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While adoptive cell therapies (ACT) have been successful as therapies for blood cancers,they have limited efficacy in treating solid tumours,where the tumour microenvironment excludes and suppresses adoptively transferred tumour-specific immune cells. A major obstacle to improving cell therapies for solid tumours is a lack of accessible and quantitative imaging modalities capable of tracking the migration and immune functional activity of ACT products for an extended duration in vivo.Methods: A high-efficiency magnetophoretic method was developed for facile magnetic labelling of hard-to-label immune cells,which were then injected into tumour-bearing mice and imaged over two weeks with a compact benchtop Magnetic Particle Imager (MPI) design.Results: Labelling efficiency was improved more than 10-fold over prior studies enabling longer-term tracking for at least two weeks in vivo of the labelled immune cells and their biodistribution relative to the tumour. The new imager showed 5-fold improved throughput enabling much larger density of data (up to 20 mice per experiment).Conclusions: Taken together,our innovations enable the convenient and practical use of MPI to visualise the localisation of ACT products in in vivo preclinical models for longitudinal,non-invasive functional evaluation of therapeutic efficacy. View Publication

Sensory neurons sense pathogenic infiltration to drive innate immune responses,but their role in humoral immunity is unclear. Here,using mouse models of Streptococcus pneumoniae infection and Alternaria alternata asthma,we show that sensory neurons are required for B cell recruitment and antibody production. In response to S. pneumoniae,sensory neuron depletion increases bacterial burden and reduces B cell numbers,IgG release,and neutrophil stimulation. Meanwhile,during A. alternata-induced airway inflammation,sensory neuron depletion decreases B cell population sizes,IgE levels,and asthmatic characteristics. Mechanistically,during bacterial infection,sensory neurons preferentially release vasoactive intestinal polypeptide (VIP). In response to asthma,sensory neurons release substance P. Administration of VIP into sensory neuron-depleted mice suppresses bacterial burden,while VIPR1 deficiency increases infection. Similarly,exogenous substance P delivery aggravates asthma in sensory neuron-depleted mice,while substance P deficiency ameliorates asthma. Our data,thus demonstrate that sensory neurons release select neuropeptides which target B cells dependent on the immunogen. Sensory neurons may regulate innate immune cells,but their roles in humoral immunity is still unclear. Here the authors show that bacterial infection and asthma induction induce sensory neuron production of distinct neurotransmitters to dampen B cell responses but differentially target IgG and IgE,respectively,to specifically modulate the symptoms. View Publication

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal),although data on this are limited. In this study,blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16− CD62L+) and aged (senescent) neutrophils (CD16+ CD62L−) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations,some of the indicators,including the flow cytometry markers (CD11b,CD62L,CD66b,CD63,apoptosis,and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence),were lower than the control. Additionally,low-density neutrophils (LDNs),which are found in the peripheral blood mononuclear cell (PBMC) fraction,were observed in Beta-thal patients but not in the control group. The expression of CD11b,CD66b,CD63,arginase I,and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion,iron overload induces neutrophil diversity along with some dysfunctions. View Publication

