bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of TRAF3 regulation of TLR signaling in B lymphocytes
Authors: Ybarra Tiffany K , Bishop Gail A
Published: August 2023

Toll-like receptors (TLRs) are pattern recognition receptors that bridge the innate and adaptive immune system and are critical for host defense. TLR signaling to B cells is important in normal immunity, but also implicated in autoimmune disease and B cell lymphomas (BCL). Most studies focus on the role of TLRs in myeloid cell populations. However, B cells express most TLRs and are highly responsive to TLR ligands, yet TLR-mediated B cell pathways are understudied. Previous studies from our lab show that TRAF3 inhibits B cell TLR functions, including cytokine production, Ig isotype switching, and antibody production in response to ligands for TLRs 3, 4, 7/8 and 9. A key knowledge gap is identifying the molecular mechanisms by which TRAF3 mediates this regulation. Our working hypothesis is that B cell-TRAF3 regulates the recruitment and/or post-translational modification of specific TLR complex signaling proteins. Our recent data show that B cell-TRAF3 forms a heterocomplex with TRAF6, associating with the tyrosine kinase Syk. In the absence of TRAF3, TRAF6 shows a greater association with several TLR signaling proteins, including MyD88, IRAK1 and Syk. This suggests that TRAF3 may inhibit TRAF6 access to the TLR signaling complex, and thus early TLR signaling. In addition, our work introduces a role for Syk in TLR signaling in B cells. In the absence of TRAF3, activation of Syk is enhanced in response to ligands for TLRs 4 and 7. Inhibition of Syk activation also revealed downstream effects on TLR-mediated NFκB activation. Further studies aim at providing new insights into molecular mechanisms contributing to the regulation of B cell-TRAF3 in TLR signaling.

VA Merit Review I01 BX001702 NIH P50 CA97274 RO1 AI5107312000000113259002001 NIH T32 Training Grant AI07485 RO1 AI1762656
SLP-76 interaction with PLC-γ1 fine-tunes TCR signal strength for appropriate thymocyte selection, T cell homeostasis, and Ag-specific T cell response
Authors: Stassenko Elizabeth , Wada Junya , Lee Mariah , Li Wenmei , Balagopalan Lakshmi , Evans Christine , Hagenau Herbert , Awasthi Parirokh , Chari Raj , Yamane Hidehiro , Samelson Lawrence E
Published: August 2023

TCR signal strength regulates T cell fate at various stages of thymocyte development and the differentiation of peripheral T cells into distinct T cell subsets. We previously showed that TCR engagement forms a heterotetrametric complex composed of LAT, Gads, SLP-76, PLC-γ1 and that this complex is essential to activate PLC-γ1 to further transduce the TCR signal. The interaction of the SLP-76 proline-rich region (PRR) with the PLC-γ1 SH3 domain is relatively weak, and these two domains are highly conserved amongst many mammalian species. Therefore, we hypothesized that the SLP-76 PRR sequence with low affinity for PLC-γ1 might be evolutionarily favored to prevent excess TCR signals. To test this hypothesis, we developed mice carrying a conditional T cell-specific knock-in mutation in the SLP-76 PRR with a 2.5-fold increased affinity for PLC-γ1 by CRISPR/Cas9-mediated gene editing and Cre/LoxP system (cKI). Notably, cKI increased the expression of active caspase-3 in signaled SP4 and SP8 thymocytes and decreased the numbers of mature medullary thymocytes, suggestive of enhanced negative selection. Under steady-state conditions, cKI enhanced the generation of CD8+ central memory T cells, implying an increased sensitivity to self-Ag/MHC I in the periphery. Moreover, cKI rendered CD8+ T cells more sensitive to OVA altered peptide ligands with a weaker affinity for OT-I TCR. Finally, cKI enhanced priming of OT-I CD8+ T cells that had not undergone massive cell divisions for IFNγ-producing effectors in vivo, implying an increased TCR signal strength in weakly activated CD8+ T cells by cognate Ag. Our data indicate that the SLP-76 PRR/PLC-γ1 SH3 interaction plays a critical role in fine-tuning TCR signal strength to avoid excess signals via TCR.

