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Cell–cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell–cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell–cell communication mechanisms.

The balance between T helper type 1 (Th1) cells and other Th cells is critical for antiviral and anti-tumor responses, but how this fine balance is achieved remains poorly understood. Here, we dissected the dynamic regulation of Th1 cell differentiation during in vitro polarization, as well as in vivo differentiation upon acute viral infection, using scRNA-seq and multiple in vitro and in vivo CRISPR screens. We confirmed the role of known regulators and identified 5 novel regulators for Th1 differentiation. Among the novel regulators the neuropeptide receptor RAMP3, which is a component of the receptor for calcitonin gene-related peptide (CGRP), plays a cell-intrinsic role in Th1 cell-fate determination. Using a unique Th1/Th2 dichotomous culture system, we identified that RAMP3-CGRP interaction directly restricted the differentiation of Th2 cells but promoted Th1 differentiation through activation of downstream cyclic AMP (cAMP) signaling in T cells. Mechanistically, RAMP3 and cAMP signaling resulted in global changes in chromatin accessibility, blocking Th2 genes and specific induction of Th1 programs through activation of IFNγ-STAT1 pathway. Furthermore, both CGRP and RAMP3 were required for inducing effective anti-viral T cell responses to control acute viral infection. Our work reveals a novel neuro-immune circuit in which tissue itself participates in T cell fate determination by producing a neuropeptide during acute viral infection, which acts on RAMP3 expressing T cells to induce an effective anti-viral Th1 response.

T follicular regulatory (TFR) cells limit Ab responses, but the underlying mechanisms remain largely unknown. In this study, we identify Fgl2 as a soluble TFR cell effector molecule through single-cell gene expression profiling. Highly expressed by TFR cells, Fgl2 directly binds to B cells, especially light-zone germinal center B cells, as well as to T follicular helper (TFH) cells, and directly regulates B cells and TFH in a context-dependent and type 2 Ab isotype–specific manner. In TFH cells, Fgl2 induces the expression of Prdm1 and a panel of checkpoint molecules, including PD1, TIM3, LAG3, and TIGIT, resulting in TFH cell dysfunction. Mice deficient in Fgl2 had dysregulated Ab responses at steady-state and upon immunization. In addition, loss of Fgl2 results in expansion of autoreactive B cells upon immunization. Consistent with this observation, aged Fgl2−/− mice spontaneously developed autoimmunity associated with elevated autoantibodies. Thus, Fgl2 is a TFR cell effector molecule that regulates humoral immunity and limits systemic autoimmunity.

The maternal immune system tolerates semi-allogeneic placental tissues during pregnancy. Fas ligand (FASLG) and tumor necrosis factor superfamily 10 (TNFSF10) are known to be components of maternal immune tolerance in humans and mice. However, the role of FASLG and TNFSF10 in the tolerance process has not been studied in pigs, which form a true epitheliochorial type placenta. Thus, the present study examined the expression and function of FASLG and TNFSF10 and their receptors at the maternal-conceptus interface in pigs. The endometrium and conceptus tissues expressed FASLG and TNFSF10 and their receptor mRNAs during pregnancy in a stage-specific manner. During pregnancy, FASLG and TNFSF10 proteins were localized predominantly to endometrial luminal epithelial cells with strong signals on Day 30 to term and on Day 15, respectively, and receptors for TNFSF10 were localized to some stromal cells. Interferon-γ (IFNG) increased the expression of TNFSF10 and FAS in endometrial tissues. Co-culture of porcine endometrial epithelial cells over-expressing TNFSF10 with peripheral blood mononuclear cells yielded increased apoptotic cell death of lymphocytes and myeloid cells. In addition, many apoptotic T cells were found in the endometrium on Day 15 of pregnancy. The present study demonstrated that FASLG and TNFSF10 were expressed at the maternal-conceptus interface and conceptus-derived IFNG increased endometrial epithelial TNFSF10, which, in turn, induced apoptotic cell death of immune cells. These results suggest that endometrial epithelial FASLG and TNFSF10 may be critical for the formation of micro-environmental immune privilege at the maternal-conceptus interface for the establishment and maintenance of pregnancy in pigs.

