Frontiers in Immunology

Chronic inflammation plays an obscure role in cancer initiation, with broad references implicating immune exhaustion (IEX) or free radical mediated cell damage. Chronic inflammation is, however, paradoxically synonymous with the term "immune tolerance" which in other cases presents itself as a therapeutic limitation to the efficacy of tumor immune therapies particularly those involving microbial -associated molecular patterns (MAMPs) in regimens. As "tolerance" remains to this day a "phenomenon" there is a pressing need to fully understand every biological aspect of this apparent negative feedback response, because doing so will serve to guide development of targeted immune therapies. In this work, we employ a rudimentary model, which can be adopted in which it is possible to provoke negative feedback through sustained antigen stimulation in immunocompetent cells, as we monitor, define and characterize phenotype evolution using next generation whole transcriptome sequencing + validation studies. This model can be used to study /create in vitro the "M2" phenotype, which is itself involved in aggressive tumors with synergistic rapid expansion of myeloid-derived suppressor cells (MDSCs), dysfunctional CD8+ T cytotoxic (CTL)/ and dysfunctional natural killer (NK) cells. Briefly, the data in this work, shows negative feedback dominates at 7 to 11 days, after acute being associated with phase specific (time dependent) elevated checkpoints; e.g. PDL1+/MSN, HAVCR2/TIM-3+, SPP1+, C3ar1+, CD73+, IL1RN+, LILRbs+, glycoproteins, integrins etc. Here we report on phase specific patterns and bidirectional changes aligning with immune escape, much centered around loss of host defense against viral infection and malignancy. Negative feedback is associated with rampant induction of degradative proteases, SOCS/JAK/STAT/IL-10, the CCL2/7 axis tantamount to sustained loss of MHC1/2 antigen recognition systems, Type I interferon response, NOD signaling, antiviral/antibacterial defense, p62/SQSTM also aligning with a disturbed metabolic signature. The data in this work demonstrates that the colloquial terms "tolerance and IEX" are somewhat flawed terminology, because this negative feedback is a potent, intentional and formidable immune offense to eradicate the active arm of immune defense. Abstract Truncated /Removed due to word countThe data show that "tolerance" aligns with six distinct chronological differential gene (DEG) expression patterns that circumscribe extensive immune suppression which dominate during chronic inflammation. These involve the following time dependent patterns: 1) transcripts overexpressed, e.g. checkpoint receptors; PDL1+/MSN, HAVCR2/TIM-3+, SPP1+, C3ar1+, CD73+, IL1RN+, LILRbs+, glycoproteins, etc.; 2) transcripts overexpressed only in chronic; e.g cytokine suppressor signaling (SOC3/Jak/Stat), cyto/chemokines (IL-10, CCL2, CCL7), proteases (cathepsins L, D, K, Adam 8, PIM2), and adhesives (TSPAN3, QSOX1, PDPN, ITGA5), (PLK2, ADGRE1, CALM1, PCNA, etc.); 3) transcripts downregulated in acute and chronic; e.g. a severe collective loss in MHC1/II antigen presentation capacity (CD74, H2-Q4, H2-Q6, etc.), NOD signaling, and interferon (IFN) type I signaling systems; 4) transcripts downregulated in chronic only, including OXPHOS/metabolic genes (Aldo A, C, Eno2, Gpi1, etc.), antiviral/antibacterial defense genes (Lyz1, Lyz2, Card19, Ninj1), and autophagy-related genes (p62/SQSTM1). In the category of Tolerance were: 5) transcripts induced sharply in acute, no longer responsive in chronic; IFN Type 1 antiviral response genes (OAS, BST2, ISG15, ISG20, IRF7, RSAD2/Viperin), TLR2, antibacterial defense genes (SAA3, SP140), and proinflammatory cytokines (CCL5, TNF, IL1a, and IL1b) along with the IL-1/TLR signaling axis. Last, 6) reverse tolerance, corresponded to restored baseline levels to maintain cell mitotic and thymosin homeostasis. In conclusion, these data suggest chronic inflammation precipitates negative feedback, aligning with the same checkpoint targets being sought after today in tumor immune therapies.