NCI/NIH Intramural Support
Tunable B cell response through allosteric modulation of Bruton’s tyrosine kinase
Authors: Reboldi Andrea , Berthelette Alyssa , Kolli Nilima , Amatya Neha , Andreotti Amy , Berg Leslie J , Der Sandy , Kang Joonsoo
Published: August 2023

The B cell receptor (BCR) is essential for both the initial burst of B cell proliferation in response to antigens, and for the differentiation of plasma cells and memory B cells during germinal center reactions. However, the BCR’s precise role in germinal center selection has been difficult to assess: mechanistic investigations into its modulation during the decision of B cell fate suffer from experimental limitations due to the fundamental role of the BCR in B cell biology.

Bruton’s tyrosine kinase (BTK), a member of the TEC family, is a key component of BCR signaling. Using a computational screening method based on small molecule docking outside of the BTK active site, we have identified putative allosteric modulators of BTK. In vitro primary B cell Nur77 upregulation, calcium flux, proliferation and plasma cell differentiation confirmed that these molecular probes alter B cell activation strength, kinetics, and fate only when the BCR is engaged. In vivo immunization using an antigen model reveled that BTK agonism strongly favors B cell differentiation into switched plasma cells. BTK agonism, either at the time of the immunization or at the peak of germinal center formation, enhanced humoral immunity, proving for the first time that transient, augmented BCR signaling during germinal center reaction has a direct impact on B cell differentiation ability.

In addition to being powerful tools for basic B cell biology investigation, allosteric agonists of BTK could be developed into antigen-licensed B cell adjuvants for vaccines in at risk populations, as well as into cancer immunotherapy drugs which hyperactivate B cells in tumor microenvironments during checkpoint inhibitor blockade therapies.
A cellular platform for the evaluation of the effect of O-glycan modification on B cell receptor signaling
Authors: Angata Takashi , Hsiao Po-Long , Tsai Ho-Yang , Huang Wan-Chen , Lin Kuo-I
Published: August 2023

The importance of N-glycosylation, a ubiquitous post-translational modification (PTM) of cell-surface and secreted proteins in vertebrates, in the regulation of B cell receptor (BCR) signaling has been well documented. On the other hand, the importance of O-glycosylation, another type of ubiquitous PTM, in B cell function is getting recognized only recently. A simplified system to study the effects of O-glycan modifications in BCR signaling would facilitate the efforts to understand the mechanism of B cell regulation by O-glycans. To this end, we reconstituted BCR components and CD45 with orthogonal self-labeling protein tags in the mouse myeloma J558L cell line, which is deficient in the components of BCR and several co-receptors. The enzymes that modify O-glycans were further introduced, and the cells were subjected to imaging-based quantitative analysis of the distance between BCR and CD45, as well as the evaluation of BCR signaling by Western blotting. Gene expression profiles among the cells were also compared by RNA sequencing. Our study revealed that core 2 branching of O-glycans has minimal effect on the distance between BCR and CD45, whereas the addition of an α2–8-linked sialic acid by ST8Sia-VI reduces the distance between them, facilitating the downstream BCR signaling. Our system may be adapted for the functional evaluation of other PTMs in BCR signaling.

Supported by a grant from the Ministry of Science and Technology (MOST)/National Science and Technology Council (NSTC) of Taiwan (MOST 109-2311-B-001-009-MY3)
QRICH1 is a negative regulator of T cell activation via the CARD11 signalosome
Authors: Carter Nicole M , Pomerantz Joel L
Published: August 2023

Controlled lymphocyte activation is crucial for defending against pathogens while preventing diseases such as autoimmunity and lymphoma. In T lymphocytes, the scaffold protein and signaling hub CARD11 mediates NF-κB, JNK, and mTOR pathway activation downstream of T cell receptor (TCR) engagement. We recently identified QRICH1 as a previously unrecognized CARD11 interactor recruited to the CARD11 complex downstream of TCR engagement and discovered that QRICH1 protein levels are upregulated in wild-type activated mouse primary CD8+ T cells. Overexpression of QRICH1 in 293T and Jurkat T cells dose-dependently inhibited NF-κB activation by wild-type CARD11 as well as by constitutively active and oncogenic CARD11 mutants. QRICH1-deficient Jurkat T cells had increased NF-κB activation and IL-2 secretion downstream of TCR stimulation. To study the in vivo role of QRICH1 in immunity, we generated a novel QRICH1 conditional KO mouse model. Compared to control mice, QRICH1 T cell KO mice had decreased splenic CD4+ and CD8+ T cells, despite no differences in thymic CD4+, CD8+, or CD4+CD8+ T cell populations. The CD8+ T cell population in QRICH1 T cell KO mice was skewed towards an effector memory phenotype. QRICH1-deficient primary CD8+ T cells had increased proliferation and production of pro-inflammatory cytokines IFN-γ and TNF-α in response to ex vivo stimulation with anti-CD3/CD28, and QRICH1-deficient OT-1 CD8+ T cells also had increased cytokine production in response to antigen-specific stimulation with Ova peptide. Our findings reveal that QRICH1 negatively regulates T cell activation by modulating CARD11’s signaling output.