BackgroundActivated fibroblast-like synoviocytes (FLS) are drivers of synovitis and structural joint damage in rheumatoid arthritis (RA). Despite the use of disease-modifying drugs,only about 50% of RA patients reach remission in real-world settings. We used an unbiased approach to investigate the effects of standard-of-care methotrexate (MTX) and a Janus kinase inhibitor,tofacitinib (TOFA),on gene expression in RA-FLS,in order to identify untargeted disease mediators.MethodsPrimary RA-FLS were activated by stimulation with interleukin-1β (IL-1β) or platelet-derived growth factor + IL-1β in the presence or absence of MTX or TOFA,with or without additional inhibitors. Co-cultures of synovial cells were performed in direct and indirect systems. Cells were collected for RNA sequencing or qPCR,and supernatants were analyzed for protein concentrations.ResultsSix thousand three hundred fifty genes were differentially expressed,the majority being upregulated,in MTX-treated activated RA-FLS and 970 genes,the majority being downregulated,in TOFA-treated samples. Pathway analysis showed that MTX had largest effects on ‘Molecular mechanisms of cancer’ and TOFA on ‘Interferon signaling’. Targeted analysis of disease-associated genes revealed that MTX increased the expression of cell cycle-regulating genes but also of pro-inflammatory mediators like IL-1α (IL1A) and granulocyte–macrophage colony-stimulating factor,GM-CSF (CSF2). The MTX-promoted expression of CSF2 in activated RA-FLS peaked at 48 h,could be mediated via either NF-κB or AP-1 transcription factors,and was abrogated by IL-1 inhibitors (IRAK4 inhibitor and anakinra). In a co-culture setting,MTX-treatment of activated RA-FLS induced IL1B expression in macrophages.ConclusionsMTX treatment induces secretion of IL-1 from activated RA-FLS which by autocrine signaling augments their release of GM-CSF. This unexpected effect of MTX might contribute to the persistence of synovitis. View Publication

Physical frailty as a sign of accelerated aging is not well characterized in breast cancer (BC) and hematopoietic cell transplant (HCT) survivors and its correlation with outcomes and quality of life (QOL) is not defined. We conducted a prospective study to determine the prevalence of frailty in adult BC and HCT survivors,examine its impact on QOL,and determine its association with p16INK4a,a molecular biomarker for biological aging. The study included 59 BC and 65 HCT survivors. Median age was 60 years (range 27-81),68.5% were female and 49.2% were 18-59 vs. 51.8% ≥60 years old. A total of 71 (57.3%) were “fit” (frailty score 0) vs. 53 (42.7%) were pre-frailty/frail (frailty scores ≥1),and of the latter 17 (32.1%) were BC and 36 (67.9%) HCT patients. On multivariate analysis,patients >60 years were twice as likely to be frail (OR 2.04,95% CI,0.96-4.33; p=0.07),HCT were more likely to be frail compared to BC patients,and female HCT had 2.43 (95% CI,0.92-6.40) and male HCT patients had 3.25 (95% CI,1.37-7.72) times higher risk of frail; p=0.02. Frailty was associated with significant decline in QOL,measured by Medical Outcomes Study (MOS) Short Form 36 (SF-36) Physical Component Summary (PCS) and Mental Component Summary (MCS),and FACT (Functional Assessment of Cancer Therapy) scores. p16INK4a expression was higher in those who were frail,older than 60,and with higher expression in frail vs. fit patients who are 18-59 years. Our study highlights the high prevalence of frailty in survivors with detrimental effects on physical and overall wellbeing,and supports an association between frailty and the senescence marker p16INK4a. View Publication

BackgroundFANCA mutations have been detected in a variety of cancers and found to be pro-carcinogenic. However,no functional studies have been identified regarding the involvement of FANCA in the occurrence and the immune response of LUAD.MethodsThe mRNA expression and overall survival rates of FANCA were evaluated by the TIMER,PrognoScan and TCGA database in LUAD tissues,and FANCA expression was further validated by clinical serum samples using ELISA. The correlation between FANCA and immune infiltration level was investigated via TISIDB database and CIBERSORT algorithm. The Kaplan–Meier plotter was used to further evaluate the prognostic value based on the expression levels of FANCA in related immune cells. Then,the influence of FANCA knockout on the proliferation,migration,and invasion of A549 and H1299 cells was validated using CCK8,cloning formation,and Transwell assays. Subsequently,HLA-A2-restricted FANCA antigenic peptides were predicted and synthesized by NetMHC4.0 and SYFPEITHI,and DCs were induced and cultured in vitro. Finally,DCs loaded with HLA-A2-restricted FANCA antigenic peptides were co-cultured with autologous peripheral blood lymphocyte to generate specific CTLs. The killing effects of different CTLs on LUAD cells were studied.ResultsThe results showed that high levels of FANCA in patients with LUAD were significantly correlated with worse OS survival,which was correlated with age,clinical stage,pathological T stage,M stage,and N stage in LUAD. Knockdown of FANCA in A549 and H1299 cells significantly inhibited proliferation,metastasis,and invasion in vitro. In addition,FANCA was significantly related to immune infiltrate,genomic alterations and TMB. FANCA expression infuenced the prognosis of LUAD patients by directly affecting immune cell infltration. Finally,HLA-A2-restricted FANCA antigenic peptides were synthesized. And FANCA 146–154 (SLLEFAQYL) antigenic peptide exhibit a stronger affinity for DCs,and induce CTLs to produce stronger targeted killing ability for LUAD cells at an effector-to-target ratio of 40:1.ConclusionThese results demonstrated that the elevation of FANCA promotes malignant phenotype of LUAD,and the potential peptide P2 (SLLEFAQYL) derived from FANCA may be used as an epitope vaccine for the treatment of LUAD. View Publication