The strength of the T cell receptor interaction with self-ligands affects antigen-specific immune responses. However, the precise function and underlying mechanisms are unclear. Here, we demonstrate that naive CD8+T cells with relatively high self-reactivity are phenotypically heterogeneous owing to varied responses to type I interferon, resulting in three distinct subsets, CD5loLy6C–, CD5hiLy6C–, and CD5hiLy6C+cells. CD5hiLy6C+cells differ from CD5loLy6C–and CD5hiLy6C–cells in terms of gene expression profiles and functional properties. Moreover, CD5hiLy6C+cells demonstrate more extensive antigen-specific expansion upon viral infection, with enhanced differentiation into terminal effector cells and reduced memory cell generation. Such features of CD5hiLy6C+cells are imprinted in a steady-state and type I interferon dependence is observed even for monoclonal CD8+T cell populations. These findings demonstrate that self-reactivity controls the functional diversity of naive CD8+T cells by co-opting tonic type I interferon signaling.

Bacteriophages, simply phages, have long been used as a potential alternative to antibiotics for livestock due to their ability to specifically kill enterotoxigenic Escherichia coli (ETEC), which is a major cause of diarrhea in piglets. However, the control of ETEC infection by phages within intestinal epithelial cells, and their relationship with host immune responses, remain poorly understood. In this study, we evaluated the effect of phage EK99P-1 against ETEC K99-infected porcine intestinal epithelial cell line (IPEC-J2). Phage EK99P-1 prevented ETEC K99-induced barrier disruption by attenuating the increased permeability mediated by the loss of tight junction proteins such as zonula occludens-1 (ZO-1), occludin, and claudin-3. ETEC K99-induced inflammatory responses, such as interleukin (IL)-8 secretion, were decreased by treatment with phage EK99P-1. We used a IPEC-J2/peripheral blood mononuclear cell (PBMC) transwell co-culture system to investigate whether the modulation of barrier disruption and chemokine secretion by phage EK99P-1 in ETEC K99-infected IPEC-J2 would influence immune cells at the site of basolateral. The results showed that phage EK99P-1 reduced the mRNA expression of ETEC K99-induced pro-inflammatory cytokines, IL-1β and IL-8, from PBMC collected on the basolateral side. Together, these results suggest that phage EK99P-1 prevented ETEC K99-induced barrier dysfunction in IPEC-J2 and alleviated inflammation caused by ETEC K99 infection. Reinforcement of the intestinal barrier, such as regulation of permeability and cytokines, by phage EK99P-1 also modulates the immune cell inflammatory response.

The gastrointestinal tract is the first organ directly affected by fasting. However, little is known about how fasting influences the intestinal immune system. Intestinal dendritic cells (DCs) capture antigens, migrate to secondary lymphoid organs, and provoke adaptive immune responses. We evaluated the changes of intestinal DCs in mice with short-term fasting and their effects on protective immunity against

STAT1 serves as a gatekeeper for naïve CD8 + T cell homeostasis by restraining type I IFN-STAT4-mTOR signaling axis.

Gastrointestinal tract is the first organ to be directly affected upon fasting. However, little is known about how the fasting influences intestinal immune system. In the present study, we focused on the changes of intestinal dendritic cells (DCs) in mice upon short-term fasting and how the changes influence protective immunity against Listeria monocytogenes (LM) infection. We found that the fasting induces an increased number of CD103+CD11b- DCs in both small intestinal lamina propria (SI LP) and mesenteric lymph nodes (mLN) and the SI LP CD103+CD11b- DCs undergo an active proliferation and migration by increased levels of GM-CSF and CCR7, respectively. At 24 hours post-infection (hpi) of LM, there was a significant reduction of bacterial burden from the spleen, liver, and mLN of the short-term fasting mice compared to those of ad libitum mice. Accordingly, short-term fasting mice showed enhanced survival against LM infection when compared with ad libitum mice. Furthermore, significantly high amount of TGF-β2 and Aldh1a2 expression from CD103+CD11b- DCs in mouse infected with LM sequentially caused the following events: the increase of Foxp3+ Tregs, preferential change in the composition of CD103+ to CD103- DCs, and the induction of IFN-γ producing cells. Collectively, increase of intestinal CD103+ DCs by short-term fasting is a key player for protection against LM infection through the changes of functional features from tolerogenic to Th1 immunogenic.