We developed a novel algorithm (DeepCAT) to perform de novo detection of cancer associated TCRs, which is based on a convolutional neural network (CNN) model. In this manuscript, we compared its performance with a similar non-deep learning approach, TCRboost, and demonstrated that DeepCAT achieved better prediction accuracy when used to distinguish cancer from non-cancer individuals. Further, although DeepCAT was trained for CDR3s with different lengths, we showed that the combined outcome does not bias the prediction accuracy. Finally, human immune repertoire is affected by many common inflammatory conditions, and our analysis demonstrated that DeepCAT predictions are minimally affected by these factors.

Macrophages are pivotal innate immune cells which exhibit high phenotypic plasticity and can exist in different polarization states dependent on exposure to external stimuli. Numerous methods have been employed to simulate macrophage polarization states to test their function in vitro. However, limited research has explored whether these polarization methods yield comparable populations beyond key gene, cytokine, and cell surface marker expression. Here, we employ an unbiased comprehensive analysis using data organized through the all RNA-seq and ChIP-seq sample and signature search (ARCHS4) database, which compiles all RNAseq data deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). In silico analyses were carried out demonstrating that commonly employed macrophage polarization methods generate distinct macrophages subsets that remained undescribed until now. Our analyses confirm existing knowledge on macrophage polarization, while revealing nuanced differences between M2a and M2c subpopulations, suggesting non-interchangeable stimuli for M2a polarization. Furthermore, we identify divergent gene expression patterns in M1 macrophages following standard polarization protocols, indicating significant subset distinctions. Consequently, equivalence cannot be assumed among polarization regimens for in vitro macrophage studies, particularly in simulating diverse pathogen responses.

Nanobodies are antigen-binding proteins of great interest as diagnostics and therapeutics. Accurate and fast characterization of their complementarity-determining regions (CDRs) is crucial to uncover the principles guiding their design. Yet, this task remains challenging, as random recombination and somatic mutations generate highly diverse CDR sequences that escape motif-based or structure-prediction approaches currently used to identify them. To overcome this hurdle, we employed two independent strategies that converged on the same conclusion. At the sequence level, we developed a deep learning model to identify nanobody CDRs directly from the primary sequence. At the structural level, we applied an energy decomposition method, revealing CDRs as residues highly uncoupled to the rest of the fold. Explainability analyses showed the network captured intrinsic CDR properties, which notably aligned with these energy values. CDRs emerge as fuzzy regions capable of adopting diverse conformational ensembles, from which a preferred state is selected upon antigen binding. This finding supports a model where chaos in both sequence and structure appears adaptive and disorder emerges as the hallmark of nanobody CDRs. This work aims to advance the definition of rules for the design of antigen binding regions, paving the way for the next-generation immune diagnostics and therapeutics.

The expression of the Nerve growth factor receptor (NGFR) has been described in follicular dendritic cells (FDCs), the major lymphoid stromal cell (LSC) compartment regulating B-cell activation within germinal centers (GCs). However, the role of NGFR in humoral response is not well defined. In this work, we have studied the effect of Ngfr KO in LNs organization and function. Ngfr KO led to spontaneous GC formation and expansion of GC B-cell compartment which were related to Ngfr depletion in non-hematopoietic radioresistant compartment. In agreement, Ngfr KO mice showed alterations in LSC with an increased frequency of FDCs harboring an activated phenotype characterized by the overexpression of CD21/35, MAdCAM-1, and VCAM-1. Moreover, Ngfr KO mice showed GC ectopic location, loss of polarization, impaired high-affinity antibody production, and increased circulating autoantibodies. In addition, Ngfr KO/Bcl2 Tg mice displayed increased levels of autoantibodies, higher incidence of autoimmunity, and decreased overall survival. Our work shows that NGFR maintains GC structure and functionality, being involved in the regulation of antibody production and immune tolerance.