BackgroundMonocytes comprise subsets of classical,intermediate and non-classical monocytes with distinct anti- or pro-tumor effects in breast cancer (BC). They are modulated by estrogen,and can contribute to BC control by endocrine therapy in preclinical models.MethodsTo elucidate whether changes in monocyte subsets are associated with treatment and response,we investigated peripheral blood samples of 73 postmenopausal women with estrogen receptor (ER) positive BC,who received aromatase inhibitor therapy with or without the mucin-1 vaccine tecemotide in the ABCSG34 trial. Blood was retrieved at baseline,midterm and end of therapy,and was analyzed for the distribution and ER expression of monocyte subsets by flow cytometry.ResultsWhen 40 healthy,age-matched women were compared with BC patients before treatment start,ER levels of monocytes did not differ,yet patients presented with a higher frequency of classical and fewer non-classical monocytes. Endocrine therapy triggered a significant increase in ER levels in all monocyte subsets,without affecting subset distribution. Vaccination had no overall impact on subset frequency and ER expression. Yet,a shift from intermediate to classical monocytes during therapy correlated with changes in plasma cytokines and chemokines and was significantly associated with low residual cancer burden in vaccinated patients. Without tecemotide,baseline ER levels in classical monocytes were significantly higher in women with good response to endocrine therapy.ConclusionsThis study identified classical monocytes to be associated with ER positive BC and with patient response to neoadjuvant endocrine treatment and cancer vaccination.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-024-05659-w. View Publication

NKG2D is a central activating receptor involved in target recognition and killing by Natural Killer and CD8+ T cells. The known role of NKG2D is to recognize a family of self-induced stress ligands that are upregulated on stressed cells such as cancerous or virally infected cells. Fungal pathogens are a major threat to human health,infecting more than a billion patients yearly and becoming more common and drug resistant. Here we show that NKG2D plays a critical role in the immune response against fungal infections. NKG2D can recognize fungal pathogens from most major families including Candida,Cryptococcus and Aspergillus species,and mice lacking NKG2D are extremely sensitive to fungal infections in models of both invasive and mucosal infections,making NKG2D an anti-fungal pattern recognition receptor. NKG2D is a central activating receptor know to recognise stress ligangs upregulated during cancer or infection. Here,Charpak-Amikam et al show that NKG2D also recognises fungal pathogens and plays a critical role in mounting an appropriate immune response to them. View Publication

