are key effector cells of innate immunity and play a critical role in the immune balance of disease pathogenesis, especially in the tumor microenvironment. In previous studies, we showed that FimH, an Escherichia coli adhesion portion, promoted dendritic cell activation. However, the effect of FimH in macrophage polarization has yet to be fully examined. In this study, we investigated the potential effect of FimH on macrophages, as well as the polarization from M2 to M1 macrophages, contributing to the overall antitumor effect. Methods: Mouse bone marrow derived macrophages and peritoneal macrophages were generated to test the effect of FimH in vitro. The expression of costimulatory molecules and production of cytokines were analyzed. The effect of FimH in the tumor-associated macrophages was examine in the B16F10-tumor bearing C57BL/6. Results: FimH was found to promote M1 macrophage activation. In addition, FimH polarized M2 macrophages, which were induced by interleukin (IL)-4 and IL-13 into M1 macrophages were dependent on toll-like receptor 4 and myeloid differentiation factor 2. Moreover, FimH reprogramed the tumor-associated macrophage (TAM) into M1 macrophages in B16 melanoma tumor-bearing mice and promoted an inflammatory reaction in the tumor microenvironment (TME). Furthermore, FimH promoted M1 macrophage activation, as well as the reversion of M2 macrophages into M1 macrophages in humans. Finally, FimH treatment was found to enhance the anti-cancer immunity of anti-PD-L1 antibody by the induction of M1 polarization from TAM. Conclusion: This study demonstrated the potential effect of FimH on the activation of macrophages, responsible for the repolarization of M2 macrophages into the M1 phenotype via the TLR4 signaling pathway. Moreover, FimH could also reprogram TAM polarization to the M1 status in the TME, as well as enhance the anti-tumor activity of immune checkpoint blockade.

Broad-spectrum vaccine via combined immunization routes triggers potent immunity to SARS-CoV-2 and its variants.

IMPORTANCE: The development of broad-spectrum SARS-CoV-2 vaccines will reduce the global economic and public health stress from the COVID-19 pandemic. The use of conserved T-cell epitopes in combination with spike antigen that induce humoral and cellular immune responses simultaneously may be a promising strategy to further enhance the broad spectrum of COVID-19 vaccine candidates. Moreover, this research suggests that the combined vaccination strategies have the ability to induce both effective systemic and mucosal immunity, which may represent promising strategies for maximizing the protective efficacy of respiratory virus vaccines.

Single-cell RNA-sequencing reveals heterogeneity and intercellular crosstalk in human tuberculosis lung.

Lung inflammation indicated by 18F-labeled fluorodeoxyglucose (FDG) in patients with tuberculosis is associated with disease severity and relapse risk upon treatment completion. We revealed the heterogeneity and intercellular crosstalk in lung tissues with 18F-FDG avidity and adjacent uninvolved tissues from 6 tuberculosis patients by single-cell RNA-sequencing. Tuberculous lungs had an influx of regulatory T cells (Treg), exhausted CD8 T cells, immunosuppressive myeloid cells, conventional DC, plasmacytoid DC, and neutrophils. Immune cells in inflamed lungs showed general up-regulation of ATP synthesis and interferon-mediated signaling. Immunosuppressive myeloid and Treg cells strongly displayed transcriptions of genes related to tuberculosis disease progression. Intensive crosstalk between IL4I1-expressing myeloid cells and Treg cells involving chemokines, costimulatory molecules, and immune checkpoints, some of which are specific in 18F-FDG-avid lungs, were found. Our analysis provides insights into the transcriptomic heterogeneity and cellular crosstalk in pulmonary tuberculosis and guides unveiling cellular and molecular targets for tuberculosis therapy.

A follow-up study on the recovery and reinfection of Omicron COVID-19 patients in Shanghai, China.