This work was supported by NIH grants R01AI148143, F31CA254167, and T32GM007445.
Understanding the involvement of sphingosine kinase-1 and two-pore channels in calcium signaling in T cells
Authors: Soares Giselle Amanda Borges E , George Emma H , Hamzah Ali H , Walseth Timothy F , Slama James T , Wall Katherine Ann
Published: August 2023

Calcium (Ca2+) signaling is an essential requirement for cell activation, proliferation, differentiation, homeostasis, etc. In the tumor microenvironment, however, an upregulation of the Ca2+ signaling pathway has been associated with cancer cell proliferation and malignancy, which further contributes to immune response suppression. Although nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ signaling secondary messenger known, the NAADP-Ca2+ signaling pathway remains elusive and controversial. Studies have so far revealed the interaction of NAADP with accessory proteins such as Jupiter microtubule-associated homolog 2 (JPT2) and Lsm12 along with the poorly characterized ‘two-pore channels’ (TPC1 and TPC2) located in the acidic endo-lysosomal compartments of cells.

In 2018, a screening campaign revealed the ability of PF-543 to inhibit NAADP-mediated Ca2+ signals in sea urchin egg homogenates and in human U2OS cells. As PF-543 is marketed as a potent sphingosine kinase 1 (SphK-1) inhibitor and is believed to possess TPC inhibitory function, the mechanism behind PF-543’s ability to inhibit NAADP-mediated Ca2+ signals (through inhibition of SphK-1 or the TPCs, or both) needed investigation. Therefore, we tested structural analogs of PF-543 and evaluated their ability to inhibit both SpK-1 and Ca2+ signals in DO.11.10 cells, a T cell hybridoma that possesses the T cell receptor (TCR). Thus, in addition to identifying novel targets for the development of Ca2+ signaling modulators and anticancer therapeutics, our results also reveal the contributions of SphK-1 and the TPCs in the NAADP-mediated Ca2+ signaling pathway in T cells.

NIH R15GM131329
Single Cell Sequencing Suggests Mucosal Origins of T Cells in HLA-B27-associated Inflammation
Authors: Paley Michael A , Hassman Lynn , Yang Xinbo , Linskey Nicole , Esaulova Ekaterina , Paley Grace L , Ruzycki Philip , Laurent Jennifer , Feigl-Lenzen Lacey , Artyomov Max , Garcia K Chris , Yokoyama Wayne M
Published: August 2023

HLA-B27 was one of the first Major Histocompatibility Complex alleles associated with an autoimmune disease, i.e., Ankylosing Spondylitis (AS) and acute anterior uveitis (B27AU), which cause joint and eye inflammation, respectively. As HLA-B27 presents antigens to CD8 T cells, an antigen-specific CD8 T cell response may be central to disease pathogenesis. Consistent with this hypothesis, our recent work identified clonal expansion of public T cell receptors (TCRs) shared across patients with AS and B27AU and enriched in the joint or eye over the blood. These TCRs recognized both bacterial and self-peptides, suggesting a role for molecular mimicry in multiple organs afflicted by HLA-B27-associated inflammation.

The main bacterial peptide recognized by these public TCRs is derived from yeiH, a gene present in multiple enteric microbiota. It remains unclear, however, whether these T cells have had a prior antigenic encounter within the gastrointestinal tract. We therefore performed single-cell RNA sequencing (scRNAseq) and T cell receptor sequencing (scTCRseq) on immune cells from the eye and blood during B27AU (n=5) and non-B27AU (n=14). In this analysis, we found that the expanded TCRs in B27AU were associated with CD161 expression and enriched for a transcriptional program from intestinal CD8 T cells. Moreover, we were able to identify an additional expanded TCR sequence that also recognized YEIH. Finally, expanded T cell clones contained higher expression of TNF and IFNG, suggestive of a pro-inflammatory transcriptional program.

