技术资料

Although chimeric antigen receptor (CAR) T cells are effective against B-lineage malignancies,post-CAR relapse is common,and efficacy in other tumors is limited. These challenges may be addressed through rational manipulations to control CAR T cell function. Here we examine the impact of cognate T cell antigen experience on subsequent CD8+ CAR T cell activity. Prior antigen encounter resulted in superior effector function against leukemia expressing low target antigen density at the expense of reduced proliferative capacity and susceptibility to dysfunction at limiting CAR doses. Distinctive temporal transcriptomic and epigenetic profiles in naive-derived and memory-derived CAR T cells identified RUNX family transcription factors as potential targets to augment the function of naive-derived CD8+ CAR T cells. RUNX2 overexpression enhanced antitumor efficacy of mouse CAR T cells,dependent on prior cell state,and heightened human CAR T cell functions. Our data demonstrate that prior antigen experience of CAR T cells determines functional attributes and amenability to transcription factor-mediated functional enhancement. Here,Fry and colleagues examine the impact of antigen experience on subsequent CD8+ CAR T cell activity during the antileukemia response and show that RUNX2 overexpression enhances antitumor activity of these cells. View Publication

Donor-specific antibodies (DSAs) targeting mismatched human leukocyte antigen (HLA) molecules are one of the principal threats to long-term graft survival in solid organ transplantation. However,many patients with long-term circulating DSAs do not manifest rejection responses,suggesting a degree of heterogeneity in their pathogenicity and related functional activity. Immunologic risk stratification of transplant recipients is complicated by challenges intrinsic to defining alloantibody responses that are potentially pathogenic versus those that are not. Thus,a comprehensive understanding of how human alloantibodies target and interact with donor HLA molecules is vital for the development and evaluation of new strategies aimed at reducing antibody-mediated rejection responses. In this study,we employ hydrogen–deuterium exchange–mass spectrometry (HDX–MS),molecular dynamics (MD) simulations,and advanced biochemical and biophysical methodologies to thoroughly characterize a panel of human monoclonal alloantibodies and define the influence of Fc-region biology,antibody binding kinetics,target antigen density,and structural characteristics on their ability to potentiate the forms of immune effector mechanisms that are strongly implicated in transplant rejection. Our findings have significant implications for our understanding of the key biological determinants that underlie the pathogenicity or lack thereof of human alloantibodies. View Publication

IntroductionUpon infection,T cell-driven B cell responses in GC reactions induce memory B cells and antibody-secreting cells that secrete protective antibodies. How formation of specifically long-lived plasma cells is regulated via the interplay between specific B and CD4+ T cells is not well understood. Generally,antibody levels decline over time after clearance of the primary infection.MethodIn this study,convalescent individuals with stable RBD antibody levels (n=14,“sustainers”) were compared with donors (n=13) with the greatest antibody decline from a cohort of 132. To investigate the role of the cellular immune compartment in the maintenance of antibody levels,SARS-CoV-2-specific responses at 4 to 6 weeks post-mild COVID-19 infection were characterized using deep immune profiling.ResultsBoth groups had similar frequencies of total SARS-CoV-2-specific B and CD4+ T cells. Sustainers had fewer Spike-specific IgG+ memory B cells early after infection and increased neutralizing capacity of RBD antibodies over time,unlike the declining group. However,declining IgG titers correlated with lower frequency of Spike-specific CD4+ T cells.ConclusionThese data suggest that “sustainers” have unique dynamics of GC reactions,yield different outputs of terminally differentiating cells,and improve the quality of protective antibodies over time. This study helps identify factors controlling formation of long-lived PC and sustained antibody responses. View Publication

IntroductionSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by an overactive immune response,particularly involving excessive production of type I interferons. This overproduction is driven by the phosphorylation of IRF7,a crucial factor in interferon gene activation. Current treatments for SLE are often not very effective and can have serious side effects.MethodsOur study introduces clobenpropit,a histamine analogue,as a potential new therapy targeting the CXCR4 receptor to reduce IRF7 phosphorylation and subsequent interferon production. We employed various laboratory techniques to investigate how clobenpropit interacts with CXCR4 and its effects on immune cells from healthy individuals and SLE patients.ResultsClobenpropit binds effectively to CXCR4,significantly inhibiting IRF7 phosphorylation and reducing interferon production. Additionally,clobenpropit lowered levels of pro-inflammatory cytokines in a mouse model of lupus,demonstrating efficacy comparable to the standard treatment,prednisolone.DiscussionThese results suggest that clobenpropit could be a promising new treatment for SLE,offering a targeted approach with potential advantages over current therapies. View Publication