Low-pathogenicity avian influenza H9N2 remains an endemic disease worldwide despite continuous vaccination, indicating the need for an improved vaccine strategy. Bacillus subtilis (B. subtilis), a gram-positive and endospore-forming bacterium, is a non-pathogenic species that has been used in probiotic formulations for both animals and humans. The objective of the present study was to elucidate the effect of B. subtilis spores as adjuvants in chickens administered inactivated avian influenza virus H9N2. Herein, the adjuvanticity of B. subtilis spores in chickens was demonstrated by enhancement of H9N2 virus-specific IgG responses. B. subtilis spores enhanced the proportion of B cells and the innate cell population in splenocytes from chickens administered both inactivated H9N2 and B. subtilis spores (Spore + H9N2). Furthermore, the H9N2 and spore administration induced significantly increased expression of the pro-inflammatory cytokines IL-1β and IL-6 compared to that in the H9N2 only group. Additionally, total splenocytes from chickens immunized with inactivated H9N2 in the presence or absence of B. subtilis spores were re-stimulated with inactivated H9N2. The subsequent results showed that the extent of antigen-specific CD4+ and CD8+ T cell proliferation was higher in the Spore + H9N2 group than in the group administered only H9N2. Taken together, these data demonstrate that B. subtilis spores, as adjuvants, enhance not only H9N2 virus-specific IgG but also CD4+ and CD8+ T cell responses, with an increase in pro-inflammatory cytokine production. This approach to vaccination with inactivated H9N2 together with a B. subtilis spore adjuvant in chickens produces a significant effect on antigen-specific antibody and T cell responses against avian influenza virus.

Porcine epidemic diarrhea virus (PEDV) invades porcine intestinal epithelial cells (IECs) and causes diarrhea and dehydration in pigs. In the present study, we showed a suppression of PEDV infection in porcine jejunum intestinal epithelial cells (IPEC-J2) by an increase in autophagy. Autophagy was activated by rapamycin at a dose that does not affect cell viability and tight junction permeability. The induction of autophagy was examined by LC3I/LC3II conversion. To confirm the autophagic-flux (entire autophagy pathway), autophagolysosomes were examined by an immunofluorescence assay. Pre-treatment with rapamycin significantly restricted not only a 1 h infection but also a longer infection (24 h) with PEDV, while this effect disappeared when autophagy was blocked. Co-localization of PEDV and autophagosomes suggests that PEDV could be a target of autophagy. Moreover, alleviation of PEDV-induced cell death in IPEC-J2 cells pretreated with rapamycin demonstrates a protective effect of rapamycin against PEDV-induced epithelial cell death. Collectively, the present study suggests an early prevention against PEDV infection in IPEC-J2 cells via autophagy that might be an effective strategy for the restriction of PEDV, and opens up the possibility of the use of rapamycin in vivo as an effective prophylactic and prevention treatment., Highlights • Rapamycin has an antiviral effect against PEDV infection. • Rapamycin prevents PEDV-induced cell death. • Rapamycin-induced autophagy restricted PEDV infection in porcine intestinal epithelial cells.

Continuous contact with self-major histocompatibility complex (MHC) ligands is essential for survival of naive T cells but not memory cells. This surprising finding implies that T cell subsets may vary in their relative T-cell receptor (TCR) sensitivity. Here we show that in CD8+T cells TCR sensitivity correlates inversely with levels of CD5, a marker for strong self-MHC reactivity. We also show that TCR sensitivity is lower in memory CD8+ T cells than naive cells. In both situations, TCR hypo-responsiveness applies only to short-term TCR signalling events and not to proliferation, and correlates directly with increased expression of a phosphatase, CD45 and reciprocal decreased expression of activated LCK. Inhibition by high CD45 on CD8+ T cells may protect against overt TCR auto-MHC reactivity, while enhanced sensitivity to cytokines ensures strong responses to foreign antigens. Naive T cells establish self-tolerance via negative selection of cells with strong reactivity for self-peptide/MHC complexes, but undergo T-cell receptor (TCR) desensitisation when leaving the thymus. Here, Choet al.show that TCR desensitisation correlates with cell-surface density of the phosphatase CD45.

