bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbiota regulate the induction and proliferation of revival intestinal stem cells through inflammatory cytokines
Authors: Tsang Derek K.L. , Wang Ryan J. , Maisonneuve Charles , Ayyaz Arshad , Trcka Daniel , Wana Jeffrey L. , Girardin Stephen E. , Philpott Dana J.
Published: February 2023

Gut microbiota are critical mediators of inflammation and regeneration of the intestinal epithelium. Intestinal restitution is a coordinated response that involves the dedifferentiation of mature epithelial cell lineages, the proliferation of Lgr5+ intestinal stem cells, and the induction of Clu+ revival stem cells (RSCs). How the gut microbiota directly impacts this regenerative process remains unclear. Using irradiation as a model for small intestinal epithelium restitution, we demonstrate that microbiota regulate the induction and subsequent proliferation of Clu+ RSCs. Our results report that specific pathogen-free (SPF) mice induce greater RSCs 3 days post-IR in comparison to germ-free (GF) mice. This microbiota-dependent increase in RSCs was matched by an increase in BrdU+ proliferating cells and an increase in TUNEL+ apoptotic cells in SPF mice. Using intestinal organoids as an in vitro model of intestinal restitution, SPF and GF intestinal crypts demonstrated equal propensity to generate mature organoids. Transcriptional analysis of GF and SPF RSCs by single-cell RNA sequencing highlighted unique regenerative and inflammatory gene signatures. The altered regeneration kinetics observed in SPF mice was accompanied by an increase in Tnfa and Cxcl1 at the peak of RSC induction and an increase in Ifnγ post RSC induction. Altogether, these findings suggest that microbiota-dependent expression of inflammatory cytokines may be key regulators in facilitating the expansion and proliferation of RSCs to efficiently repair the intestinal epithelium following damage.
Defective Humoral Immunity Disrupts Bile Acid Homeostasis Which Promotes Inflammatory Disease of the Small Bowel
Authors: Mohammed Ahmed Dawood , Kubinak Jason L.
Published: February 2023

Mucosal antibodies maintain gut homeostasis by promoting spatial segregation between host tissues and luminal microbes. Whether and how mucosal antibody responses influence gut health through modulation of microbiota composition is unclear. One critical metabolic function carried out exclusively by bacteria in the gut is bio-transformation of host bile acids (BAs), and dysregulation of BAs metabolism has been linked to numerous inflammatory and metabolic diseases in humans. Here, we use a CD19−/− mouse model of antibody-deficiency to demonstrate that a relationship exists between dysbiosis, defects in BA homeostasis, and enteropathy of the small intestine (SI). SI enteropathy that develops in CD19−/− mice is associated with alterations to the luminal BA pool in the SI, marked by significant reductions in the abundance of conjugated BAs. Manipulation of BA availability, adoptive transfer of functional B cells, and ablation of bacterial bile salt hydrolase (bsh) activity all influence the severity of SI enteropathy in CD19−/− mice. Collectively, results from our experiments support a model whereby mucosal humoral immune responses limit inflammatory disease of the small bowel by regulating bacterial BA metabolism.
Lack of bone marrow stromal cell antigen-1 (BST-1)/CD157 ameliorated colitis induced by dextran sodium sulfate (DSS)
Authors: Ishihara Katsuhiko , Yahagi Ayano , Katsumata Ryo , Shiotani Akiko , Iseki Masanori
Published: February 2023

Bone marrow stromal cell antigen-1(BST-1)/CD157 is GPI-anchored ectoenzyme having adenosine diphosphate(ADP)-ribosyl cyclase and cyclic ADP-ribose hydrolase activities. BST-1 also functions as a receptor for signal transduction. Recently, various groups reported novel functions of BST-1, such as regulation of intestinal or mesenchymal stem cells. Re-analyses of BST-1 deficient mice (Bst1KO) in C57BL/6 background revealed roles in defense against tuberculosis, and prevention of depressive or anxious behaviors. Thus, BST-1 is now appreciated as an entero-neuro-immune regulator. Since expression of BST-1 in the intestines is common in human and mouse, we examined the roles for BST-1 in DSS-induced colitis using Bst1KO.

Drinking water containing 3% DSS was supplied ad libitum to Bst1KO and C57BL/6 (WT) in SPF condition for 5 days, and then water without DSS for 2 days. Decreases of body weights in Bst1KO were similar to WT until day 5, but since then they became significantly slower than WT. Clinical scores based on bleeding and diarrhea were similar in both genotypes. Histological analyses with HE staining revealed the ratio of destroyed epitheliums in the total colon of Bst1KO was 15% whereas that of WT was 50%. Flow cytometrical analyses revealed no difference in the cell numbers of neutrophils and macrophages increased in the colon at day 7. Quantitative real-time PCR revealed that increases of Bst1 expression in the colons of WT started on day 1 and reached to the level more than thousand times at day 5. In the colons of Bst1KO, increases of Il6 expression at day 5 were markedly decreased compared with those of WT.

