Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in hematologic malignancies, but its efficacy in solid tumors remains limited, primarily due to the immunosuppressive tumor microenvironment (TME) that hinders CAR-T cell trafficking and function. NKG2D CAR-T cells, which target stress-induced NKG2D ligands (NKG2DLs) broadly expressed on tumor cells, have shown promising potential in overcoming the immunosuppressive barriers of the solid TME. This review highlights recent advances in NKG2D CAR-T cell strategies for solid tumors, including innovations in CAR architecture, signaling pathway engineering, combination immunotherapy, and the development of armored CAR constructs. We further discuss the therapeutic potential, current challenges, and future directions of these approaches to inform the design of more effective and durable CAR-T cell therapies for solid tumors.
ABSTRACT Background Sustained pulmonary inflammation contributes significantly to lung carcinogenesis. Macrophages play a pivotal role in perpetuating inflammatory responses, undergoing a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis upon activation. The interplay between metabolic reprogramming and macrophage polarization remains poorly defined. The objective of this study is to examines the anti‐inflammatory mechanism of lipoic acid (LA), focusing on its ability to modulate immunometabolism in activated macrophages. Methods We utilized lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophages and a murine acute lung injury (ALI) model to evaluate the anti‐inflammatory effects of LA. Inflammatory cytokine expression was assessed by qPCR, ELISA, and Western blot. Metabolic profiling was performed using Seahorse XF technology to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), evaluating glycolytic and oxidative metabolic functions. Results This study systematically elucidates the molecular mechanism by which LA modulates macrophage inflammatory responses through targeting the HIF1α/glycolysis axis. The main findings are as follows: (1) In LPS‐induced RAW264.7 macrophages, LA treatment significantly inhibited the expression of M1 macrophage markers (iNOS, CD86) and the secretion of proinflammatory cytokines (IL‐1β, IL‐6, etc.). (2) LA effectively reduced the expression of GSDMD, the key executor of pyroptosis, demonstrating its inhibitory effect on macrophage pyroptosis. (3) Metabolic analysis revealed that LA reversed LPS‐induced metabolic reprogramming by decreasing the ECAR and increasing the OCR, thereby suppressing glycolysis. (4) Mechanistic studies showed that siRNA‐mediated knockdown of HIF1α replicated both the anti‐inflammatory and metabolic regulatory effects of LA, confirming HIF1α as the key target in this pathway. (5) In an ALI mouse model, LA treatment significantly reduced HIF1α expression in lung tissues and effectively alleviated inflammatory responses, further validating the proposed mechanism. Conclusion LA exerts potent anti‐inflammatory effects by targeting HIF1α‐mediated metabolic reprogramming in macrophages. Our results highlight the therapeutic potential of targeting immunometabolic pathways in inflammatory lung diseases, providing new insights into the mechanism by which LA ameliorates pulmonary inflammation.
Raja Rajalingam, Pawan Kumar Raghav, Neelam Sharma
et al.