Modern livestock production became highly intensive and large scaled to increase production efficiency. This production environment could add stressors affecting the health and growth of animals. Major stressors can include environment (air quality and temperature), nutrition, and infection. These stressors can reduce growth performance and alter immune systems at systemic and local levels including the gastrointestinal tract. Heat stress increases the permeability, oxidative stress, and inflammatory responses in the gut. Nutritional stress from fasting, antinutritional compounds, and toxins induces the leakage and destruction of the tight junction proteins in the gut. Fasting is shown to suppress pro-inflammatory cytokines, whereas deoxynivalenol increases the recruitment of intestinal pro-inflammatory cytokines and the level of lymphocytes in the gut. Pathogenic and viral infections such as Enterotoxigenic E. coli (ETEC) and porcine epidemic diarrhea virus can lead to loosening the intestinal epithelial barrier. On the other hand, supplementation of Lactobacillus or Saccharaomyces reduced infectious stress by ETEC. It was noted that major stressors altered the permeability of intestinal barriers and profiles of genes and proteins of pro-inflammatory cytokines and chemokines in mucosal system in pigs. However, it is not sufficient to fully explain the mechanism of the gut immune system in pigs under stress conditions. Correlation and interaction of gut and systemic immune system under major stressors should be better defined to overcome aforementioned obstacles.

Developing effective mucosal subunit vaccine for the Streptococcus pneumoniae has been unsuccessful mainly because of their poor immunogenicity with insufficient memory T and B cell responses. We thus address whether such limitation can be overcome by introducing effective adjuvants that can enhance immunity and show here that polysorbitol transporter (PST) serves as a mucosal adjuvant for a subunit vaccine against the Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) with PST adjuvant induced protective immunity against S. pneumoniae challenge, especially long-term T and B cell immune responses. Moreover, we found that the PST preferentially induced T helper (Th) responses toward Th2 or T follicular helper (Tfh) cells and, importantly, that the responses were mediated through antigen-presenting cells via activating a peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway. Thus, these data indicate that PST can be used as an effective and safe mucosal vaccine adjuvant against S. pneumoniae infection. STATE OF SIGNIFICANCE: In this study, we suggested the nanoparticle forming adjuvant, PST works as an effective adjuvant for the pneumococcal vaccine, PspA. The PspA subunit vaccine together with PST adjuvant efficiently induced protective immunity, even in the long-term memory responses, against Streptococcus pneumoniae lethal challenge. We found that PspA with PST adjuvant induced dendritic cell activation followed by follicular helper T cell responses through PPAR-γ pathway resulting long-term memory antibody-producing cells. Consequently, in this paper, we suggest the mechanism for safe nanoparticle forming subunit vaccine adjuvant against pneumococcal infection.

Respiratory syncytial virus (RSV) is a major pathogen that infects lower respiratory tract and causes a common respiratory disease. Despite serious pathological consequences with this virus, effective treatments for controlling RSV infection remain unsolved, along with poor innate immune responses induced at the initial stage of RSV infection. Such a poor innate defense mechanism against RSV leads us to study the role of alveolar macrophage (AM) that is one of the primary innate immune cell types in the respiratory tract and may contribute to protective responses against RSV infection. As an effective strategy for enhancing anti-viral function of AM, this study suggests the intranasal administration of Bacillus subtilis spore which induces expansion of AM in the lung with activation and enhanced production of inflammatory cytokines along with several genes associated with M1 macrophage differentiation. Such effect by spore on AM was largely dependent on TLR-MyD88 signaling and, most importantly, resulted in a profound reduction of viral titers and pathological lung injury upon RSV infection. Taken together, our results suggest a protective role of AM in RSV infection and its functional modulation by B. subtilis spore, which may be a useful and potential therapeutic approach against RSV.

Ginsenosides, the major active component of ginseng, are traditionally used to treat various diseases, including cancer, inflammation, and obesity. Among these, compound K (CK), an intestinal bacterial metabolite of the ginsenosides Rb1, Rb2, and Rc from Bacteroides JY-6, is reported to inhibit cancer cell growth by inducing cell-cycle arrest or cell death, including apoptosis and necrosis. However, the precise effect of CK on breast cancer cells remains unclear. MCF-7 cells were treated with CK (0-70 micrometer) for 24 or 48 h. Cell proliferation and death were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Changes in downstream signaling molecules involved in cell death, including glycogen synthase kinase 3β (GSK3β), GSK3β, β-catenin, and cyclin D1, were analyzed by western blot assay. To block GSK3β signaling, MCF-7 cells were pretreated with GSK3β inhibitors 1 h prior to CK treatment. Cell death and the expression of β-catenin and cyclin D1 were then examined. CK dose- and time-dependently inhibited MCF-7 cell proliferation. Interestingly, CK induced programmed necrosis, but not apoptosis, via the GSK3β signaling pathway in MCF-7 cells. CK inhibited GSK3β phosphorylation, thereby suppressing the expression of β-catenin and cyclin D1. Our results suggest that CK induces programmed necrosis in MCF-7 breast cancer cells via the GSK3β signaling pathway.