Steadily accumulating evidence supports the concept that the outputs of immune responses are influenced by local nutrient and metabolite conditions or concentrations, as well as by the molecular programming of intermediary metabolism within immune cells. Humoral immunity and germinal center reactions are one setting in which these factors are under active investigation. Hypoxia has been highlighted as one example of how a particular nutrient is distributed in primary and secondary follicles during an antibody response, and how its sensors could impact the qualities of antibody output after immunization. Based on a bio-informatic analysis of mRNA levels in germinal center and other B cells, recently published work challenges the concept that there is any hypoxia or that it has any influence. In this perspective, we perform new analyses of published genomics data to explore potential sources of disparity and elucidate aspects of what on the surface might seem to be conflicting conclusions. In particular, the replicability and variance among data sets derived from different naive as well as germinal center B cells are considered. The results of the investigation highlight several broader issues that merit consideration, especially at a time of heightened focus on scientific reports in the realm of immunity and antibody responses. From one finding of this re-analysis, it is proposed that a standard should be expected in which the relationship of new data sets compared to prior "fingerprints" of cell types should be reported transparently to referees and readers. In light of the strong evidence for diversity in the constituencies within germinal centers elicited by protein immunization, it also is proposed that a core practice should be to avoid overly broad conclusions about germinal centers in general when experimental systems are subject to substantial constraints imposed by technical features.

The sympathetic nervous system (SNS) contributes to immune balance by promoting anti-inflammatory B cells. However, whether B cells possess a self-regulating mechanism by which they modulate regulatory B cell (Breg) function is not well understood. In this study, we investigated the ability of B cells to synthesize catecholamines upon stimulation with different B cell activators. We found, that expression of the enzymes required to generate catecholamines, is upregulated by TLR9. TLR-9-specific expression of tyrosine hydroxylase (TH) correlated with upregulation of adrenergic receptors, enhanced IL-10 production, and with an overexpression of the co-inhibitory ligands PD-L1 and FasL. Moreover, concomitant stimulation of {beta}1-3-adrenergic receptors together with a BCR/TLR9 stimulus enhances the anti-inflammatory potential of Bregs to suppress CD4 T cells, a crucial population in the pathogenesis of autoimmune diseases, like rheumatoid arthritis. In conclusion, our data show that B cells possess autonomous mechanisms to modulate their regulatory function. These findings help to better understand the function of Bregs in autoimmune diseases and the interplay of sympathetic nervous system and B cell function.

Regulatory T cells (Treg) are crucial in the proper balance of the immune system. A better characterization of Treg-specific genes should extend our knowledge on their complex biology. However, to date there is no consensual Treg signature in the literature. Here, we extracted a molecular Treg meta-signature relative to CD4+ conventional T cell from 8 different but comparable publicly available microarray datasets. We confirmed the validity of our result using the much larger but less stringent Immuno-Navigator database. However, many genes of the Treg meta-signature were also expressed at the protein level by other immune cell subsets, as assessed by mass cytometry, with the noticeable exceptions of Il2ra, Ctla4, and Tnfrsf9. Surprisingly, the proenkephalin (Penk) gene was a prominent member of this restricted Treg meta-signature. Further analysis of public datasets and of our own RNA sequencing experiments confirms that Penk was over expressed by Treg in various murine tissues, including thymic Treg. Interestingly, Penk expression was increased in intra tumoral Treg whereas it was down modulated in the central nervous system of mice suffering from EAE. Finally, we propose a mechanistic model linking TNFR signaling and the transcription factor Batf in the regulation of Penk expression in Treg. Altogether, our results provide the first Treg meta-signature in mice and identifies Penk as a novel and unexpected Treg marker.