The lungs of people with cystic fibrosis (PwCF) are characterized by recurrent bacterial infections and inflammation. Infections in cystic fibrosis (CF) are left unresolved despite excessive neutrophil infiltration. The role of CFTR in neutrophils is not fully understood. In this study,we aimed to assess which antimicrobial functions are directly impaired by loss of CFTR function in neutrophils. In order to do so,we used a specific inhibitor of CFTR ion channel activity,inh-172. CF neutrophils from PwCF harboring severe CFTR mutations were additionally isolated to further discern CFTR-specific functional defects. We evaluated phagocytosis,reactive oxygen species (ROS) production,neutrophil elastase (NE) and myeloperoxidase (MPO) exocytosis and bacterial killing. The inh-172 model identified decreased acidification of the phagosome,increased bacterial survival and decreased ROS production upon stimulation. In PwCF neutrophils,we observed reduced degranulation of both NE and MPO. When co-culturing neutrophils with CF sputum supernatant and airway epithelial cells,the extent of phagocytosis was reduced,underscoring the importance of recreating an inflammatory environment as seen in PwCF lungs to model immune responses in vitro. Despite low CFTR expression in blood neutrophils,functional defects were found in inh-172-treated and CF neutrophils. The inh-172 model disregards donor variability and allows pinpointing neutrophil functions directly impaired by dysfunctional CFTR.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-82535-z. View Publication

Achieving the precise targeting of lentiviral vectors (LVs) to specific cell populations is crucial for effective gene therapy,particularly in cancer treatment where the modulation of the tumor microenvironment can enhance anti-tumor immunity. Programmed cell death protein 1 (PD-1) is overexpressed on activated tumor-infiltrating T lymphocytes,including regulatory T cells that suppress immune responses via FOXP3 expression. We developed PD1-targeted LVs by incorporating the anti-PD1 nanobody nb102c3 into receptor-blinded measles virus H and VSV-Gmut glycoproteins. We assessed the retargeting potential of nb102c3 and evaluated transduction efficiency in activated T lymphocytes. FOXP3 expression was suppressed using shRNA delivered by these LVs. Our results demonstrate that PD1-targeted LVs exerted pronounced tropism towards PD1+ cells,enabling the selective transduction of activated T lymphocytes while sparing naive T cells. The suppression of FOXP3 in Tregs reduced their suppressive activity. PD1-targeted glycoprotein H provided greater specificity,whereas the VSV-Gmut,together with the anti-PD1 pseudoreceptor,achieved higher viral titers but was less selective. Our study demonstrates that PD1-targeted LVs may offer a novel strategy to modulate immune responses within the tumor microenvironment with the potential for developing new therapeutic strategies aimed at enhancing anti-tumor immunity. View Publication

Pathobionts have evolved many strategies to coexist with the host,but how immune evasion mechanisms contribute to the difficulty of developing vaccines against pathobionts is unclear. Meanwhile,Staphylococcus aureus (SA) has resisted human vaccine development to date. Here we show that prior SA exposure induces non-protective CD4+ T cell imprints,leading to the blunting of protective IsdB vaccine responses. Mechanistically,these SA-experienced CD4+ T cells express IL-10,which is further amplified by vaccination and impedes vaccine protection by binding with IL-10Rα on CD4+ T cell and inhibit IL-17A production. IL-10 also mediates cross-suppression of IsdB and sdrE multi-antigen vaccine. By contrast,the inefficiency of SA IsdB,IsdA and MntC vaccines can be overcome by co-treatment with adjuvants that promote IL-17A and IFN-γ responses. We thus propose that IL-10 secreting,SA-experienced CD4+ T cell imprints represent a staphylococcal immune escaping mechanism that needs to be taken into consideration for future vaccine development. Mechanisms of inefficient vaccine protection against pathobionts such as S. aureus (SA) are still unclear. Here the authors show that prior SA exposure induces non-protective CD4+ T cells,which impair IsdB vaccine protection by IL-10 secretion and IL-17A suppression,whereas IL-17A promoting adjuvant CAF01 overcomes this dilemma. View Publication

AbstractChlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. A mouse model has been developed in our laboratory to better understand the effect of cold-induced stress on chlamydia genital infection and immune response. However,the stress mechanism affecting the host response to Chlamydia muridarum genital infection remains unclear. Here,we demonstrate a role for the beta2-adrenergic receptor (β2-AR),which binds noradrenaline and modulates the immune response against chlamydia genital infection in a mouse model. A successful β2-AR homozygous knockout (KO) mouse model was used to study the infection and analyze the immune response. Our data show that stressed mice lacking the β2-AR are less susceptible to C. muridarum genital infection than controls. A correlation was obtained between lower organ load and higher interferon-gamma production by CD4+ and CD8+ cells of the KO mice. Furthermore,exposure of CD4+ T cells to noradrenaline alters the production of cytokines in mice during C. muridarum genital infection. This study suggests that the blockade of β2-AR signaling could be used to increase resistance to chlamydia genital infection. We value the β2-AR KO as a viable model that can provide reproducible results in investigating medical research,including chlamydia genital infection. Deficiency in a receptor leads to a reduced disease of chlamydia in a mouse model. View Publication

Eosinophils,a type of granulocyte derived from myeloid precursors in the bone marrow,are distinguished by their cytoplasmic granules. They play crucial roles in immunoregulation,tissue homeostasis,and host defense,while also contributing to the pathogenesis of various inflammatory diseases. Although long non-coding RNAs (lncRNAs) are known to be involved in eosinophilic conditions,their specific expression and functions within eosinophils have not been thoroughly investigated,largely due to the reliance on tissue homogenates. In an effort to address this gap,we analyzed publicly available high-throughput RNA sequencing data to identify lncRNAs associated with eosinophilic conditions. Among the identified lncRNAs,ITGB2 antisense RNA 1 (ITGB2-AS1) was significantly downregulated in blood eosinophils from patients with hypereosinophilia. To further explore its role in eosinophil biology,we generated a stable ITGB2-AS1 knockdown in the HL-60 cell line. Interestingly,ITGB2-AS1 deficiency led to impaired eosinophil differentiation,as evidenced by a reduction in cytoplasmic granules and decreased expression of key eosinophil granule proteins,including eosinophil peroxidase (EPX) and major basic protein-1 (MBP-1). Additionally,ITGB2-AS1-deficient cells exhibited compromised eosinophil effector functions,with reduced degranulation and impaired production of reactive oxygen species (ROS). These findings suggest that ITGB2-AS1 plays a pivotal role in eosinophil differentiation and function,positioning it as a novel regulator in eosinophil biology. View Publication

Elevated neutrophil counts in broncho-alveolar lavage (BAL) fluids of lung transplant (LTx) patients with chronic lung allograft dysfunction (CLAD) are associated with disease pathology. However,phenotypical characteristics of these cells remained largely unknown. Moreover,despite enhanced levels of the most potent human neutrophil-attracting chemokine CXCL8 in BAL fluid,no discrimination had been made between natural NH2-terminally truncated CXCL8 proteoforms,which exhibit up to 30-fold differences in biological activity. Therefore,we aimed to characterize the neutrophil maturation and activation state,as well as proteolytic activation of CXCL8,in BAL fluids and peripheral blood of LTx patients with CLAD or infection and stable LTx recipients. Flow cytometry and microscopy revealed a high diversity in neutrophil maturity in blood and BAL fluid,ranging from immature band to hypersegmented aged cells. In contrast,the activation phenotype of neutrophils in BAL fluid was remarkably homogeneous. The highly potentiated NH2-terminally truncated proteoforms CXCL8(6–77),CXCL8(8–77) and CXCL8(9–77),but also the partially inactivated CXCL8(10–77),were detected in BAL fluids of CLAD and infected LTx patients,as well as in COVID-19 and influenza patient cohorts by tandem mass spectrometry. Moreover,the most potent proteoform CXCL8(9–77) specifically correlated with the neutrophil counts in the LTx BAL fluids. Finally,rapid proteolysis of CXCL8 in BAL fluids could be inhibited by a combination of serine and metalloprotease inhibitors. In conclusion,proteolytic activation of CXCL8 promotes neutrophilic inflammation in LTx patients. Therefore,application of protease inhibitors may hold pharmacological promise for reducing excessive neutrophil-mediated inflammation and collateral tissue damage in the lungs.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05500-z. View Publication