These data indicate that BST-1 has pro-inflammatory roles in DSS-induced colitis through positive regulation of IL-6 production.
Lymph node stromal cells drive MHCI-restricted CD8 T cell lineage plasticity
Authors: Honan Amanda , Vazquez Emily , Chen Zhibin
Published: February 2023

CD8+ T cells have been shown to convert to CD4+ T cells in the gut environment producing a unique population of MHCI-restricted CD4+ T cells. MHCI-restricted CD4+ T cells then have the ability to further differentiate into MHCI-restricted CD4+Foxp3+ T cells (CI-Treg). Here, we show several factors which facilitate the conversion from the CD8+ T celllineage to the CD4+ T cell lineage in the gut environment. Intrinsic MHCII expression by lymph node stromal cells (LNSC) is an important factor in this process. Both the conversion from CD8+ T cells to CD4+ T cells and the differentiation of CI-Treg are impaired when MHCII is deleted. Furthermore, in vitro co-culture experiments show that LNSC are vital in the expansion of both MHCI-restricted CD4+ T cells and CI-Treg. This population of regulatory T cells also exhibits an atypical phenotype which may contribute to the ability to control inflammation within the gut environment and beyond, and thus may add a new possibility to the arsenal of immune cell therapy for tolerance induction.
Evolution of immunogenetic components used to form ultralong VH CDR3 antibodies in Bovidae
Authors: Mitchell Christian D. , Marchand Ashley J , Head Kelly , Haakenson Jeremy K , Kelley Abigail , Deiss Thaddeus C , Derr James N , Smider Vaughn V , Criscitiello Michael F
Published: February 2023

Cattle has a restricted repository of immunoglobulin heavy chain gene segments with only one family of V, 4 J and 16 D segments functional. Among these segments are a V and D that, together, encode a heavy chain with an ultralong CDR3 region which manifests as a “stalk” supporting a “knob” domain of which only the knob has antigen binding capability. This antibody structure has boundless therapeutic potential, including for treatment of HIV and other antigens with veiled epitopes. Upon the discovery of this ultralong antibody in Bos taurus, speculation of its origin began. The components that should facilitate ultralong CDR3H antibodies appear in more species than just Bos taurus; they expand throughout the bovine subfamily as evidence by the presence of a V motif “TTVHQ” crested by an 8 base-pair (bp) duplication, and a comparatively long D segment. Several closely-related species encode the ultralong V segments gene in their germline IgH locus. Phylogenetically bookending the cluster that is genomically equipped for the ultralong cattlebody are the species Bos taurus and Bison bison with Bos mutus, indicus, and grunniens in between. The added length of the D segment is less consistent throughout the group as none of the species encode as long of segment as Bos taurus at 50 amino acids. Two species studied, Bos mutus and Bos grunniens, have 15 and 22 amino acids, respectively, encoded by their longest D segment, and Bison bison has 23. Though we do know the germline encodes the ultralong antibody, it is imperative to know if the gene rearrangement is actually expressed and employed in the mature repertoire.
SARS-CoV-2 spike binding peptides: A potential therapeutic approach for COVID-19
Authors: Ramasamy Santhamani , Zain Siddiqui , Roy Abhishek , Kumar Ranjeet , Kolloli Afsal , Chang Theresa Li-Yun , Kumar Vivek , Subbian Selvakumar
Published: February 2023

SARS-CoV-2, the causative agent of COVID-19, has infected about 100 million people and resulted in 2.6% mortality worldwide since late 2019. Various anti-viral agents, such as Remdesivir and hydroxychloroquine, have been used empirically to treat COVID-19. However, efficient therapeutics for COVID-19 are yet to be developed. SARS-CoV-2 enters the host cells by binding its envelope spike protein to the cellular receptors, like the ACE2. Here, we report the assessment of a panel of SARS-CoV-2 spike-binding synthetic, self-assembling peptides for their therapeutic potential. A HIV-1 pseudo typed virus (pNL-Luc.R-E-) expressing full-length SARS-CoV-2 spike protein was used to evaluate the anti-viral effect of these peptides. Briefly, pseudoviruses were treated with nine peptides at 0.01μM, 0.1μM, 1μM, 10μM concentrations before infecting 293T cells overexpressing ACE2. Viral infection was determined by measuring the luciferase activity of the infected cells. The anti-SARS-CoV2 activity of tested peptides was also evaluated by plaque-forming assay using replication-competent viruses. All the tested peptides showed SARS-CoV-2 neutralization, and five of the nine peptides showed IC50 values ≤ 0.01μM. In a rat model, we showed that the peptides, administered at 1mM daily-dosing for 10 days, was non-toxic and well-tolerated, indicating their potential therapeutic use for COVID-19. Further study warrants in vivo efficacy testing of selected peptides in preclinical animal models.
DEC-205 is essential for control of WNV infection and immunity in the CNS during WNV encephalitis
Authors: Ho Justin , Khatri Hemisha , Santana Anthony , Durrant Douglas
Published: February 2023

West Nile Virus (WNV) is the leading cause of mosquito-borne neuroinvasive disease in the United States. Upon entry into the central nervous system (CNS), WNV infects neurons, causing neuronal injury and inflammation, potentially resulting in long-term sequelae and death. Studies have shown that infiltrating virus-specific CD8+ T cells are dependent on dendritic cell-mediated reactivation in the CNS to restrict viral replication and pathogenesis within the CNS. Previous reports have shown that CD8+DEC-205+ dendritic cells (DCs) rapidly accumulate within the CNS during WNV encephalitis; however, their specific role in host defense has not been demonstrated. DEC-205, an endocytic receptor expressed by DC subsets, is responsible for cross-presentation and can promote either antigen (Ag) specific effector T cell activation or immunological T cell tolerance. Using a mouse model of WNV encephalitis, we show that DEC-205 critically regulates effector T cell responses within the CNS. DEC-205−/− mice exhibited enhanced mortality compared to wild-type (WT) controls. The enhanced susceptibility of the DEC-205−/− mice arose from an increased viral burden; however, this was associated with increased numbers of infiltrating and activated T cells and increased inflammatory myeloid cells suggesting that DEC-205 promotes protective immune responses within the CNS that limit immunopathology during a neurotropic viral infection. These data clarify the role of DEC-205+ DCs in the restriction of viral pathogenesis and implicate DEC-205+ DCs as a key regulator of protective immune responses within the CNS.
Crucial roles of XCR1-expressing dendritic cells and the XCR1-XCL1 chemokine axis in protection against West Nile virus within the CNS
Authors: Durrant Douglas M. , Cortes Alejandro , Alvarado Marina O
Published: February 2023