BackgroundMolecular HLA mismatch at the epitope level is strongly associated with donor-specific antibody formation, antibody-mediated rejection, and graft failure after transplantation. Accurate molecular mismatch assessment requires high-resolution HLA typing; however, in clinical practice—particularly in deceased donor transplantation—typing is often performed at low or intermediate resolution. Haplotype-based imputation methods have been developed to infer high-resolution alleles but show variable accuracy, particularly in underrepresented populations. We evaluated whether the first allele listed in the reverse sequence-specific oligonucleotide (rSSO)–derived National Marrow Donor Program (NMDP) multiple allele code (MAC) string can serve as a practical surrogate for high-resolution typing in molecular mismatch assessment.MethodsWe analyzed 4,738 individuals who underwent dual HLA typing by rSSO and next-generation sequencing (NGS) across 11 classical HLA loci. Concordance between the first allele of the rSSO MAC string and the corresponding two-field NGS allele was assessed. For discordant allele pairs, molecular disparity was quantified by calculating eplet mismatches using the HLA Eplet Mismatch Calculator.ResultsAllele-level concordance exceeded 94% for 9 of 11 loci, including HLA-A (97.9%), -B (98.5%), -C (95.2%), -DRB1 (96.8%), -DRB3 (94.5%), -DRB5 (99.6%), -DQB1 (98.3%), -DPB1 (96.8%), and -DPA1 (98.7%). Lower concordance was observed for HLA-DQA1 (86.7%) and -DRB4 (72.0%). Most discordant NGS alleles, except for the DPB1 locus, were present in the rSSO MAC string. Among discordant allele pairs, 30.4% had zero eplet mismatches and 77.6% had two or fewer mismatched eplets. Allele pairs with more than two mismatched eplets accounted for less than 0.3% of all comparisons across loci. No significant differences in concordance were observed across racial or ethnic groups.ConclusionThe first allele listed in the rSSO-derived MAC string provides a reliable, population-agnostic surrogate for high-resolution HLA typing in molecular mismatch assessment. Although allele-level discordance occurs, it rarely results in clinically meaningful increases in eplet mismatch burden. This laboratory-based approach enables real-time molecular mismatch estimation in time-sensitive settings and facilitates retrospective analysis of cohorts lacking sequencing-based HLA data, supporting broader integration of molecular mismatch into precision transplantation practice.
IntroductionGlucose Regulated Proteins/Binding protein (GRP78/Bip), a representative molecular chaperone, effectively influences and actively participates in the replication processes of many viruses. Little is known, however, about the functional involvement of GRP78 in the replication of Newcastle disease virus (NDV) and the underlying mechanisms.MethodsThe method of this study are to establish protein interactomes between host cell proteins and the NDV Hemagglutinin-neuraminidase (HN) protein, and to systematically investigate the regulatory role of the GRP78-HN protein interaction during the NDV replication cycle.ResultsOur study revealed that GRP78 is upregulated during NDV infection, and its direct interaction with HN is mediated by the N-terminal 326 amino acid region. Knockdown of GRP78 by small interfering RNAs (siRNAs) significantly suppressed NDV infection and replication. Conversely, overexpression of GRP78 resulted in a significant increase in NDV replication, demonstrating its role as a positive regulator in the NDV replication cycle. We further showed that the direct interaction between GRP78 and HN protein enhanced the attachment of NDV to cells, and masking of GRP78 expressed on the cell surface with specific polyclonal antibodies (pAbs) inhibited NDV attachment and replication.DiscussionThese findings highlight the essential role of GRP78 in the adsorption stage during the NDV infection cycle, and, importantly, identify the critical domain required for GRP78-HN interaction, providing novel insights into the molecular mechanisms involved in NDV replication and infection.
Tuberculosis (TB) is an infectious disease that seriously affects human health. Until now, the only anti-TB vaccine approved for use is the live attenuated Mycobacterium bovis (M. bovis) vaccine — BCG vaccine, but its protective efficacy is relatively low and does not provide satisfactory protection against TB in adults. Therefore, there is an urgent need for more effective vaccines to reduce the global TB epidemic. In this study, ESAT-6, CFP-10, two antigens full-length and the T-cell epitope polypeptide antigen of PstS1, named nPstS1, were selected to form one multi-component protein antigens, named ECP001, which include two types, one is a mixed protein antigen named ECP001m, the other is a fusion expression protein antigen named ECP001f, as candidates for protein subunit vaccines. were prepared by constructing one novel subunit vaccine by mixing or fusing the three proteins and combining them with aluminum hydroxide adjuvant, and the immunogenicity and protective properties of the vaccine was evaluated in mice. The results showed that ECP001 stimulated mice to produce high titre levels of IgG, IgG1 and IgG2a antibodies; meanwhile, high levels of IFN-γ and a broad range of specific cytokines were secreted by mouse splenocytes; in addition, ECP001 inhibited the proliferation of Mycobacterium tuberculosis in vitro with a capacity comparable to that of BCG. It can be concluded that ECP001 is a novel effective multicomponent subunit vaccine candidate with potential as BCG Initial Immunisation-ECP001 Booster Immunisation or therapeutic vaccine for M. tuberculosis infection.