γδ T cells, known to be an important source of innate IL-17 in mice, provide critical contributions to host immune responses. Development and function of γδ T cells are directed by networks of diverse transcription factors (TFs). Here, we examine the role of the zinc finger TFs, Kruppel-like factor 10 (KLF10), in the regulation of IL-17-committed CD27− γδ T (γδ27−-17) cells. We found selective augmentation of Vγ4+ γδ27− cells with higher IL-17 production in KLF10-deficient mice. Surprisingly, KLF10-deficient CD127hi Vγ4+ γδ27−-17 cells expressed higher levels of CD5 than their wild-type counterparts, with hyper-responsiveness to cytokine, but not T-cell receptor, stimuli. Thymic maturation of Vγ4+ γδ27− cells was enhanced in newborn mice deficient in KLF10. Finally, a mixed bone marrow chimera study indicates that intrinsic KLF10 signaling is requisite to limit Vγ4+ γδ27−-17 cells. Collectively, these findings demonstrate that KLF10 regulates thymic development of Vγ4+ γδ27− cells and their peripheral homeostasis at steady state.

Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, South Korea In a steady-state, naïve CD8+ T cells are kept to be quiescent in response to homeostatic cues such as self-ligands and cytokines while preserving a robust responsiveness to foreign antigens. Although such a steady-state quiescence involves multiple regulators, an exact nature of the regulators and underlying mechanisms are still incompletely understood. Here we showed that the signal transducer and activator of transcription 1 (STAT1), a key transcription factor downstream of type I and II interferon receptor signaling, plays a cell-intrinsic role in regulating naïve CD8+ T cell responsiveness to tonic homeostatic cytokines. Thus, mice lacking STAT1 led to abnormalities in the composition and number of naïve and effector/memory phenotype among CD8+ T cells. The observed alterations were not due to dysregulation in thymic development but rather resulted from enhanced proliferative capacity of peripheral mature STAT1-deficient naïve CD8+ T cells in a cell-autonomous manner. In vitro analyses revealed that two key cytokines, IL-7 and type I interferon, serve as a major driver to induce abnormal proliferation of STAT1-deficient but not wild-type CD8+ T cells depending on a pathway that is sensitive to rapamycin, indicating a role of mTORC1 signaling. Based on these findings, we suggest that, under steady-state, naïve CD8+ T cells that are continuously exposed to homeostatic cytokines actively restrain their responsiveness to the “Goldilocks” levels – that is, not too strong to induce activation/proliferation and not too weak to lose a survival ability – presumably via blocking mTORC1 pathway, therefore maintaining a functional state of quiescence before encountering a cognate foreign antigen.

Cross-presentation of epitope from exogenous antigen onto MHC I molecule enhances CD8+ T cell activity is important in host protective immune responses against virus infections and cancer. We previously reported that polysorbitol transpoter (PST), prepared from sorbitol diacrylate and low-molecular-weight polyethylenimine, induces a long-term antigen specific B cell response and phagolysosomal escape of antigens in macrophages via mechanism called ‘proton-sponge effect’. Therefore, we hypothesize that PST could enhance the cross-presentation and antigen-specific CD8+ T cell immune responses. In the present study, we tried to determine whether PST could enhance cross-presentation of DC using model antigen Ovalbumin (OVA). PST formed a nano-sized complex with OVA (OVA-PST) enhanced SIINFEKL peptide loading onto MHC I molecules. The results further showed that it enhanced OVA-specific CD8+ T cell proliferation and activation. However, PST was not able to upregulate co-stimulatory molecules and pro-inflammatory cytokines, which are necessary to induce adequate protective immune response. Together with OVA-PST, Monophospholipid A (MPLA), a nontoxic TLR4 agonist which is analog of lipid A also used as co-delivery agent in nanoparticle peptide vaccine, upregulated the effects of OVA-PST complexes. Finally, when PST-OVA and MPLA applied to DC-based vaccine for tumor therapy, survival enhanced in mouse engrafted with tumor. Collectively, these results suggested that the PST enhances cross-presentation in DCs together with antigen-specific CD8+ T cell immune responses. Therefore PST with MPLA could act to improve DC-based vaccine.