West Nile Virus (WNV) is a leading cause of mosquito-borne neuroinvasive disease in the US. Following infection, WNV can cause neuronal injury and disease. Previous studies have shown that the proper activation of virus-specific T cells results in protection from WNV infection and any associated disease. Dendritic cells (DCs) are often defined as pivotal inducers of immunity due to their role in activating immune defense mechanisms against invading pathogens. XCR1, a chemokine receptor expressed predominantly by a subset of DCs, has been shown to be essential in cross-presenting antigen and in activating anti-viral immunity. However, DCs are not readily present within the CNS and it remains unknown how DCs are recruited to the CNS during WNV encephalitis to fully activate antiviral immunity. XCL1, the ligand for XCR1, is expressed by activated T cells, DCs, macrophages, and NK cells. We hypothesize that following WNV infection, virus-specific T cells within the CNS produce XCL1 in order to recruit and stabilize their interaction with XCR1+ DCs thus ensuring their proper activation and subsequent protection against WNV encephalitis. Here we demonstrate that mice lacking XCR1+ DCs are more susceptible to WNV encephalitis, display increased neuronal cell death, as well as increased viral burden compared to control animals. In addition, in the absence of XCR1+ DCs, WNV-infected animals displayed decreased parenchymal localization of T cells within the brain compared to controls. These data indicate that XCL1-XCR1 interactions are crucial in the development of efficient anti-viral immunity. The knowledge gained from this work may then be applicable to other viral diseases of the CNS.
An interferon-stimulated transcriptionally independent isoform of ACE2 inhibits SARS-CoV-2 infection
Authors: Onabajo Olusegun O , Stanifer Megan , Banday A. Rouf , Vargas Joselin , Yan Wusheng , Obajemu Adeola , Ring Timothy , Kee Carmon , Doldan Patricio , Boulant Steeve , Prokunina-Olsson Ludmila
Published: February 2023

Regulation of ACE2, the SARS-CoV-2 receptor, could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a transcriptionally independent truncated isoform of ACE2, designated as deltaACE2 (dACE2). dACE2 starts from a new exon in intron 9 of ACE2, and is highly conserved in primates. dACE2, but not ACE2, is induced by interferons and viruses including SARS-CoV-2. In-vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Endogenous dACE2 protein appears to be unstable and poorly detectable. However, using our custom generated antibodies, we detected dACE2 in nasopharyngeal normal and tumor tissues by immunohistochemistry. In-vitro, overexpression of dACE2 inhibited SARS-CoV-2 infection, possibly by blocking internalization of the virus. Our results suggest that dACE2 might be an antiviral mechanism that evolved in primates to defend against certain viruses that utilize the ACE2 receptor for entry.
Distinct peripheral inflammatory profiles distinguish COVID-19 from influenza infection, with limited contributions from cytokine storm
Authors: Crawford Jeremy Chase , Mudd Philip A. , Turner Jackson S. , Souquette Aisha , Reynolds Daniel , Bender Diane , Bosanquet James , Anand Nitin J. , Striker David A. , Martin R. Scott , Boon Adrianus C.M. , House Stacey L. , Remy Kenneth E. , Hotchkiss Richard , Presti Rachel , O’Halloran Jane , Powderly William G. , Thomas Paul Glyndwr , Ellebedy Ali H.
Published: February 2023

We developed a prospective observational cohort of COVID-19 and influenza patients to assess the quality and magnitude of their immune responses at the cellular and protein levels. Although COVID-19 patients exhibited equivalent lymphocyte counts compared to influenza patients, they had fewer monocytes and lower surface HLA-class II expression on select monocyte populations compared to influenza patients and healthy controls. Decreased HLA-DR on intermediate monocytes was a significant predictor of COVID-19 disease severity. Protein cytokine levels were measured in two distinct COVID-19 cohorts, composed of 73 and 89 patients, revealing multiple inflammatory phenotypes. Only four percent of patients (7 of 162) exhibited a distinct Cytokine Storm Syndrome (CSS) phenotype. Furthermore, COVID-19 patients generally exhibited lower cytokine levels than influenza patients. Upregulation of a few innate inflammatory mediators, including IL-6, GCSF, IL-1RA, and MCP1, predicted death from acute respiratory failure among COVID-19 patients but were not statistically higher than those of influenza patients. Single-cell transcriptional profiling of 2 healthy controls as well as 3 COVID-19 and 3 influenza subjects with respiratory failure was concordant with the profound suppression in type I and type II interferon signaling in COVID-19 patients across multiple cell types. In contrast, COVID-19 cells were enriched for alterations in metabolic, stress, and apoptotic pathways. When considered across the spectrum of peripheral innate and adaptive immune profiles, the immune pathologies underlying severe influenza and COVID-19 are substantially distinct, with COVID-19 patients generally less inflamed than those with influenza.
Interferon signature in cutaneous lesion of COVID toes
Authors: Komura Kazuhiro , Ichimura Yuki , Okiyama Naoko , Watanabe Kazuyoshi , Muramoto Hiroaki , Matsushita Takashi
Published: February 2023