Complement activation has been verified in COVID-19 patients by both increased serum levels of complement factors C3a and C5b-9 and increased complement deposition at the tissue levels. Complement regulatory proteins (CRPs) CD55, CD46, CD59 and CR1 act to control complement overactivation and eliminate complement deposition and cell lysis. The aim of the study was to investigate the expression of CRPs in COVID-19 in order to identify potential dysregulated expression patterns of CRPs and address whether these may contribute to disease pathogenesis. Single cell RNA-sequencing (scRNA-seq) analysis performed on isolated PBMCs revealed an increase of CD55 expression in severe and critical COVID-19 patients compared to healthy controls. This increase was also detected upon integrated subclustering analysis of the monocyte, T cell and B cell populations. Flow cytometric analysis verified the distinct pattern of upregulated CD55 expression in monocyte and T cell sub populations of severe COVID-19 patients. This upregulation was associated with decreased expression of interferon stimulated genes (ISGs) in patients with severe COVID-19 suggesting a potential suppressor effect of CD55 on interferon responses. The present study identifies a COVID-19 specific CD55 expression pattern in PBMC subpopulations that coincides with reduced interferon responses thus indicating that the complement regulator CD55 may contribute to COVID-19 pathogenesis.

ObjectiveThis study aims to explore the causal relationship between 731 immune cell traits and vitiligo using Mendelian randomization. MethodsWe used a two-sample Mendelian randomization (MR) analysis, employing genetic variations extracted from genome-wide association studies (GWAS) as instrumental variables (IVs). Data sources included immune phenotype data from 3,757 European individuals and data from FinnGen, comprising a total of 385,538 samples (292 cases and 385,509 controls). The study used the inverse-variance weighted (IVW) method as the primary analysis method and conducted various robustness tests through median-based weighted analysis, mode-based weighted analysis, and the MR-Egger method to control for false positive results in multiple hypothesis testing. ResultsOur study found that the pathogenesis of vitiligo may significantly reduce the levels of the following immune cells: TD CD4+ %T cells (b=-0.458, 95% CI=0.17-0.76, PFDR=0.015, P=0.000), CD25 on CD39+ CD4+ cells (b=-0.155, 95% CI=0.78-0.94, PFDR=0.166, P=0.769), and CD4 on HLA DR+ CD4+ cells (b=-0.431, 95% CI=0.50-0.85, PFDR=0.166, P=0.001). Additionally, the causal effect estimate of vitiligo on CCR2 on monocyte is 0.75 (b=-0.290, 95% CI=0.64-0.88, PFDR=0.114, P=0.828), and a negative association was also found on FSC-A on NK cells (b=-0.481, 95% CI=0.47-0.82, PFDR=0.142, P=0.197). On the other hand, our study suggests that the occurrence of vitiligo may increase the levels of CD28 on CD28+ CD45RA+ CD8br cells (b=0.311, 95% CI=1.13-1.65, PFDR=0.166, P=0.393). ConclusionThe results of this study reveal causal links between vitiligo and multiple immune cell traits, highlighting the important role of the immune system in the pathogenesis of vitiligo. These findings provide new research directions for controlling vitiligo through immune regulation and may pave the way for early intervention and treatment strategies.

Ex-vivo stimulation of dendritic cells (DCs) is a critical step in DC-based cancer immunotherapies. In humans, conventional type 1 dendritic cells (cDC1s) are a rare myeloid dendritic cell (mDC) subset that express BDCA-3 (CD141). cDC1s promote CD8+ T cell cross-priming against tumor antigens and are therefore being explored for use in immunotherapy. We evaluated the impact of ex-vivo stimulation on human peripheral blood cDC1s. In contrast to routine evaluation, which focuses on pre-defined surface maturation markers or soluble factors released from the activated cells, we investigated the impact of stimulation on the transcriptome using both RNA-sequencing (RNA-seq) and microarrays. Specifically, we analyzed the mRNA of cDC1s upon activation with two clinical-grade adjuvants, Hiltonol (poly IC, a TLR3 ligand) and protamine-stabilized RNA (pRNA, a TLR7/8 ligand) compared to unstimulated controls. Both RNA-seq and microarray analysis showed profound and similar effects of both Hiltonol and pRNA on the transcriptome of cDC1s. A gene ontology (GO) analysis suggested that these changes were mainly related to activation and maturation pathways, including induction of type-I interferon (IFN) and interleukin (IL)-12 transcription, while pathways related to adverse effects or cell damage did not appear to be affected. Combination of both reagents did not appear to have a synergistic effect, as the transcriptome changes were similar to those induced by each stimulus alone. Together, our results indicate that both adjuvants have comparable effects on cDC1 maturation within an immunogenic short-term culture as performed in immunotherapy.

