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SummaryDrug resistance threatens the effective control of infections,including parasitic diseases such as leishmaniases. Neutrophils are essential players in antimicrobial control,but their role in drug-resistant infections is poorly understood. Here,we evaluated human neutrophil response to clinical parasite strains having distinct natural drug susceptibility. We found that Leishmania antimony drug resistance significantly altered the expression of neutrophil genes,some of them transcribed by specific neutrophil subsets. Infection with drug-resistant parasites increased the expression of detoxification pathways and reduced the production of cytokines. Among these,the chemokine CCL3 was predominantly impacted,which resulted in an impaired ability of neutrophils to attract myeloid cells. Moreover,decreased myeloid recruitment when CCL3 levels are reduced was confirmed by blocking CCL3 in a mouse model. Collectively,these findings reveal that the interplay between naturally drug-resistant parasites and neutrophils modulates the infected skin immune microenvironment,revealing a key role of neutrophils in drug resistance. Graphical abstract Highlights•Drug-resistant parasites induce distinct neutrophil transcriptional programs•Meglumine-antimoniate-resistant (MAR) Leishmania limits neutrophil chemokine release•Infection with MAR parasites impairs CCL3-driven early myeloid cell recruitment Immunology; Parasitology View Publication

SummaryThe viral vector-based COVID-19 vaccine Ad26.COV2.S has been recommended by the WHO since 2021 and has been administered to over 200 million people. Prior studies have shown that Ad26.COV2.S induces durable neutralizing antibodies (NAbs) that increase in coverage of variants over time,even in the absence of boosting or infection. Here,we studied humoral responses following Ad26.COV2.S vaccination in individuals enrolled in the initial Phase 1/2a trial of Ad26.COV2.S in 2020. Through 8 months post vaccination,serum NAb responses increased to variants,including B.1.351 (Beta) and B.1.617.2 (Delta),without additional boosting or infection. The level of somatic hypermutation,measured by nucleotide changes in the VDJ region of the heavy and light antibody chains,increased in Spike-specific B cells. Highly mutated mAbs from these sequences neutralized more SARS-CoV-2 variants than less mutated comparators. These findings suggest that the increase in NAb breadth over time following Ad26.COV2.S vaccination is mediated by affinity maturation. Graphical abstract Highlights•Ad26.COV2.S induced neutralizing antibodies increase in breadth over 8 months•Somatic hypermutation in spike specific B cells also increases over 8 months•Highly mutated monoclonal antibodies neutralize more variants than less mutated•Ad26.COV2.S induces long term affinity maturation Health sciences; Immunity; Immune response; Virology View Publication

SummaryHispanic/Latino children have the highest risk of acute lymphoblastic leukemia (ALL) in the US compared to other racial/ethnic groups,yet the basis of this remains incompletely understood. Through genetic fine-mapping analyses,we identified a new independent childhood ALL risk signal near IKZF1 in self-reported Hispanic/Latino individuals,but not in non-Hispanic White individuals,with an effect size of ∼1.44 (95% confidence interval = 1.33–1.55) and a risk allele frequency of ∼18% in Hispanic/Latino populations and <0.5% in European populations. This risk allele was positively associated with Indigenous American ancestry,showed evidence of selection in human history,and was associated with reduced IKZF1 expression. We identified a putative causal variant in a downstream enhancer that is most active in pro-B cells and interacts with the IKZF1 promoter. This variant disrupts IKZF1 autoregulation at this enhancer and results in reduced enhancer activity in B cell progenitors. Our study reveals a genetic basis for the increased ALL risk in Hispanic/Latino children. Graphical abstract Highlights•IKZF1 variants contribute to the increased risk of ALL in Hispanic/Latino children•Risk allele is associated with Indigenous American ancestry and underwent selection•Risk variant impacts IKZF1 enhancer that is selectively active in B cell development•Risk allele reduces enhancer activity,chromatin accessibility,and IKZF1 expression Genetic fine-mapping across the IKZF1 gene revealed a variant associated with childhood ALL that contributes to the increased risk of this disease in Hispanic/Latino individuals. The ALL risk allele reduces enhancer activity and IKZF1 expression specifically in B cell progenitors,likely resulting in stalled B cell development and an increased risk of ALL. View Publication