Increasing reports have showed several kinds of dermatological manifestation in Coronavirus disease 2019 (COVID-19). Some of them may occur as a direct implication of coronavirus infection. Within those, acral skin changes, recently called COVID toes, have a potential to provide a diagnostic value. We herein described a 49-year old Japanese man with COVID toes. The skin lesions appeared simultaneously with fever. The rash was similar for cold-induced erythema multiforme of the patients with familial Chilblain; hereditary type I interferonopathy. Histopathological staining indicated up-regulated expression of MxA, effected by local enhancement of type I interferon. These speculated that the specificity of COVID toes may depend on interferon up-regulated by specific mechanisms.
ARID3a Defines a Novel Subset of Naïve B Cells in SLE Patients
Authors: Garton Joshua W , Shankar Malini , Guthridge Joel , James Judith A , Webb Carol
Published: February 2023

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by overproduction of autoantibodies by B cells and the loss of tolerance to nucleic acids. Our previous studies show that AT-rich interacting domain 3a (ARID3a), a DNA-binding protein, is more abundant in SLE B cells than in healthy control B cells. Increased numbers of ARID3a+ B cells in SLE correlated with increased disease activity in patients. ARID3a expression in SLE patient blood occurred in naïve B cell subsets which do not express ARID3a in healthy controls. Studies by the Sanz laboratory revealed that activated naïve B cells and may be precursors for the double negative (DN) B cells linked to pathogenic responses in SLE. We hypothesize that ARID3a expression in naïve B cells also defines precursors of pathogenic B cells. In individual patients, ARID3a is bimodally expressed, such that only a fraction of the naïve B cells express ARID3a at any given time. We took advantage of this bimodal expression using single cell technology to perform RNA-seq analyses of ARID3a+ and ARID3a− SLE B cells to identify differentially expressed genes associated with ARID3a. PCA analyses of scRNA seq data show that ARID3a+ naïve B cells cluster together and away from ARID3a− cells. This allows for analyses within and among individual samples by binning cells based on ARID3a expression. Preliminary data indicate that ARID3a expression is correlated with innate immune response genes, transcription factors, and epigenetic regulators. These studies will be informative regarding how ARID3a expression contributes to disease pathogenesis in patients with SLE and will provide novel information defining a new subset of naive B cells that may be directly linked to breaches in tolerance.
CD8 T follicular cell function in autoimmune disease
Authors: Mullins Genevieve N , Valentine Kristen M , Davalos Oscar , Hoyer Katrina K.
Published: February 2023

Autoimmune disease comprises more than 80 well characterized disorders that affect 24 million Americans and many of these disorders are characterized by the presence of high-affinity auto-antibodies. The production of these high-affinity antibodies requires activation, affinity maturation, and differentiation of B cells into class-switched plasma cells. This process occurs within the germinal center and requires the help of specialized T follicular helper (Tfh) cells. Traditionally, Tfh are CD4 T cells that become activated and differentiate, acquiring the surface proteins required to migrate into the germinal center and interact directly with B cells to induce germinal center responses. Recently, a population of CD8 T follicular (Tfc) cells that are phenotypically similar to CD4 Tfh have been described in chronic viral infection, cancer, and autoimmune disease. These cells have been shown to function similarly to CD4 Tfh cells as well as cytotoxic CD8 T cells under various conditions. Further, the mechanism and localization of CD8 Tfc cell function in autoimmune disease has not yet been investigated. Using immunofluorescence CD8 Tfc cells were found to localize and produce cytokines in the B cell follicle and germinal center in autoimmune disease. Indirect co-culture of CD8 Tfc cells with B cells show that while CD8 produced IL-21 does not influence B cell function, CD8 produced IL-4 does. This data indicates that CD8 Tfc and CD4 Tfh cells may rely on slightly different mechanisms to influence B cell function within and without the germinal center.
Leukemic Cytokines co-opt Regulatory T cell and Th17 immunity to promote chronic myeloid leukemia
Authors: Camacho Virginia , Matkins Victoria , Patel Sweta , Kuznetsova Valeriya , Anderson Ashlyn E , Harrington Laurie E , Welner Robert S
Published: February 2023

Non-immune roles of Regulatory T cells (Tregs) have been described across various tissues. However, the mechanisms by which T cells regulate the bone marrow in myeloproliferative neoplasms lack resolution. A hallmark of leukemic progression is the increase in pro-inflammatory cytokines, cellular inflammation, and an expansion of CD4 T cells. In this study, we demonstrate that this inflammatory milieu promotes pathogenic skewing of both regulatory and effector T cells. We characterized the altered CD4+ T cell compartment in chronic myeloid leukemia (CML) and we found that Tregs are unstable and dysregulated across leukemic tissues. Specifically, the CML environment effectively co-opts Treg function and stability. This contributes to a phenotype of immune activation as well as an increase of pathogenic inflammatory T cells. Notably, our findings reveal that Treg/Th17 ratios are disrupted in leukemia and that loss of Tregs and activation of Th17 cells influence disease progression. Additionally, we found that Treg-secreted IL-10 directly influences myeloid and neutrophil differentiation of leukemic progenitors has beneficial anti-inflammatory effects. We demonstrate that restoring Treg function and expanding their IL-10 production can be used therapeutically to limit leukemic progression. Our results support the hypothesis that Tregs are compromised during leukemic progression as a consequence of pro-inflammatory cytokines from the myeloid associated disease. These findings highlight how the Treg/Th17 balance regulates disease progression. Furthermore, these studies provide an avenue by which Treg secreted IL-10 can be used for therapeutic intervention in the context of hematopoietic malignancies.
Intestinal interleukin (IL)-22Ra1 signaling mediates the onset of systemic metabolic disorders
Authors: Gaudino Stephen , Huang Huakang , Jean-Pierre Makheni , Lin Xun , Kumar Pawan
Published: February 2023