Jutamas Shaughnessy, Aleyo Chabeda, Lisa A. Lewis
et al.
SummaryThe alternative pathway (AP) is the phylogenetically oldest arm of the complement system and may have evolved to mark pathogens for elimination by phagocytes. Studies using purified AP proteins or AP‐specific serum showed that C3b amplification on bacteria commenced following a lag phase of about 5 min and was highly dependent on the concentration of complement. Most pathogens have evolved several elegant mechanisms to evade complement, including expressing proteases that degrade AP proteins and secreting proteins that block function of C3 convertases. In an example of convergent evolution, many microbes recruit the AP inhibitor factor H (FH) using molecular mechanisms that mimic FH interactions with host cells. In most instances, the AP serves to amplify C3b deposited on microbes by the classical pathway (CP). The role of properdin on microbes appears to be restricted to stabilization of C3 convertases; scant evidence exists for its role as an initiator of the AP on pathogens in the context of serum. Therapeutic complement inhibition carries with it an increased risk of infection. Antibody (Ab)‐dependent AP activation may be critical for complement activation by vaccine‐elicited Ab when the CP is blocked, and its molecular mechanism is discussed.
Gustavo Ferreira Alves, Gustavo Ferreira Alves, Eleonora Aimaretti
et al.
Sepsis and septic shock are associated with high mortality and are considered one of the major public health concerns. The onset of sepsis is known as a hyper-inflammatory state that contributes to organ failure and mortality. Recent findings suggest a potential role of two non-receptor protein tyrosine kinases, namely Focal adhesion kinase (FAK) and Proline-rich tyrosine kinase 2 (Pyk2), in the inflammation associated with endometriosis, cancer, atherosclerosis and asthma. Here we investigate the role of FAK-Pyk2 in the pathogenesis of sepsis and the potential beneficial effects of the pharmacological modulation of this pathway by administering the potent reversible dual inhibitor of FAK and Pyk2, PF562271 (PF271) in a murine model of cecal ligation and puncture (CLP)-induced sepsis. Five-month-old male C57BL/6 mice underwent CLP or Sham surgery and one hour after the surgical procedure, mice were randomly assigned to receive PF271 (25 mg/kg, s.c.) or vehicle. Twenty-four hours after surgery, organs and plasma were collected for analyses. In another group of mice, survival rate was assessed every 12 h over the subsequent 5 days. Experimental sepsis led to a systemic cytokine storm resulting in the formation of excessive amounts of both pro-inflammatory cytokines (TNF-α, IL-1β, IL-17 and IL-6) and the anti-inflammatory cytokine IL-10. The systemic inflammatory response was accompanied by high plasma levels of ALT, AST (liver injury), creatinine, (renal dysfunction) and lactate, as well as a high, clinical severity score. All parameters were attenuated following PF271 administration. Experimental sepsis induced an overactivation of FAK and Pyk2 in liver and kidney, which was associated to p38 MAPK activation, leading to increased expression/activation of several pro-inflammatory markers, including the NLRP3 inflammasome complex, the adhesion molecules ICAM-1, VCAM-1 and E-selectin and the enzyme NOS-2 and myeloperoxidase. Treatment with PF271 inhibited FAK-Pyk2 activation, thus blunting the inflammatory abnormalities orchestrated by sepsis. Finally, PF271 significantly prolonged the survival of mice subjected to CLP-sepsis. Taken together, our data show for the first time that the FAK-Pyk2 pathway contributes to sepsis-induced inflammation and organ injury/dysfunction and that the pharmacological modulation of this pathway may represents a new strategy for the treatment of sepsis.