IntroductionSystemic sclerosis (SSc) is a complex autoimmune connective tissue disease characterized by microvascular damage,immune system reactivity and progressive fibrosis of skin and internal organs. Interstitial lung disease is the leading cause of death for SSc patients (SSc-ILD),and the process of lung fibrosis involves also circulating monocytes and alveolar macrophages.MethodsCurrent study aimed to identify monocyte/macrophage phenotypes in lung and peripheral blood of SSc-ILD patients by immunostaining and flow cytometry,respectively. Single immunostaining was performed using primary antibodies against CD68 (pan-macrophage marker),CD80,CD86,TLR4 (M1 markers),CD163,CD204,and CD206 (M2 markers). Flow cytometry analysis included the evaluation of CD45,CD14,CD16 (monocyte lineage),CD1c (dendritic lineage),together with M1 and M2 activation markers on circulating monocytes. Protein synthesis of TLR4 and M2 markers was also investigated in cultured monocytes-derived macrophages (MDMs) from SSc-ILD patients by Western Blotting.ResultsLung samples were obtained from 9 SSc-ILD patients (50 ± 9 years old) and 5 control non-SSc patients without lung fibrosis (58 ± 23 years old). Alveolar macrophages (CD68+ cells) showed a significantly higher positivity of M1 and M2 markers in SSc-ILD lung samples than in controls (p<0.05 for CD80,p<0.01 for CD86,p<0.001 for CD68,p<0.0001 for TLR4,CD163,CD204 and CD206). In CD68 positive areas of SSc-ILD samples,a significantly higher percentage of TLR4,CD163,CD204,and CD206 positive cells was observed compared to CD80 and CD86 positive cells (p<0.001 in both cases),suggesting the possible presence of hybrid TLR4+M2 macrophages (CD68+CD80-CD86-TLR4+CD163+CD204+CD206+cells) in SSc-ILD samples. A second cohort of 26 SSc-ILD patients (63 ± 14 years old) and 14 SSc patients without ILD (63 ± 19 years old) was recruited for flow cytometry analysis of circulating monocytes. Again,a significantly higher percentage of hybrid TLR4+M2 monocytes (CD1c-CD80-TLR4+CD163+CD204+CD206+cells) was found in SSc-ILD positive than SSc-ILD negative patients (p<0.05). Moreover,the protein synthesis of TLR4 and M2 markers was also found higher in cultured MDMs obtained from SSc-ILD patients than in MDMs from SSc patients without ILD and this increase was significantly higher for CD163 (p<0.05) and CD206 (p<0.01).ConclusionsThe presence of hybrid TLR4+M2 markers on both circulating monocytes and resident lung macrophages in SSc-ILD patients,is reported for the first time. Therefore,the detection of circulating hybrid TLR4+M2 monocytes in SSc-ILD might represent a further potential biomarker of progressive organ fibrosis,to be searched in blood samples of SSc patients. View Publication

ABSTRACT T cell therapies,such as chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) T cells,are a growing class of anti-cancer treatments. However,expansion to novel indications and beyond last-line treatment requires engineering cells’ dynamic population behaviors. Here we develop the tools for cellular behavior analysis of T cells from live-cell imaging,a common and inexpensive experimental setup used to evaluate engineered T cells. We first develop a state-of-the-art segmentation and tracking pipeline,Caliban,based on human-in-the-loop deep learning. We then build the Occident pipeline to collect a catalog of phenotypes that characterize cell populations,morphology,movement,and interactions in co-cultures of modified T cells and antigen-presenting tumor cells. We use Caliban and Occident to interrogate how interactions between T cells and cancer cells differ when beneficial knock-outs of RASA2 and CUL5 are introduced into TCR T cells. We apply spatiotemporal models to quantify T cell recruitment and proliferation after interactions with cancer cells. We discover that,compared to a safe harbor knockout control,RASA2 knockout T cells have longer interaction times with cancer cells leading to greater T cell activation and killing efficacy,while CUL5 knockout T cells have increased proliferation rates leading to greater numbers of T cells for hunting. Together,segmentation and tracking from Caliban and phenotype quantification from Occident enable cellular behavior analysis to better engineer T cell therapies for improved cancer treatment. View Publication