Interleukin (IL)-22 has been shown to protect against detrimental high fat diet (HFD)-induced phenotypes. However, it is unknown where IL-22Ra1 signaling specifically occurs to regulate HFD-induced metabolic disorders. To examine this, we utilized intestinal epithelium-specific Il22Ra1fl/fl;Villin-cre+ (IL22Ra1ΔIEC), white adipose tissue (WAT)-specific Il22Ra1fl/fl;Adipoq-cre+ (IL22Ra1ΔWAT), and liver-specific Il22Ra1fl/fl;Albumin-cre+ (IL22Ra1ΔLiver) knockout mice as well as their respective littermate cre-(IL22Ra1fl/fl), mice. When placed on long term HFD, IL22Ra1ΔIECand IL22Ra1ΔLiver mice but not IL22Ra1ΔWAT mice displayed impaired systemic glucose metabolism. We specifically observed that impaired glucose metabolism of IL22Ra1ΔIEC mice was microbiota dependent. IL22Ra1ΔIEC mice also possessed altered lipid metabolism since their intestinal tissues expressed increased levels of peroxisomal β-oxidation genes. Furthermore, extra-intestinal tissues from IL22Ra1ΔIEC mice displayed altered metabolism. Liver tissue of IL22Ra1ΔIEC mice displayed decreased expression of G6PC, a key glycolytic enzyme, and WAT displayed increased expression of peroxisomal Acox1 and decreased levels of certain fatty acids. These liver- and WAT-specific changes were dependent on IL-22 since Il22−/− mice displayed opposite trends in gene expression. We decided to examine where intestinal IL-22Ra1 signaling may specifically occur to mediate these effects. Interestingly, we observed a unique role of Paneth cell-specific IL-22Ra1 signaling in mediating systemic glucose metabolism. Overall, our data highlight a specific importance of intestinal IL-22Ra1 signaling in regulating HFD-induced metabolic disorders.
Neurokinin A blocks the ABCA1 channel to promote IL-10-dependent mast cell regulation
Authors: Williams Li’an , Hawse William F , Cattley Richard , Carey Cara D , Zhang Jiying , Falo Louis D , Larregina Adriana T , Sumpter Tina L
Published: February 2023

Cutaneous mast cells (MCs) reside in close proximity to peripheral sensory nerve fibers and are susceptible to regulation by neuropeptides. The tachykinin family member, Neurokinin A (NKA) binds with highest affinity to the Neurokinin 2 Receptor, expressed on MCs. MCs are poised to respond to NKA, but its role in the MC biology is, to our knowledge, under-explored. The objective of this study was to evaluate the effects of NKA on bone marrow (BM) MCs and peritoneal (P)MCs in vitro and in the murine model of passive cutaneous anaphylaxis (PCA) in vivo. Neurokinin A inhibited FceRI-initiated phosphorylation and nuclear localization of STAT5, and release of TNF and IL-13 in an IL-10-dependent manner. NKA affected the MC secretome, abrogating ABCA1-dependent release of the cysteine protease, calpain. Extracellular calpain, in turn, degraded IL-10. In vivo, NKA administration reduced PCA, inhibiting edema, and induction of inflammatory cytokines and in a manner that relied on MC derived-IL-10. Likewise, direct inhibition of the ABCA1 phenocopied the effects of NKA, reducing IgE-initiated MC responses in vitro and in vivo. In conclusion, NKA minimizes IgE-initiated inflammation though an IL-10-dependent mechanism and inhibits ABCA1-dependent release of the IL-10 degrading enzyme, calpain. This study illuminates a novel tier of neuropeptide mediated MC regulation through the ABCA1. Strategies interfering with the ABCA1 may be promising therapeutics for targeting MCs in cutaneous inflammatory diseases.
Glutamine metabolism drives NKT cell homeostasis and function
Authors: Kumar Ajay , Yarosz Emily L , Hong Hanna , Lyssiotis Costas , Chang Cheong-Hee
Published: February 2023

Cellular metabolism is essential in dictating T cell development and function, but its role in natural killer T (NKT) cells has not been well studied. Activated CD4 T cells undergo a metabolic switch from oxidative to glycolytic metabolism to fuel their proliferation and effector function. We have previously shown that NKT cells operate distinctly different metabolic programming from CD4 T cells. We found that, unlike CD4 T cells, NKT cells rely more on glutamine for their homeostasis. In fact, we observed that resting NKT cells have higher levels of glutamine-derived metabolites than conventional CD4 T cells. However, the precise mechanisms by which NKT cells use glutamine for their proliferation and cytokine production remain unknown. Glutamine is metabolized into glutamate and then into alpha-ketoglutarate (α-KG) and glutathione (GSH). Using the pathway specific inhibitors revealed that NKT cells rely on both pathways for cell homeostasis, but cytokine production is associated with α-KG production. These glutamine oxidation processes control optimal mTORC1 activity and mitochondrial functions. Importantly, we observed a novel role of 5′ AMP-activated protein kinase (AMPK) in regulating glutamine metabolism in NKT cells. Taken together, glutamine metabolism is essential for NKT cell homeostasis and cytokine production.
A B cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation
Authors: Wang Jia C , Yim Yang-in , Wu Xufeng , Hammer John A
Published: February 2023