AbstractIntestinal chronic inflammation is associated with microbial dysbiosis and accumulation of various immune cells including myeloid-derived suppressor cells (MDSC),which profoundly impact the immune microenvironment,perturb homeostasis and increase the risk to develop colitis-associated colorectal cancer (CAC). However,the specific MDSCs–dysbiotic microbiota interactions and their collective impact on CAC development remain poorly understood. In this study,using a murine model of CAC,we demonstrate that CAC-bearing mice exhibit significantly elevated levels of highly immunosuppressive MDSCs,accompanied by microbiota alterations. Both MDSCs and bacteria that infiltrate the colon tissue and developing tumors can be found in close proximity,suggesting intricate MDSC-microbiota cross-talk within the tumor microenvironment. To investigate this phenomenon,we employed antibiotic treatment to disrupt MDSC–microbiota interactions. This intervention yielded a remarkable reduction in intestinal inflammation,decreased MDSC levels,and alleviated immunosuppression,all of which were associated with a significant reduction in tumor burden. Furthermore,we underscore the causative role of dysbiotic microbiota in the predisposition toward tumor development,highlighting their potential as biomarkers for predicting tumor load. We shed light on the intimate MDSCs-microbiota cross-talk,revealing how bacteria enhance MDSC suppressive features and activities,inhibit their differentiation into mature beneficial myeloid cells,and redirect some toward M2 macrophage phenotype. Collectively,this study uncovers the role of MDSC-bacteria cross-talk in impairing immune responses and promoting tumor growth,providing new insights into potential therapeutic strategies for CAC.Significance:MDSCs–dysbiotic bacteria interactions in the intestine play a crucial role in intensifying immunosuppression within the CAC microenvironment,ultimately facilitating tumor growth,highlighting potential therapeutic targets for improving the treatment outcomes of CAC. View Publication

During the humoral immune response,B cells undergo rapid metabolic reprogramming with a high demand for nutrients,which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3),members of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However,how Rag-GTPases coordinate amino acid sensing,mTORC1 activation,and TFEB/TFE3 activity in humoral immunity remains undefined. Here,we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs,produce antibodies,and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically,the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells,which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development,GC formation in Peyer’s patches and TI humoral immunity,but not TD humoral immunity in the absence of Rag-GTPases. Collectively,our data establish Rag-GTPase-TFEB/TFE3 axis as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells. View Publication

This study describes clinical features and cellular and molecular mechanisms underlying immune deficiency in seven patients with biallelic germline variants in CD4. The data reveal important roles for CD4 in host defense against a range of pathogens,particularly human papilloma virus. CD4+ T cells are vital for host defense and immune regulation. However,the fundamental role of CD4 itself remains enigmatic. We report seven patients aged 5–61 years from five families of four ancestries with autosomal recessive CD4 deficiency and a range of infections,including recalcitrant warts and Whipple’s disease. All patients are homozygous for rare deleterious CD4 variants impacting expression of the canonical CD4 isoform. A shorter expressed isoform that interacts with LCK,but not HLA class II,is affected by only one variant. All patients lack CD4+ T cells and have increased numbers of TCRαβ+CD4−CD8− T cells,which phenotypically and transcriptionally resemble conventional Th cells. Finally,patient CD4−CD8− αβ T cells exhibit intact responses to HLA class II–restricted antigens and promote B cell differentiation in vitro. Thus,compensatory development of Th cells enables patients with inherited CD4 deficiency to acquire effective cellular and humoral immunity against an unexpectedly large range of pathogens. Nevertheless,CD4 is indispensable for protective immunity against at least human papillomaviruses and Trophyrema whipplei. View Publication

BackgroundT cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however,the distinct signaling pathway that regulates T cell activation remains unclear. In this study,we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation.MethodsWhole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation,lipid raft isolation,and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells.ResultsTRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells,using high throughput mass spectrometry-based proteomics analysis. Furthermore,Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells,leading to the interruption of proximal TCR signaling and subsequent T cell activation.ConclusionsTRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus,our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation,and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-024-01023-8. View Publication