V{gamma}9V{delta}2 T cells play a key role in the innate immune response to viral infections, including SARS-CoV-1 and 2, and are activated through butyrophilin (BTN)-3A. Here, the objectives were to: 1) characterize the effects of SARS-CoV-2 infection on the number, phenotype, and activation of V{gamma}9V{delta}2 T cells in infected patients, and 2) assess the effects of in vitro SARS-CoV-2 infection on the expression of BTN3A and its impact on the activation and response of V{gamma}9V{delta}2 T cells to an anti-BTN3A antibody. Blood V{gamma}9V{delta}2 T cells decreased in clinically mild SARS-CoV-2 infections compared to healthy volunteers (HV). This decrease was maintained up to 28 days and in the recovery period. Terminally differentiated V{gamma}9V{delta}2 T cells tend to be enriched on the day of diagnosis, 28 days after and during the recovery period compared to HV. Furthermore, these cells showed cytotoxic and inflammatory activities as shown by TNF, IFN{gamma} and CD107a/b increase following anti-BTN3A activation. Moreover, BTN3A upregulation and V{gamma}9V{delta}2 T cell infiltration were observed in a lung biopsy from a fatal SARS-CoV-2 infection, as compared to HV. In vitro, SARS-CoV-2 infection significantly increased BTN3A expression in macrophages and lung cell lines. The activation via BTN3A enhanced the anti-SARS-CoV-2 V{gamma}9V{delta}2 T cells cytotoxicity and IFN-{gamma} and TNF in SARS-CoV-2 infected patient. Increasing concentrations of anti-BTN3A were accompanied by an inhibition of viral replication. Altogether, these data suggest that V{gamma}9V{delta}2 T cells are important in the immune response against SARS-CoV-2 infection and that activation by an anti-BTN3A antibody may enhance their response. KEY POINTSO_LISARS-CoV-2 mediates upregulation of the key receptor of V{gamma}9V{delta}2 T cells BTN3A on lung tissues and cell lines as well as monocytes C_LIO_LIDuring SARS-CoV-2 infection, V{gamma}9V{delta}2 are differentiated and efficiently degranulate and secrete cytokines upon activation with BTN3A mAb C_LI