B cells that are activated by antigen-presenting cells form an immune synapse (IS), which supports B cell activation and a robust antibody response. The organization and dynamics of the actin cytoskeleton are essential for centralizing antigen and promoting the B cell receptor signaling that are central to IS formation. When antigen is limiting, B cells require integrin co-stimulation to form the IS. Given the indispensable function of actin in forming the IS, we tested the role of the actin cytoskeleton in integrin-dependent IS formation and B cell activation. Here we show, using live-cell super-resolution imaging of primary B cells, the formation of a dynamic actin arc network, which dominated the synaptic actin and appeared only when B cells bound both antigen and the integrin ligand, ICAM-1. Actin arcs were created by the formin mDia1 and were decorated with the motor protein myosin 2A, which organized actin arcs into a concentric, contractile network. Dynamic imaging of the actin in primary B cells on planar lipid bilayers showed that actin arcs moved centripetally together with antigen microclusters and that these microclusters were swept inward by individual actin arcs. Critically, integrin-dependent IS formation under limiting antigen conditions required myosin activity as blocking myosin with Blebbistatin prevented antigen centralization. Consistently, myosin inhibition also diminished B cell receptor signaling and disrupted the distribution of key signaling proteins at the IS. Together, these results argue that a contractile actomyosin arc network created downstream of integrin co-stimulation plays an important role in the mechanism by which integrin co-stimulation promotes B cell activation and IS formation.
Metabolic Control of T cell responses by HDAC3
Authors: Wilfahrt Drew Nicholas , Philips Rachael , Kizerwetter Monika , Lama Jyoti K , Rajcula Matthew , Kennedy Madeleine , Shapiro Virginia Smith
Published: February 2023

After activation, CD4+ T cells undergo metabolic modifications in order to orchestrate successful immune responses. In particular, naïve T cells shift to a more glycolysis-heavy metabolic program that produces the necessary metabolites for growth, division, and exertion of effector function. Histone Deacetylase 3 (HDAC3) has been shown to epigenetically regulate many T cell fate decisions and is also capable of post-translationally modifying non-histone proteins. Deletion of HDAC3 in mature CD4+ T cells using a distal Lck-Cre HDAC3 cKO mouse revealed a reduction in differentiated T-helper cell populations in vivo. In contrast, in vitro differentiation of HDAC3-deficient CD4+ T cells occurred normally, however, the numbers of T cells present within each culture was greatly diminished. CD3/CD28-stimulated HDAC3-deficient cells exhibited poor proliferation with decreased blastogenesis, decreased CD25 expression, and decreased IL-2 production. Signaling downstream of TCR is disrupted in the HDAC3-deficient T cells. HDAC3-deficient CD4+ T cells have an overabundance of Foxo1 protein, and diminished signaling downstream of mTOR, including reduced phosphorylation of Ribosomal Protein S6, and decreased expression of Egr2. Key transporters involved in cholesterol efflux, ABCA1 and ABCG1, were upregulated in HDAC3-deficient CD4+ T cells. Strikingly, the proliferation and blasting defects of HDAC3-deficient CD4+ T cells are restored with the addition of exogenous cholesterol, but the signaling defects remain. Thus, the failure of HDAC3-deficient CD4+ T cells to proliferate is due to alterations in cholesterol homeostasis.
Protein immunization induces T follicular helper memory cells and nonpolarized T helper (Tnp) memory cells
Authors: Sircy Linda M , Harrison-Chau Malia , Novis Camille , Baessler Andrew , Nguyen Jacklyn , Hale J. Scott
Published: February 2023

Acute viral infection generates lineage-committed T helper 1 (Th1) and T follicular helper (Tfh) memory cells that recall their lineage-specific functions following secondary challenge with virus. However, the lineage commitment of effector and memory T helper cells in vivo following protein vaccination is poorly understood. In this study, we analyzed effector and memory CD4+ T cell differentiation following adjuvanted glycoprotein immunization compared to acute lymphocytic choriomeningitis virus (LCMV) infection. Glycoprotein immunization-induced CXCR5-non-Tfh effector and memory CD4+ T cells that surprisingly had not undergone polarization toward any particular T helper cell lineage but had undergone memory differentiation. However, upon challenge with virus, these T helper lineage-nonpolarized memory CD4+ T cells were able to generate Th1 secondary effector cells, demonstrating their lineage plasticity. In addition, Tfh and memory Tfh cells were generated in response to protein immunization, and these cells differed from infection-induced Tfh cells by their lack of the transcription factor Tbet. Rechallenge experiments demonstrated that viral infection, but not protein immunization, during either the primary or secondary immune response, restricts the recall of Bcl6 expression and the generation of germinal center Tfh cells. Together, these data demonstrate that protein immunization generates a combination of nonpolarized memory cells that are highly plastic and memory Tfh cells that can undergo further Th1-like modulation during a secondary response to viral infection.
Dominant interfering CARD11 variants disrupt JNK signaling in T cells
Authors: Bauman Bradly M. , Chan Alice Y. , Snow Andrew L.
Published: February 2023