Immunological non-responders (InRs) are HIV-infected individuals in whom the administration of combination antiretroviral therapy (cART), although successful in suppressing viral replication, cannot properly reconstitute patient circulating CD4+ T-cell number to immunocompetent levels. The causes for this immunological failure remain elusive, and no therapeutic strategy is available to restore a proper CD4+ T-cell immune response in these individuals. We have recently demonstrated that platelets harboring infectious HIV are a hallmark of InR, and we now report on a causal connection between HIV-containing platelets and T-cell dysfunctions. We show here that in vivo, platelet–T-cell conjugates are more frequent among CD4+ T cells in InRs displaying HIV-containing platelets (<350 CD4+ T cells/μl blood for >1 year) as compared with healthy donors or immunological responders (IRs; >350 CD4+ T cells/μl). This contact between platelet containing HIV and T cell in the conjugates is not infectious for CD4+ T cells, as coculture of platelets from InRs containing HIV with healthy donor CD4+ T cells fails to propagate infection to CD4+ T cells. In contrast, when macrophages are the target of platelets containing HIV from InRs, macrophages become infected. Differential transcriptomic analyses comparing InR and IR CD4+ T cells reveal an upregulation of genes involved in both aerobic and anaerobic glycolysis in CD4+ T cells from InR vs. IR individuals. Accordingly, InR platelets containing HIV induce a dysfunctional increase in glycolysis-mediated energy production in CD4+ T cells as compared with T cells cocultured with IR platelets devoid of virus. In contrast, macrophage metabolism is not affected by platelet contact. Altogether, this brief report demonstrates a direct causal link between presence of HIV in platelets and T-cell dysfunctions typical of InR, contributing to devise a platelet-targeted therapy for improving immune reconstitution in these individuals.
Wanghong Xiao, Wanghong Xiao, Wanghong Xiao
et al.
Viviparity has originated independently more than 150 times in vertebrates, while the male pregnancy only emerged in Syngnathidae fishes, such as seahorses. The typical male pregnancy seahorses have closed sophisticated brood pouch that act as both uterus and placenta, representing an excellent model system for studying the evolutionary process of paternal immune protection. Phylogenetic analysis indicated that the hampII gene family has multiple tandem duplicated genes and shows independent lineage-specific expansion in seahorses, and they had the highest ratio of nonsynonymous substitutions to synonymous substitutions (dN/dS) in the seahorse phylogenetic branch. The expression levels of hampIIs in the brood pouch placenta were significantly higher during pregnancy than non-pregnancy. Both LPS stimulation test in vivo and cytotoxicity test in vitro proved the immunological protection function of hampIIs against pathogen infection in seahorse. Besides, seahorse hampII peptides exhibit weaker antibacterial function, but stronger agglutination and free endotoxin inhibition. We assumed that the modified immunological function seemed to be a trade-off between the resistance to microbial attack and offspring protection. In brief, this study suggests that the rapid co-option of hampIIs contributes to the evolutionary adaption to paternal immune care during male pregnancy.
Staphylococcal protein A (SpA) is a multifunctional, highly conserved virulence factor of Staphylococcus aureus. By binding the Fc portion of all human IgG subclasses apart from IgG3, SpA interferes with antibody and complement deposition on the bacterial surface, impairing staphylococcal clearance by phagocytosis. Because of its anti-opsonic properties, SpA is not investigated as a surface antigen to mediate bacterial phagocytosis. Herein we investigate human sera for the presence of SpA-opsonizing antibodies. The screening revealed that sera containing IgG3 against SpA were able to correctly opsonize the target and drive Fcγ receptor-mediated interactions and phagocytosis. We demonstrated that IgG3 Fc is significantly more efficient in inducing phagocytosis of SpA-expressing S. aureus as compared to IgG1 Fc in an assay resembling physiological conditions. Furthermore, we show that the capacity of SpA antibodies to induce phagocytosis depends on the specific epitope recognized by the IgGs on SpA molecules. Overall, our results suggest that anti-SpA IgG3 antibodies could favor the anti-staphylococcal response in humans, paving the way towards the identification of a correlate of protection against staphylococcal infections.