Collectively, our data suggest that, in HLA-B27-associated inflammation, the early antigen exposure and differentiation of putative pathogenic CD8 T cells may have occurred in enteric organs.

This study received support from NIH 5R01AI103867 (KCG), U19AI057229 (KCG), the Howard Hughes Medical Institute (KCG), the Rheumatology Research Foundation (MAP), Arthritis National Research Foundation (MAP), the Rheumatic Diseases Research Resource-based Center at Washington University (NIH P30-AR073752, MAP, WMY), the Bursky Center for Human Immunology and Immunotherapy Programs (WMY), and the Barnes-Jewish Hospital Foundation (WMY).
T cell subsets in Systemic Sclerosis patients show features of exhaustion and reduced anti-fibrotic activities
Authors: Dooms Hans , Volfson-Sedletsky Victoria , Belkina Anna C , Bujor Andreea
Published: August 2023

Systemic Sclerosis (SSc) or Scleroderma is a chronic autoimmune disease where immune cells contribute to the initiation and/or progression of microvascular damage and fibrosis, leading to devastating tissue malfunction often resulting in high morbidity and mortality. We recently discovered that PBMCs from SSc patients show increased expression of various co-inhibitory receptors, consistent with the presence of a state of chronic immune activation and dysfunction. Here, we sought to identify and functionally characterize dysregulated T cell subsets in SSc, and determine their role in pathogenic fibroblast activation. We used multi-parameter spectral flow cytometry and in vitro T cell/fibroblast co-culture assays to understand the role of T cell dysfunction and exhaustion in fibrosis, using clinical samples from a well-defined cohort of SSc patients. Our immunophenotyping analysis revealed that T cell populations in SSc patients show features of T cell exhaustion, such as enhanced expression of the co-inhibitory receptors PD-1 and TIGIT, and reduced production of the anti-fibrotic cytokine IFN-γ. PD-1+TIGIT+ T cells were found in peripheral blood as well as lung and skin of SSc patients. In addition, we found a significant reduction of immune cell subsets expressing the transcription factor T-bet in the blood of SSc patients, suggesting a reduced presence of immune cells that counteract a pro-fibrotic Type 2 immune response in SSc. In support of this, isolated T cell subsets from SSc patients showed reduced capacity to limit collagen production in T cell/fibroblast co-cultures. Together, our data suggest that T cells with anti-fibrotic activities are driven into a state of exhaustion in SSc, enabling the progression of fibrosis.

Supported by grants from NIH (R21 AR071580) and the National Scleroderma Foundation (Walter & Marie Coyle Research Grant)
Distinct PD-1hi CD4+ T helper cell populations promote CD11c+ T-bet+ B cell expansion in liver and spleen during obesity
Authors: Enslow Benjamin , Vanz Carlo , Dudley Elizabeth , Hägglöf Thomas , Leadbetter Elizabeth A
Published: August 2023

Obesity is associated with increased deposition of fat in ectopic tissues, such as within the liver and visceral adipose depots, including the omentum and mesentery. Increased visceral fat is an accompanying feature of the metabolic syndrome and is associated with increased end-organ damage in NAFLD. Emerging evidence implicates intrahepatic B cells in the progression of NAFLD, and increased number of proinflammatory B cells have been found in the steatotic livers of HFD-fed mice. Our preliminary studies demonstrate accumulation of inflammatory T-bet+ CD11c+ B cells in the subcutaneous adipose tissue of obese humans and perigonadal adipose tissue of mice on HFD. We now extend those findings into other sites of ectopic fat deposition to determine the obesity-related changes in T-bet+ B cell frequencies in the liver, omenta, and MAT. Flow cytometry identified increased frequencies of T-bet+ B cells in the spleen and livers, but not visceral adipose, of HFD mice. Furthermore, HFD-associated expansion of T-bet+ B cells in the spleen and liver correlated with a rise of TFH and TPH frequencies, respectively. Future studies will determine the mechanism of tissue-specific T and B cell interactions associated with obesity-induced chronic inflammation.