Human milk oligosaccharides (HMOs) can modulate the intestinal barrier and regulate immune cells to favor the maturation of the infant intestinal tract and immune system,but the precise functions of individual HMOs are unclear. To determine the structure-dependent effects of individual HMOs (representing different structural classes) on the intestinal epithelium as well as innate and adaptive immune cells,we assessed fucosylated (2′FL and 3FL),sialylated (3′SL and 6′SL) and neutral non-fucosylated (LNT and LNT2) HMOs for their ability to support intestinal barrier integrity,to stimulate the secretion of chemokines from intestinal epithelial cells,and to modulate cytokine release from LPS-activated dendritic cells (DCs),M1 macrophages (MØs),and co-cultures with naïve CD4+ T cells. The fucosylated and neutral non-fucosylated HMOs increased barrier integrity and protected the barrier following an inflammatory insult but exerted minimal immunomodulatory activity. The sialylated HMOs enhanced the secretion of CXCL10,CCL20 and CXCL8 from intestinal epithelial cells,promoted the secretion of several cytokines (including IL-10,IL-12p70 and IL-23) from LPS-activated DCs and M1 MØs,and increased the secretion of IFN-γ and IL-17A from CD4+ T cells primed by LPS-activated DCs and MØs while reducing the secretion of IL-13. Thus,3′SL and 6′SL supported Th1 and Th17 responses while reducing Th2 responses. Collectively,our data show that HMOs exert structure-dependent effects on the intestinal epithelium and possess immunomodulatory properties that confer benefits to infants and possibly also later in life. View Publication

Graphical abstract Summary of the study. Peripheral blood neutrophils from >200 hospitalised patients across three patient groups (coronavirus disease 2019 (COVID-19),non-COVID-19 lower respiratory tract infection (LRTI) and matched controls) were comprehensively profiled using mass spectrometry,revealing novel proteomic changes in acute and convalescent COVID-19. DIA: data-independent acquisition; TLR: Toll-like receptor; ARG: arginase; TGF: transforming growth factor; IFN: interferon. BackgroundNeutrophils are important in the pathophysiology of coronavirus disease 2019 (COVID-19),but the molecular changes contributing to altered neutrophil phenotypes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We used quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes immediately following acute SARS-CoV-2 infection and during recovery.MethodsProspective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May to December 2020). Patients were enrolled within 96 h of admission,with longitudinal sampling up to 29 days. Control groups comprised non-COVID-19 acute lower respiratory tract infection (LRTI) and age-matched noninfected controls. Neutrophils were isolated from peripheral blood and analysed using mass spectrometry. COVID-19 severity and recovery were defined using the World Health Organization ordinal scale.ResultsNeutrophil proteomes from 84 COVID-19 patients were compared to those from 91 LRTI and 42 control participants. 5800 neutrophil proteins were identified,with >1700 proteins significantly changed in neutrophils from COVID-19 patients compared to noninfected controls. Neutrophils from COVID-19 patients initially all demonstrated a strong interferon signature,but this signature rapidly declined in patients with severe disease. Severe disease was associated with increased abundance of proteins involved in metabolism,immunosuppression and pattern recognition,while delayed recovery from COVID-19 was associated with decreased granule components and reduced abundance of metabolic proteins,chemokine and leukotriene receptors,integrins and inhibitory receptors.ConclusionsSARS-CoV-2 infection results in the sustained presence of circulating neutrophils with distinct proteomes suggesting altered metabolic and immunosuppressive profiles and altered capacities to respond to migratory signals and cues from other immune cells,pathogens or cytokines. Tweetable abstractHigh-resolution mass spectrometry analysis of peripheral blood neutrophils from >200 individuals provides novel insights into neutrophil phenotypes during acute COVID-19 and reveals that altered neutrophils persist in convalescent COVID-19 patients https://bit.ly/3QSSq9W View Publication