Colorectal cancer (CRC) resulting from chronic inflammation is a crucial issue in patients with inflammatory bowel disease (IBD). Although many reports established that intestinal resident CX3CR1high macrophages play an essential role in suppressing intestinal inflammation,their function in colitis-related CRC remains unclear. In this study,we found that colonic CX3CR1high macrophages,which were positive for MHC-II,F4/80 and CD319,promoted colitis-associated CRC. They highly expressed Col1a1,Tgfb,II10,and II4,and were considered to be fibrocytes with an immunosuppressive M2-like phenotype. CX3CR1 deficiency led to reductions in the absolute numbers of CX3CR1high fibrocytes through increased apoptosis,thereby preventing the development of colitis-associated CRC. We next focused statins as drugs targeting CX3CR1high fibrocytes. Statins have been actively discussed for patients with IBD and reported to suppress the CX3CL1/CX3CR1 axis. Statin treatment after azoxymethane/dextran sulfate sodium-induced inflammation reduced CX3CR1high fibrocyte counts and suppressed colitis-associated CRC. Therefore,CX3CR1high fibrocytes represent a potential target for carcinogenesis-preventing therapy,and statins could be safe therapeutic candidates for IBD. View Publication

IntroductionCytokine release syndrome (CRS) is one of the leading causes of mortality in patients with COVID-19 caused by the SARS-CoV-2 coronavirus. However,the mechanism of CRS induced by SARS-CoV-2 is vague.MethodsUsing spike protein combined with IL-2,IFN-γ,and TNF-α to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete CRS-related cytokines,the content of cytokines in the supernatant was detected,and the effects of NK,T,and monocytes were analyzed.ResultsThis study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more interleukin-2 (IL-2,) which subsequently cooperates with spike protein to facilitate PBMCs to release IL-1β,IL-6,and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ),as well as T cells to release IFN-γ Mechanistically,IFN-γ and TNF-α enhance the transcription of CD40,and the interaction of CD40 and its ligand stabilizes the membrane expression of toll-like receptor 4 (TLR4) that serves as a receptor of spike protein on the surface of monocytes. As a result,there is a constant interaction between spike protein and TLR4,leading to continuous activation of nuclear factor-κ-gene binding (NF-κB). Furthermore,TNF-α also activates NF-κB signaling in monocytes,which further cooperates with IFN-γ and spike protein to modulate NF-κB–dependent transcription of CRS-related inflammatory cytokines.DiscussionTargeting TNF-α/IFN-γ in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19. View Publication

Mature erythrocytes are known to lack major histocompatibility complex (MHC) proteins. However,the presence of MHC molecules on erythrocytes has been occasionally reported,though without a defined function. In this study,we designed erythrocyte conjugated solely with a fusion protein consisting of an antigenic peptide linked to MHC class I (MHC-I) protein,termed MHC-I‒Ery. The modified erythrocyte,decorated with the peptide derived from human papillomavirus (HPV) 16 oncoprotein E6/E7,effectively activated antigen-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from HPV16+ cervical cancer patients. Additionally,MHC-I‒Ery monotherapy was shown to inhibit antigen-positive tumor growth in mice. This treatment immediately activated CD8+ T cells and reduced suppressive myeloid cells in the spleen,leading to systemic anti-tumor activity. Safety and tolerability evaluations of MHC-I‒Ery in non-human primates further supported its clinical potential. Our results first demonstrated that erythrocytes equipped solely with antigen peptide‒MHC-I complexes can robustly stimulate the immune system,suggesting a novel and promising approach for advancing cancer immunotherapy. View Publication

Single-cell RNA sequencing (scRNA-seq) can resolve transcriptional features from individual cells,but scRNA-seq techniques capable of resolving the variable regions of B cell receptors (BCRs) remain limited,especially from widely-used 3′-barcoded libraries. Here,we report a method that can recover paired,full-length variable region sequences of BCRs from 3′-barcoded scRNA-seq libraries. We first verify this method (B3E-seq) can produce accurate,full-length BCR sequences. We then apply this method to profile B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in five infant rhesus macaques. We identify BCR features associated with specificity for the ST3 antigen which are present in multiple vaccinated monkeys,indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and profile antigen-specific responses elicited by vaccination. A method that recovers full-length,paired heavy- and light-chain variable regions of B cell receptor transcripts from 3’barcoded scRNA-seq libraries reveals a convergent response to pneumococcus vaccination in rhesus macaques. View Publication