B.T.E. is supported by the NIH award (K12GM111726). T.H. is supported by a postdoctoral fellowship from the Swedish Research Council. This study was supported by NIH R01 AI32798-01A1 and the Voelcker Fund Young Investigator Awards (E.A.L).
Role of complement activation in cellular activation by systemic lupus erythematosus (SLE) immune complexes (ICs)
Authors: Khandelwal Sanjay , Myoung Sooho S , Lambris John D , Rogers Jennifer L , Pisetsky David S , Arepally Gowthami M
Published: August 2023

Complement (C’) activation is a prominent feature of SLE ICs, but the mechanism by which C’ mediates cellular activation by ICs is not well understood. To investigate this question, we formed SLE ICs in situ in healthy donor (HD) whole blood (WB) by adding SLE plasma (n=1–3;10% v/v) or HD plasma (as a control) along with apoptotic bodies (generated from RAW 264.7 cells) or RNP-Sm antigen (Arotec Diagnostics, New Zealand) with and without C’ inhibitors (CI). For cellular binding studies, WB was incubated for 45 minutes at 37°C and deposition of C3c and human IgG (ICs) on blood cells was examined by flow cytometry. In these studies, we noted that C’ inhibition significantly reduced deposition of C3c and ICs on B cells {% reduction: C3c (97%) and IgG (97%)}, monocytes {% reduction: C3c (40%) and IgG (49%)} and neutrophils {% reduction: C3c (69%) and IgG (70%)}. To investigate cellular activation, WB was incubated for 22 hours and cellular activation was measured by HLA-DR expression on monocytes by flow cytometry and cytokine release (IL-8, IL-6 and TNF) by ELISA. Incubation of WB with SLE ICs, but not ICs made with HD plasma (control ICs), resulted in increased HLA-DR expression on monocytes (MFI control ICs: 1609 V SLE ICs: 11096), which was susceptible to C’ inhibition (MFI SLE ICs with CI: 4975). Similarly, inflammatory cytokine release by SLE ICs was also significantly inhibited with C’ inhibition {% inhibition: IL-8 (75 %), IL-6 (99%) and TNF (99 %)}. Taken together, our results show significant C’-dependent effects of SLE ICs on cellular binding and activation. Additional studies are underway to investigate the prevalence of C’ activating ICs in SLE patient and correlate their presence with clinical disease.

Wendell F. Rosse Complement Research Award, Duke University Medical Center (SK) and Duke Scholars Award, Duke University Medical Center (GMA)
CX3CR1 modulates SLE-associated cardiovascular disease in MRL/lpr mice
Authors: Lu Ran , Geng Shuo , Cabana-Puig1 Xavier
Published: August 2023

Systemic lupus erythematosus (SLE) is a chronic and complex autoimmune disease that can cause multi-organ damages. Patients with SLE often have higher risk for heart disease, with poorly defined underlying mechanisms. We generated a Cx3cr1-deficient MRL/lpr lupus-prone mouse which exhibits a distinct phenotype of exacerbated glomerulonephritis. Upon feeding with a high-fat diet, Cx3cr1−/− MRL/lpr mice developed significantly more atherosclerotic plaques that were promoted by Cx3cr1 deficiency-mediated activation of Ly6C++ monocytes. The activated monocytes expressed ICOS-L that interacted with ICOS-expressing follicular T helper cells, which in turn facilitated a germinal center reaction to produce more autoantibodies. Through a positive feedback mechanism, the increased circulatory autoantibodies further promoted the activation of Ly6C-expressing monocytes and their display of ICOS-L. Together, we uncovered a novel, Cx3cr1 deficiency-mediated pathogenic mechanism contributing to SLE-associated atherosclerosis in MRL/lpr mice. The results of these studies will lead to the identification of new therapeutic targets for SLE patients at risk of developing cardiovascular disease.
Silencing of aryl hydrocarbon receptor repressor modulates Th17-cell immunity in human autoimmune hepatitis
Authors: Longhi Maria Serena , Gao Li , Zhang Wei , Zhang Lina , Nastasio Silvia , Graham Jonathon J , Bonder Alan , Patwardhan Vilas , Robson Simon C
Published: August 2023