Phagocytic responses by effector cells to antibody or complement-opsonised viruses have been recognized to play a key role in anti-viral immunity. These include antibody dependent cellular phagocytosis mediated via Fc-receptors, phagocytosis mediated by classically activated complement-fixing IgM or IgG1 antibodies and antibody independent phagocytosis mediated via direct opsonisation of viruses by complement products activated via the mannose-binding lectin pathway. Limited data suggest these phagocytic responses by effector cells may contribute to the immunological and inflammatory responses in SARS-CoV-2 infection, however, their development and clinical significance remain to be fully elucidated. In this cohort of 62 patients, acutely ill individuals were shown to mount phagocytic responses to autologous plasma-opsonised SARS-CoV-2 Spike protein-coated microbeads as early as 10 days post symptom onset. Heat inactivation of the plasma prior to use as an opsonin caused 77-95% abrogation of the phagocytic response, and pre-blocking of Fc-receptors on the effector cells showed only 18-60% inhibition. These results suggest that SARS-CoV-2 can provoke early phagocytosis, which is primarily driven by heat labile components, likely activated complements, with variable contribution from anti-Spike antibodies. During convalescence, phagocytic responses correlated significantly with anti-Spike IgG titers. Older patients and patients with severe disease had significantly higher phagocytosis and neutralisation functions when compared to younger patients or patients with asymptomatic, mild, or moderate disease. A longitudinal study of a subset of these patients over 12 months showed preservation of phagocytic and neutralisation functions in all patients, despite a drop in the endpoint antibody titers by more than 90%. Interestingly, surface plasmon resonance showed a significant increase in the affinity of the anti-Spike antibodies over time correlating with the maintenance of both the phagocytic and neutralisation functions suggesting that improvement in the antibody quality over the 12 months contributed to the retention of effector functions. Author SummaryLimited data suggest antibody dependent effector functions including phagocytosis may contribute to the immunological and inflammatory responses in SARS CoV-2 infection, however, their development, maintenance, and clinical significance remain unknown. In this study we show: O_LIPatients with acute SARS CoV-2 infection can mount phagocytic responses as early as 10 days post symptom onset and these responses were primarily driven by heat labile components of the autologous plasma. These results indicate that the current approach of studying phagocytosis using purified or monoclonal antibodies does not recapitulate contribution by all components in the plasma. C_LIO_LIIn convalescent patients, high phagocytic responses significantly correlated with increasing age, increasing disease severity, high neutralisation functions and high anti-Spike antibody titers, particularly IgG1. C_LIO_LILongitudinal study of convalescent patients over a 12-month period showed maintenance of phagocytic and neutralisation functions, despite a drop in the anti-Spike endpoint antibody titers by more than 90%. However, we found significant increase in the affinity of the anti-Spike antibodies over the 12-month period and these correlated with the maintenance of functions suggesting that improvement in the antibody quality over time contributed to the retention of effector functions. Clinically, measuring antibody titers in sera but not the quality of antibodies is considered a gold standard indicator of immune protection following SARS-CoV 2 infection or vaccination. Our results challenge this notion and recommends change in the current clinical practice. C_LI

Transfer factors are known since 1955 due to their activities on the immune system. Although the reports on the effects on diverse immune mechanisms, their role on Th1, Th2, Th17 and Treg responses was still not described. In this sense, the present work focused on the evaluation of such immune responses. For that, human lymphocytes, and mice thymic, splenic and Peyers cells were stimulated with Lipopolysaccharides and Concanavalin A, and then treated with isolated transfer factors (Imuno TF(R)). The culture medium was harvested and the quantification of Th1 cytokines (IL-2 and IFN-{gamma}), Th2 cytokines (IL-4, IL-5, and IL-13), Th17 cytokine (IL- 17), Treg cytokine (IL-35), inflammatory cytokines (IL-6 and TNF-), and anti-inflammatory cytokine (IL-10) was performed, as well as the quantification of mRNA levels. Imuno TF(R) positively regulated Th1 cytokines, while decreased Th2 cytokines. It also increased levels of mRNA and secretion of the anti-inflammatory cytokine IL-10, whereas it reduced levels of mRNA and the secretion of pro-inflammatory cytokines IL-6 and TNF-. Finally, it reversed the hypersecretion of IL-17 and did not promote significant changes in IL-35 secretion. This highlights the role of Imuno TF(R) in the regulation of the immune responses.