Limited follow-up data is available on the recovery of Omicron COVID-19 patients after acute illness. It is also critical to understand persistence of neutralizing antibody (NAb) and of T-cell mediated immunity and the role of hybrid immunity in preventing SARS-CoV-2 reinfection. This prospective cohort study included Omicron COVID-19 individuals from April to June 2022 in Shanghai, China, during a large epidemic caused by the Omicron BA.2 variant. A total of 8945 patients from three medical centres were included in the follow up programme from November 2022 to February 2023. Of 6412 individuals enrolled for the long COVID analysis, 605 (9.4%) individuals experienced at least one sequelae, mainly had fatigue and mental symptoms specific to Omicron BA.2 infection compared with other common respiratory tract infections. During the second-visit, 548 (12.1%) cases of Omicron reinfection were identified. Hybrid immunity with full and booster vaccination had reduced risk of SARS-CoV-2 reinfection by 0.29-fold (95% CI: 0.63-0.81) and 0.23-fold (95% CI: 0.68-0.87), respectively. For 469 participants willing to the hospital during the first visit, those who received full (72 [IQR, 36-156]) or booster (64 [IQR, 28-132]) vaccination had significantly higher neutralizing antibody titers than those with incomplete vaccination (36 [IQR, 16-79]). Moreover, non-reinfection cases had higher neutralizing antibody titers (64 [IQR, 28-152]) compared to reinfection cases (32 [IQR, 20-69]).

CD160 Signaling Is Essential for CD8+ T Cell Memory Formation via Upregulation of 4-1BB.

A better understanding of the regulatory mechanisms governing the development of memory CD8+ T cells could provide instructive insights into vaccination strategies and T cell-based immunotherapies. In this article, we showed that CD160 surface protein is required for CD8+ T cell memory formation. In the response to acute lymphocytic choriomeningitis virus infection in a mouse model, CD160 ablation resulted in the failure of the development of all three memory CD8+ T cell subsets (central, effective, and tissue-resident memory), concomitant with a skewed differentiation into short-lived effector T cells. Such memory-related defect was manifested by a diminished protection from viral rechallenge. Mechanistically, CD160 deficiency led to downregulation of 4-1BB in activated CD8+ T cells, which contributes to the impaired cell survival and decreased respiratory capacity. The nexus between CD160 and 4-1BB was substantiated by the observation that ectopic introduction of 4-1BB was able to largely complement the loss of CD160 in memory CD8+ T cell development. Collectively, our studies discovered that CD160, once thought to be a coinhibitor of T cell signaling, is an essential promoter of memory CD8+ T cell development via activation of the costimulatory molecule 4-1BB.

Scopoletin Reactivates Latent HIV-1 by Inducing NF-κB Expression without Global T Cell Activation.

Reversing HIV-1 latency promotes the killing of infected cells and is essential for cure strategies. However, current latency-reversing agents (LRAs) are not entirely effective and safe in activating latent viruses in patients. In this study, we investigated whether Scopoletin (6-Methoxy-7-hydroxycoumarin), an important coumarin phytoalexin found in plants with multiple pharmacological activities, can reactivate HIV-1 latency and elucidated its underlying mechanism. Using the Jurkat T cell model of HIV-1 latency, we found that Scopoletin can reactivate latent HIV-1 replication with a similar potency to Prostratin and did so in a dose- and time-dependent manner. Moreover, we provide evidence indicating that Scopoletin-induced HIV-1 reactivation involves the nuclear factor kappa B (NF-κB) signaling pathway. Importantly, Scopoletin did not have a stimulatory effect on T lymphocyte receptors or HIV-1 receptors. In conclusion, our study suggests that Scopoletin has the potential to reactivate latent HIV-1 without causing global T-cell activation, making it a promising treatment option for anti-HIV-1 latency strategies.

Transferrin receptor modulated by microRNA-497-5p suppresses cervical cancer cell malignant phenotypes.