Previous studies in mice implicated c-Jun N-terminal kinase 1 and 2 (JNK1/2) as important determinants of helper CD4+ T cell differentiation through their influence on AP-1 and NF-AT-dependent gene transcription. However, mechanisms governing the JNK signaling pathway in human lymphocytes remain nebulous. CARD11 is a critical scaffold protein in T and B cells that connects antigen receptor (AgR) engagement to downstream transcriptional programs responsible for effector cell survival, proliferation and differentiation. Although CARD11 is best known for facilitating AgR-induced NF-κB activation, a handful of studies have shown it is also required for AgR-induced mTORC1 and JNK2 signaling. Using both CARD11-deficient Jurkat T cells and primary human T cells, we found that phosphorylation of both JNK1 and JNK2 in response to T cell receptor (TCR)/CD28 activation is dependent on CARD11. Furthermore, we demonstrate that heterozygous dominant negative (DN) CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease also disrupt TCR-dependent JNK1/2 phosphorylation and c-Jun accumulation with variable potency, closely mirroring the extent of impaired NF-κB activation. Considering that loss of either JNK1 or JNK2 in mice contributes to reduced IFN-γ production and skewed Th2 phenotypes, our findings suggest that defective JNK signaling in CD4+ T cells may contribute to severe atopic disease manifestations noted in most CADINS patients. Ongoing studies will define differences in specific JNK-dependent gene signatures that correlate with specific CARD11 DN variants and clinical phenotypes.
Intravital three-photon microscopy of T-cell migration in the T-cell zones over the entire depth of mouse popliteal lymph node.
Authors: Choe Kibaek , Hontani Yusaku , Wang Tianyu , Hebert Eric , Ouzounov Dimitre , Xu Chris
Published: February 2023

Most T cells in a popliteal lymph node are located in T-cell zones at ~150 to ~600 μm below the cortical surface of the lymph node. For the last 20 years, intravital two-photon microscopy has been a powerful tool for observing the three dimensional T-cell migration in the T-cell zones. However, two-photon imaging can only visualize superficial T-cell zones to ~300 μm depth. In this work, we successfully visualized T-cell migration in T-cell zones over the entire depth of a mouse popliteal lymph node with intravital three-photon microscopy. We measured the change in lymphocyte velocity with increasing laser powers at 600 μm depth to determine the safe laser power range for visualizing T-cell migration. We found that, with less than 80 mW at 1300 nm excitation, the migration of EGFP-expressing lymphocytes were not perturbed by the laser illumination. Using the safe power of 1300 nm excitation, we observed dynamic migration of EGFP-expressing naïve CD4 T cells and DsRed-expressing naïve CD8 T cells simultaneously in T-cell zones at various depths. The naïve T cells were sparse at 0 ~150 μm depth in cortical side (follicular and interfollicular regions), dense in the T-cell zones, and less dense in the medulla, which was identified by labeling with eFluor615-conjugated LYVE-1 antibody in vivo. We found that the density distributions of CD4 and CD8 T cells were different in the T-cell zones. We also measured the migration velocity and motility coefficient for naïve CD4 and CD8 T cells in the T-cell zones over the entire depth of the lymph node. This work shows that three-photon microscopy has the potential to shed light on immune cell dynamics in the deep regions of lymph nodes that are typically inaccessible to conventional two-photon microscopy.
EasySep&[trade] T75 Magnet: A Novel Magnetic Platform for Large-Volume Cell Isolation from Whole Blood and Leukapheresis Packs
Authors: MacDonald G. Neil , Toombs Eric , de Jong Susan , McQueen Karina L , Eaves Allen C , Louis Sharon A. , Kokaji Andy I
Published: February 2023

Isolating cells from large volume-samples such as leukapheresis or whole blood bags is a common procedure that precedes many immunological studies. Working with large volumes can be challenging as the sample often needs to be split and processed in parallel, which can be tedious and may delay downstream studies. The EasySep™ T75 Magnet simplifies cell separation procedures when processing up to 225 mL of leukapheresis sample or 125 mL of whole blood, and is based on column-free immunomagnetic EasySep™ cell isolation technology.

Negative selection protocols have been optimized for isolation of human T cells (purity: 95.3 ± 2.5%, n=5), CD4+ T cells (purity: 97.2 ± 1.4%, n=4), CD8+ T cells (purity: 91.9 ± 2.2%, n=5), and monocytes (purity: 91.5 ± 1.0%, n=3). Positive selection protocols have been optimized for isolation of CD3+ cells (purity: 95.4 ± 2.9%, n=9), CD4+ cells (purity: 88.8 ± 3.2%, n=3), CD8+ cells (purity: 95.1 ± 2.4%, n=6), and CD14+ cells (purity: 96.2 ± 0.7%, n=3). Protocols for depletion of red blood cells (RBCs) (residual RBCs: 2.5 ± 1.7%, n=5) and enrichment of peripheral blood mononuclear cells from whole blood (purity: 99.2 ± 0.5%, n=5) have also been developed.