BackgroundImmune activities of monocytes (MOs) can be altered within the microenvironment of solid malignancies, including breast cancer. Metformin (1,1-dimethylbiguanide hydrochloride, MET), has been shown to decrease tumor cell proliferation, but its effects have yet to be explored with respect to the crosstalk between monocytes and breast cancer cells. Here, we investigated the effects of MET on overall phenotypic functional activities of autologous MOs during the interplay with primary breast cancer cells. MethodsHuman primary breast cancer cells were either cultured alone or co-cultured with autologous MOs before treatment with MET. ResultsMET downregulated both breast cancer cell proliferation and the ratio of phosphorylated Akt (p-Akt)-to-Akt in breast cancer cells. Additionally, we observed that, in the absence of MET treatment, the levels of LDH-based cytotoxicity, catalase, intracellular free calcium ions (ifCa2+), IL-10 and arginase activity were significantly reduced in co-cultures compared to those of MOs cultivated alone whereas levels of iNOS were significantly increased (for all comparisons, p < 0.05). In contrast, MET upregulated breast cancer cell LDH-based cytotoxicity levels when co-cultured with MO. MET also induced upregulation of both the inducible enzymatic activity of nitric oxide synthase (iNOS) and arginase activity in MO cells and co-culture systems, although these differences did not reach significant levels for iNOS activity (p > 0.05). MET greatly decreased phagocytic activity in isolated MOs while inducing a robust increase of catalase activity in co-culture systems and of superoxide dismutase (SOD) activity in MOs, but not in MOs co-cultured with breast cancer cells. MET strongly upregulated the levels of ifCa2+ in co-culture systems and IFN-{gamma} production in both isolated MOs and co-culture systems. Moreover, MET treatment markedly downregulated IL-10 production in MOs, while inducing a slight increase in co-cultures (p > 0.05). ConclusionsOur results show that the phenotypic functional activities of MOs change when co-cultured with primary human breast cancer cells. Furthermore, treatment with MET induced enhancing effects on the production of antitumor cytokine IFN-{gamma} and ifCa2+, as well as cytotoxicity during breast cancer cell-MO crosstalk. Novel Highlights includeO_LIFirst analysis of the anti-tumoral effects of Metformin on primary human breast cancer cells and the crosstalk with autologous monocytes. C_LIO_LIPhenotypic functional activities of monocytes change during their interplay with breast cancer cells, which is improved by upregulation of IFN-{gamma} after Metformin treatment. C_LIO_LIMetformin induces downregulation of phosphorylated-Akt1/2-to-Akt1/2 ratio in breast cancer cells. C_LIO_LIMetformin downregulates phagocytic capacity of monocyte from breast cancer patients. C_LI

Osteopontin is a protein with many physiological roles and is widely expressed by many cell types, tissues, and bodily fluids, including breastmilk. The functions of breastmilk osteopontin are not clearly defined, however, it is known to impact intestinal and brain development in infants. Although it is known that endogenous osteopontin influences the survival of intestinal intraepithelial lymphocytes (IEL)2, the impact of breastmilk osteopontin on developing intestinal immune cells remains unclear. In this report, mouse models lacking expression of osteopontin were used to demonstrate that milk-derived osteopontin is important for the development of IELs, with observed effects in both juvenile and adult mice. These changes are most prevalent in IELs expressing CD8: however, the impact of these alterations is unclear, as mice with disrupted IEL compartments are not more susceptible to DSS-induced colitis or infection by Citrobacter rodentium.

Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from the blood of two horses. All main DC subsets were detected by key gene expression, including conventional DC type 1 (cDC1; XCR1) and type 2 (cDC2; FCER1A, CD1E) as well as plasmacytoid DC (pDC; TCF4). In addition, we detected a small cluster of hematopoietic progenitors, as well as transitional DC (tDC; FCER1A, TCF4) and putative DC type 3 (DC3; FLT3, CD163). Our data confirms the previously reported phenotype of equine pDC (Flt3+MHC-IIlowCADM1lowCD172aint), cDC1 (Flt3+MHC-IIhighCADM1highCD172alow-int) and cDC2 (Flt3+MHC-IIhighCADM1intCD172ahigh), while also highlighting considerable CD14 expression for cDC2. Two subclusters of equine cDC2 were found to be enriched in FCER1A or CX3CR1 transcripts (cDC2.1 and cDC2.2, respectively), with suggested enhanced extravasation and T-cell stimulatory capacities of the latter. Conservation of DC subsets across species (horse, pig, human, mouse) was illustrated by enrichment analyses with subset-specific gene signatures and by cross-species data integration with publicly available scRNA-seq datasets. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for understanding the involvement of distinct DC subsets in infections and immune-mediated pathologies.