SummaryMesenchymal stromal cells (MSCs) have been modified via genetic or pharmacological engineering into potent antigen-presenting cells-like capable of priming responding CD8 T cells. In this study,our screening of a variant library of Accum molecule revealed a molecule (A1) capable of eliciting antigen cross-presentation properties in MSCs. A1-reprogrammed MSCs (ARM) exhibited improved soluble antigen uptake and processing. Our comprehensive analysis,encompassing cross-presentation assays and molecular profiling,among other cellular investigations,elucidated A1’s impact on endosomal escape,reactive oxygen species production,and cytokine secretion. By evaluating ARM-based cellular vaccine in mouse models of lymphoma and melanoma,we observe significant therapeutic potency,particularly in allogeneic setting and in combination with anti-PD-1 immune checkpoint inhibitor. Overall,this study introduces a strong target for developing an antigen-adaptable vaccination platform,capable of synergizing with immune checkpoint blockers to trigger tumor regression,supporting further investigation of ARMs as an effective and versatile anti-cancer vaccine. Graphical abstract Highlights•Treatment with A1/antigen mix reprograms MSCs into antigen-presenting cells•The antigen cross-presenting ability of ARM cells require ROS and UPR•ARMs synergize with immune-checkpoint inhibitors in priming potent antitumoral activity Classification Description: Immunology; Pharmaceutical engineering; Cancer View Publication

Background:Throughout HIV infection,productively infected cells generate billions of viral particles and are thus responsible for body-wide HIV dissemination,but their phenotype during AIDS is unknown. As AIDS is associated with immunological changes,analyzing the phenotype of productively infected cells can help understand HIV production during this terminal stage.Methods:Blood samples from 15 untreated viremic participants (recent infection,n=5; long-term infection,n=5; active opportunistic AIDS-defining disease,n=5) and 5 participants virologically controlled on antiretroviral therapy (ART) enrolled in the Analysis of the Persistence,Reservoir and HIV Latency (APRIL) study (NCT05752318) were analyzed. Cells expressing the capsid protein p24 (p24+ cells) after 18 hours of resting or 24 hours of stimulation (HIV-Flow) revealed productively infected cells from viremic participants or translation-competent reservoir cells from treated participants,respectively.Results:The frequency of productively infected cells tended to be higher during AIDS in comparison with recent and long-term infections (median,340,72,and 32/million CD4+ T cells,respectively) and correlated with the plasma viral load at all stages of infection. Altogether,these cells were more frequently CD4low,HLA-ABClow,CD45RA-,Ki67+,PD-1+,with a non-negligible contribution from pTfh (CXCR5+PD-1+) cells,and were not significantly enriched in HIV coreceptors CCR5 nor CXCR4 expression. The comparison markers expression between stages showed that productively infected cells during AIDS were enriched in memory and exhausted cells. In contrast,the frequencies of infected pTfh were lower during AIDS compared to non-AIDS stages. A UMAP analysis revealed that total CD4+ T cells were grouped in 7 clusters and that productive p24+ cells were skewed to given clusters throughout the course of infection. Overall,the preferential targets of HIV during the latest stages seemed to be more frequently highly differentiated (memory,TTD-like) and exhausted cells and less frequently pTfh-like cells. In contrast,translation-competent reservoir cells were less frequent (5/million CD4+ T cells) and expressed more frequently HLA-ABC and less frequently PD-1.Conclusions:In long-term infection and AIDS,productively infected cells were differentiated and exhausted. This could indicate that cells with these given features are responsible for HIV production and dissemination in an immune dysfunction environment occurring during the last stages of infection. View Publication

Regulated cell death is a key component of the innate immune response,which provides the first line of defense against infection and homeostatic perturbations. However,cell death can also drive pathogenesis. The most well-defined cell death pathways can be categorized as nonlytic (apoptosis) and lytic (pyroptosis,necroptosis,and PANoptosis). While specific triggers are known to induce each of these cell death pathways,it is unclear whether all cell types express the cell death proteins required to activate these pathways. Here,we assessed the protein expression and compared the responses of immune and non-immune cells of human and mouse origin to canonical pyroptotic (LPS plus ATP),apoptotic (staurosporine),necroptotic (TNF-α plus z-VAD),and PANoptotic (influenza A virus infection) stimuli. When compared to fibroblasts,both mouse and human innate immune cells,macrophages,expressed higher levels of cell death proteins and activated cell death effectors more robustly,including caspase-1,gasdermins,caspase-8,and RIPKs,in response to specific stimuli. Our findings highlight the importance of considering the cell type when examining the mechanisms regulating inflammation and cell death. Improved understanding of the cell types that contain the machinery to execute different forms of cell death and their link to innate immune responses is critical to identify new strategies to target these pathways in specific cellular populations for the treatment of infectious diseases,inflammatory disorders,and cancer. View Publication