Forkhead box protein 3 (Foxp3+)-expressing regulatory T (Treg) cells are a unique CD4+T cell subset that suppresses excessive immune responses. The epigenetic plasticity and metabolic traits of Treg cells are crucial for the acquisition of their phenotypic and functional characteristics. Therefore, alterations to the epigenetics and metabolism affect Treg cell development and function. Recent evidence reveals that altering the metabolic pathways and generation of metabolites can regulate the epigenetics of Treg cells. Specifically, some intermediates of cell metabolism can directly act as substrates or cofactors of epigenetic-modifying enzymes. Here, we describe the metabolic and epigenetic features during Treg cell development, and discuss how metabolites can contribute to epigenetic alterations of Treg cells, which affects Treg cell activation, differentiation, and function.
Laura Pandolfi, Sara Bozzini, Vanessa Frangipane
et al.
The release of neutrophil extracellular traps (NETs), a process termed NETosis, avoids pathogen spread but may cause tissue injury. NETs have been found in severe COVID-19 patients, but their role in disease development is still unknown. The aim of this study is to assess the capacity of NETs to drive epithelial-mesenchymal transition (EMT) of lung epithelial cells and to analyze the involvement of NETs in COVID-19. Bronchoalveolar lavage fluid of severe COVID-19 patients showed high concentration of NETs that correlates with neutrophils count; moreover, the analysis of lung tissues of COVID-19 deceased patients showed a subset of alveolar reactive pneumocytes with a co-expression of epithelial marker and a mesenchymal marker, confirming the induction of EMT mechanism after severe SARS-CoV2 infection. By airway in vitro models, cultivating A549 or 16HBE at air-liquid interface, adding alveolar macrophages (AM), neutrophils and SARS-CoV2, we demonstrated that to trigger a complete EMT expression pattern are necessary the induction of NETosis by SARS-CoV2 and the secretion of AM factors (TGF-β, IL8 and IL1β). All our results highlight the possible mechanism that can induce lung fibrosis after SARS-CoV2 infection.
Jaryd R Sullivan, Andréanne Lupien, Elias Kalthoff
et al.
Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis.
Chiara Niespolo, Jessica M. Johnston, Sumeet R. Deshmukh
et al.
The pseudokinase TRIB1 controls cell function in a range of contexts, by regulating MAP kinase activation and mediating protein degradation via the COP1 ubiquitin ligase. TRIB1 regulates polarization of macrophages and dysregulated Trib1 expression in murine models has been shown to alter atherosclerosis burden and adipose homeostasis. Recently, TRIB1 has also been implicated in the pathogenesis of prostate cancer, where it is often overexpressed, even in the absence of genetic amplification. Well described TRIB1 effectors include MAP kinases and C/EBP transcription factors, both in immune cells and in carcinogenesis. However, the mechanisms that regulate TRIB1 itself remain elusive. Here, we show that the long and conserved 3’untranslated region (3’UTR) of TRIB1 is targeted by miRNAs in macrophage and prostate cancer models. By using a systematic in silico analysis, we identified multiple “high confidence” miRNAs potentially binding to the 3’UTR of TRIB1 and report that miR-101-3p and miR-132-3p are direct regulators of TRIB1 expression and function. Binding of miR-101-3p and miR-132-3p to the 3’UTR of TRIB1 mRNA leads to an increased transcription and secretion of interleukin-8. Our data demonstrate that modulation of TRIB1 by miRNAs alters the inflammatory profile of both human macrophages and prostate cancer cells.
Information about human papillomavirus (HPV) disease and its vaccination has been increasingly communicated and discussed on social media platforms. The current study aims to investigate the coverage of HPV-related information on one of the most popular Chinese social media – Zhihu. Data for this study were user-generated articles, which were identified and collected by a Python web crawler with keyword searching. The final sample included a total of 115 articles, covering a two-year period between 2017 and 2018. Each article was coded for several key characteristics, including HPV epidemiological information, health belief model (HBM) constructs, framing strategy, and responses to the article. Results suggested that most of the articles reported HPV’s relationship to cervical cancer, HPV severity, and vaccination benefits. Thematic framing was more often used by the Zhihu articles to disseminate HPV-related issues, and a significant relationship between framing strategy and information coverage was found. The study has not only theoretically extended the literature of online HPV vaccine information coverage, but also practically suggested the needs to communicate comprehensive and detailed knowledge about HPV vaccination on Chinese social media.