TLR7 and 8 regulate B cell immunity, but the precise details of the mechanism are still unclear. Here, we studied the kinetics of both heavy and light chains (IgKL) of B-cell receptor (BCR) repertoire responding to the TLR7/8 stimulation in two geniuses of non-human primates (NHPs), African green monkeys (AGMs) and rhesus macaques (RMs). We evaluated the activation of lymphocytes by flow cytometry, and studied characteristics of BCR repertoire in terms of gene usage, repertoire diversity, and the number of lineages. Although AGMs had a weaker activation than RMs, and a different responding kinetic, both AGMs and RMs presented an increased IgKL repertoire diversity and lineages expansion. It suggested that the responding time rather than initiation of TLR7/8-induced IgKL repertoire response related to B cell activation. Expanded IgKL lineages with frequency from 0.001% to 1% had an elevated mutation rate and expanded IgH lineages used more IgA/G/E, suggesting that the TLR7/8 stimulation expanded low-frequent but high-mutated lineages. Besides, most of expanded IgKL lineages were {lambda} isotype. In conclusion, TLR7/8 selectively expands IgKL lineages with a high mutation rate, low frequency, and {lambda} isotype. The selective effect of TLR7/8 on BCR repertoire allows TLR7/8 agonists to be adjuvant for selectively accelerating antibody maturation.

Marginal zone (MZ) B cells are considered to be innate-like immune cells, and follicular (FO) B cells are considered to be adaptive immune B cells. Yet, the proliferative response of MZ and FO B cells to different innate stimuli remains unclear. Here, we investigated cell growth, division, and death to determine the collective proliferative response of MZ and FO B cells in response to innate stimuli, LPS and CpG. We show that the growth rate of FO B cells is higher than that of MZ B cells in response to CpG, though MZ B cells acquire a higher mass at division, whereas both the growth rate and mass at division for MZ and FO B cells remain similar in response to LPS stimulation. We show that MZ B cells divide faster and induce a higher cRel expression in response to both CpG and LPS stimulation than FO B cells. A higher proportion of MZ B cells enter first division in response to LPS stimulation, not in response to CpG stimulation, than FO B cells. Interestingly, CpG stimulation, not LPS stimulation, leads to higher cell death in MZ B cells than FO B cells. In response to LPS stimulation, MZ B cells show a higher cell number at early time and a reduced/ similar cell number at late time, whereas in response to CpG stimulation, MZ B cells show a lower cell number at both early and late time. Our study suggests that LPS and CpG stimulation impact cell growth, division, and death differently, which in turn regulate different proliferative responses of MZ and FO B cells. Thus, our study offers a new perspective that different innate stimuli regulate different features of proliferative responses.

The chemokine CXCL4 activates myeloid cells and contributes to the pathogenesis of inflammatory and fibrotic diseases. One mechanism of CXCL4 action is binding of nucleic acids to promote their internalization and activation of endosomal TLRs. However, the signaling pathways and receptors that mediate myeloid cell responses to CXCL4 alone are not well characterized. Here, we report that in primary human monocytes, CXCL4 activated NF-{kappa}B and a TBK1-JNK signaling axis that drive the expression of inflammatory, fibrotic and neutrophil chemokine genes, and also RIPK3-dependent necroptosis. Surprisingly, six distinct lines of evidence targeting TLR4 expression and function suggested a role for TLR4 in CXCL4 responses. However, we were not able to completely dissect the contributions of CXCL4 alone to the observed results versus a contribution from endotoxin contamination. Our findings suggest that the interactions between CXCL4 and TLR4 merit further study.