Notably, these protocols can be completed in 20 – 26 minutes, making the EasySep™ T75 Magnet the fastest and simplest method for isolating highly purified immune cells from large-volume whole blood and leukapheresis samples.
Leveraging high-dimensional flow cytometry to reveal the human immune system at a population-wide scale
Authors: Liechti Thomas Andreas , Beddall Margaret , Van Gassen Sofie , Novák David , Ballard Reid , Iftikhar Yaser , Mangino Massimo , Saeys Yvan , Spector Tim , Roederer Mario
Published: February 2023

Population-wide assessment of the human immune system is needed to better understand immune homeostasis, immune responses against pathogens or the efficacy of vaccines. High-dimensional flow cytometry is the gold standard for in-depth immunophenotyping but the assessment of large cohorts requires multiple experiments over a prolonged period of time and bear multiple sources of error, reducing precision required for sensitive analyses. We performed a large immunophenotyping study using 28-color flow cytometry to investigate the heritability and homeostasis of the immune system and T cell functions in 3000 individuals. Here, we describe our high-throughput sample and data processing pipeline (200 samples/day) in combination with high dimensional flow cytometry to dramatically reduce the number of experiment-days in our study, resulting in low variation and high reproducibility. We demonstrate several carefully optimized strategies to reduce experimental errors and inter-assay variation at every level. In addition, we discuss strategies for pre-processing and analysis of large flow cytometry datasets using manual gating and unsupervised clustering analyses, such as FlowSOM, in view of the fact that most current strategies are highly limited for large datasets.

In conclusion, we established a high-throughput high-dimensional flow cytometry pipeline enabling the analysis of thousands of samples with low technical and experimental variation. Our pipeline in combination with our three 28-color flow cytometry panels is adaptable to other large cohort studies for which precision and accuracy over time are important.
ctDNA technology for early cancer detection in the Pacific setting – the importance of Talanoa
Authors: Sika-Paotonu Dianne , Walton Mischa , Faavae Sepola , Guilford Parry
Published: February 2023

For Pacific Peoples living in Aotearoa/New Zealand, the burden of cancer remains a serious health concern. A promising early cancer detection approach involves the use of circulating tumour DNA (ctDNA) found in the blood circulation. Current ctDNA approaches focus on the analysis of ctDNA derived from established tumours in patients, to help provide information relating to the cancer’s size and progression using specific laboratory isolation and quantification techniques. Adaptation of these current ctDNA technologies used in the cancer monitoring setting, for early diagnosis, would generate a cancer detection approach easily initiated by obtaining ctDNA from DNA fragments found within a blood sample. To understand the potential utility of ctDNA technology for early cancer detection within the Pacific setting in Aotearoa/New Zealand, engagement was sought with Pacific leaders using Talanoa, focused on cancer and early cancer detection. Interim analyses highlighted factors considered directly relevant to Pacific Communities and the cancer setting including the need for health technologies and systems that align with cultural contexts for Pacific Peoples and their families. Support was also expressed for the development of an early cancer detection tool utilising ctDNA as a simple methodological approach, with acknowledgement of the potential for this method to address current barriers to cancer care and services. This work highlights the importance of ensuring cultural context is understood and respected when engaging with Pacific Communities, and also within the cancer and health care setting.
Micro-structure Proteins Characterization in Tissue Sections and Clinical Biopsy Samples by a Novel Spatial Proteomic Method Named AutoSTOMP
Authors: Yin Bocheng , Ewald Sarah , Holmes Jeff , McGowan Emily
Published: February 2023

We aim at structure determining immunological machinery using a novel spatial selective proteomic tool, autoSTOMP, developed in Ewald lab. AutoSTOMP comprises of UV initiated biotin tagging by confocal microscopy, affinity isolation, and peptide identification by the mass spectrometer, rendering a precise characterization of micro-structures of interest (mSOIs) at intracellular organelle scale excluding surrounding area with the decent signal-to-noise ratio. Previously, autoSTOMP was employed to investigate the parasitophorous vacuole membrane, a unique intracellular membrane structure in apicomplexan infected cells. We broaden the application of autoSTOMP to respond to the calls from the research or clinical laboratory for a reliable, repeatable, and re-constructible protocol to explore the mSOIs of an animal specimen or a patient biopsy. We demonstrated, in the cardiac infarcted tissue in ischemic injured rat, the mSOIs stained against the macrophage/monocyte marker, CD68, are enriched in lysosomal proteins, and characteristic proteins involved in the Fcγ receptor-activated macrophage deformation, migration, and phagocytosis. Furthermore, in eosinophilic esophagitis (EoE) biopsy, we identify the lgG4+ cell-cluster structures are enriched in eosinophil-derived granule proteins and lgE receptor FcɛRI signaling associated proteins as well as food allergens to build up the correlation between patient’s diet with EoE progression.
Multiomic characterization of T-cell populations at the single-cell level utilizing sensitive dextramers and BD® AbSeq on the BD Rhapsody™ Single-Cell Analysis system
Authors: Lopez-Salmeron Vadir , Sakofsky Cynthia , Jacobsen Kivin , Nakamoto Margaret , Brix Liselotte , Mortimer Stefanie
Published: February 2023

Adoptively transferred antigen-specific T cells have shown great efficacy in treatment of some virus-associated diseases and malignancies. A major driver of the development of adoptive T-cell therapy has been our ability to successfully characterize the functional status and antigen specificity of T cells. However, this has been limited by inefficient detection of antigen-specific T cells possibly due to their low frequency and low binding affinities to known MHC-peptide complexes. Here, we aim to combine two powerful technologies, advanced dCODE™ Dextramer® from Immudex and single-cell multiomics analysis using the BD Rhapsody™ Single-Cell Analysis system, to detect and characterize disease-specific CD8+ T cells within thousands of PBMCs. Currently, we are able to identify over 350 mRNAs alongside a panel of over 20 BD® AbSeq cell surface protein markers which can be associated with T cell activation states. These data can be used to define T-cell phenotypes alongside antigen specificity of enriched CD8+ dextramer+ cells from a PBMC population. This study outlines our ability for high-resolution T-cell profiling that has broader implications and utility in immuno-oncology, infectious diseases and autoimmunity.

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