BACKGROUND: Cervical cancer is prevalent throughout the world, and microRNA-497-5p (miR-497-5p) plays an important role in its development. However, the specific mechanism by which miR-497-5p targets the transferrin receptor (TFRC) during cervical cancer development has not been clarified. OBJECTIVES: The aim of the study was to unravel TFRC expression and its role in cervical cancer cells, as well as the impact of the miR-497-5p/TFRC axis on cervical cancer cells. MATERIAL AND METHODS: The target mRNA was determined through differential analysis, followed by the evaluation of its impact on survival and clinical staging. Then, quantitative real-time polymerase chain reaction (qPCR) was conducted to analyze the TFRC mRNA level in cervical cancer cells and normal cervical epithelial cells. Western blot (WB) was utilized to examine the expression levels of TFRC, cleaved caspase-3, cleaved caspase-9, and epithelial-mesenchymal transition (EMT)-related proteins. The miRNAs upstream of the target mRNA were predicted, and Pearson correlation analysis was performed, followed by the validation through the dual-luciferase reporter assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays were performed to analyze cancer cell viability, followed by a transwell assay aimed at measuring cell migratory and invasive abilities. Finally, flow cytometry was conducted to examine cell apoptosis and cell cycle. RESULTS: The transferrin receptor was significantly increased in cervical cancer cells and positively associated with clinical T and N stages. Silencing TFRC could constrain cell proliferative, migratory and invasive abilities, arrest the cell cycle and facilitate cell apoptosis in cervical cancer cells. The bioinformatics analysis showed a significantly negative correlation between miR-497-5p and TFRC in cervical cancer. Moreover, upregulated miR-497-5p hampered cervical cancer progression and decreased TFRC expression. The overexpression of TFRC reversed the suppressive impact of miR-497-5p overexpression on cervical cancer progression. CONCLUSIONS: The modulatory role of the miR-497-5p/TFRC axis was confirmed in cervical cancer cells. This axis may present a new avenue for the diagnosis of cervical cancer and provide a novel target for cervical cancer treatment.

Safety and Clinical Response to Combined Immunotherapy with Autologous iNKT Cells and PD-1+CD8+ T Cells in Patients Failing First-line Chemotherapy in Stage IV Pancreatic Cancer.

Purpose: A phase I clinical trial was conducted to assess the safety and feasibility of invariant natural killer T (iNKT) cells combined with PD-1+CD8+ T cells in patients with advanced pancreatic cancer and failing the first-line chemotherapy. Patients and Methods: Fifteen eligible patients were enrolled, of whom 9 received at least three cycles of treatment each. In total, 59 courses were administered. Results: Fever was the most common adverse event, peaking at about 2-4 hours after cell infusion and reverting within 24 hours without treatment in all patients. Influenza-like reactions such as headache, myalgia, and arthralgia were also observed in 4, 4, and 3 of the patients, respectively. In addition, vomiting and dizziness were prevalent, while abdominal pain, chest pain, rash, and stuffy nose were rare adverse events, each reported in 1 patient. Side effects above grade 2 were not observed. Two patients achieved partial regression, while 1 patient experienced disease progression assessed 4 weeks after the third course. Three patients are still alive at the time of writing and have progression-free survival longer than 12 months. The overall survival time has been extended to over 12 months in 6 of the 9 patients. No constant changes of CD4+ T, B, and NK cells were recorded except for elevated CD8+ T cells after the first course. Conclusions: The combination of autologous iNKT cells and PD-1+CD8+ T cells was a safe therapeutic strategy against advanced pancreatic cancer. The patients exhibited a potentially promising prolonged survival time. Further study appears warranted to evaluate the efficacy of these combined cell infusions in pancreatic cancer. Trial registration: This trial was included in the clinical trial which was registered in ClinicalTrials.gov (ID:NCT03093688) on March 15, 2017. Significance: There is an unmet need for novel, more effective, and tolerable therapies for pancreatic cancer. Here we present a phase I clinical trial employing iNKT cells combined with PD-1+CD8+ T cells in 9 patients with advanced pancreatic cancer and failing the first-line chemotherapy. The combined immunotherapy was shown to be feasible in the enrolled patients with limited side effects and optimistic clinical responses, which could bring opportunity of therapeutic advancement.

A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response.

A major theme of host against invading pathogens lies in multiple regulatory nodes that ensure sufficient signals for protection while avoiding excessive signals toward over-inflammation. The TLR4/MD-2/CD14 complex receptor-mediated response to bacterial lipopolysaccharide (LPS) represents a paradigm for understanding the proper control of anti-pathogen innate immunity. In this study, we studied the mechanism by which the glycosylphosphatidylinositol (GPI)-linked LY6E protein constrains LPS response via downregulating CD14. We first showed that LY6E downregulated CD14 via ubiquitin-dependent proteasomal degradation. The subsequent profiling of LY6E protein interactome led to the revelation that the degradation of CD14 by LY6E requires PHB1, which interacts with CD14 in a LY6E-dependent manner. Finally, we identified the PHB1-interacting TRIM21 as the major ubiquitin E3 ligase for the LY6E-mediated ubiquitination of CD14. Together, our study elucidated the molecular basis of LY6E-mediated governance of LPS response, alongside providing new insights to regulatory mechanisms controlling the homeostasis of membrane proteins.