Th17-cells play a key role in the pathogenesis of autoimmune hepatitis (AIH), a severe hepatopathy characterized by hypergammaglobulinemia, seropositivity for autoantibodies and interface hepatitis on histology. Aberrant Th17-cell response in AIH is linked to alterations of aryl-hydrocarbon-receptor (AHR) signaling. These result in Th17-cell inability to respond to AHR activation. Since AHR can be regulated upon binding to aryl-hydrocarbon-receptor-repressor (AHRR), we investigated whether defective Th17-cell response to AHR derives from aberrant AHRR regulation in AIH. Th17-cells obtained from AIH patients and healthy controls (HC) were exposed to the AHR endogenous ligands unconjugated bilirubin and L-kynurenine (L-kyn), with or without AHRR siRNA.

Levels of AHRR mRNA were similar in AIH and HC Th17-cells. Silencing of AHRR by siRNA boosted Th17-cell response to L-kyn, as reflected by increased CYP1A1, this effect being noted in AIH but not in HC-derived Th17-cells. AHRR blockade did not change the levels of AHR-regulated genes like CD39, IL10, IL17, and IL22, but decreased the levels of IL1b, in the absence and presence of L-kyn in AIH Th17-cells. Silencing of AHRR limited the differentiation of naïve CD4-cells into Th17-cells and modulated Th17-cell transcriptome by downregulating SOCS2, CISH and IL2RA.

Overall, these data indicate that blockade of AHRR restores Th17-cell response to AHR activation and modulates Th17-cell differentiation by decreasing IL1b along with genes involved in cell activation and linked to JAK/STAT signaling. Silencing of AHRR might represent a novel therapeutic strategy to modulate effector Th17-cell immunity and restore immune homeostasis in AIH.

Supported by grants from NIH (R01DK124408 and R01DK108894)
Single-cell sequencing of PBMC characterizes a distinct inflammatory monocyte subset in Lyme disease patients with Post-Treatment Lyme Disease Syndrome (PTLD)
Authors: Chen Xin , Zhou Xin , Ji Xuhuai , Miranda Molly , Rebman Alison , Kathuria Karan , Robinson William H , Aucott John , Davis Mark M
Published: August 2023

Lyme disease (LD) is the most common tick-borne disease in the United States. Most LD patients can return to healthy (RTH) status after antibiotic treatment. Still, 20% of LD patients develop Post-Treatment Lyme Disease Syndrome (PTLD), including fatigue and musculoskeletal pain that persist for a long time. Previous RNA-seq studies showed that most individuals with PTLD had an inflammatory gene expression compared to RTH, but the specific immune components that contribute to PTLD are not well understood. Here, we performed the single-cell sequencing analysis of PBMCs from 6 RTH and 4 PTLD patients collected during multiple clinic visits after antibiotic treatment over two years. We identified a CD14+ monocyte cluster significantly enriched in 4/4 PTLD patients and 2/6 RTH patients from the beginning to 6 months post-treatment. This monocyte cluster displayed distinct inflammatory gene signatures such as CCL3, CCL4, IL1B, CXCL2, CXCL8, and others. RTH patients with such inflammatory monocytes showed low expression of CXCR2 compared to PTLD, suggesting that higher CXCR2 signaling in inflammatory monocytes may contribute to the development of PTLD. In addition, pathway analysis of upregulated genes in PTLD inflammatory monocytes indicated that this subset was induced by molecules of bacterial origin and involved in the regulation of lymphocyte activation. In conclusion, our single-cell study identified a distinct inflammatory monocyte that was enriched and may contribute to PTLD. Tracking this inflammatory monocyte subset could lead to a more accurate prediction of PTLD and identify molecular biomarkers and pathways for therapeutic intervention.
Boosting NAD blunts TH17 inflammation via arginine biosynthesis and redox regulatory control in healthy control and psoriasis human subjects
Authors: Han Kim K , Singh Komudi , Meadows Allison , Sharma Rahul , Hassanzadeh Shahin , Wu Jing , Goss-Holmes Haley , Huffstutler Rebecca , Teague Heather , Mehta Nehal , Griffin Julian , Tian Rong , Traba Javier , Sack Michael N
Published: August 2023

Although NAD+ levels modulate metabolism, how this regulates immunometabolism/inflammation remains unclear. Employing Nicotinamide Riboside (NR) to boost NAD, we explored TH17-linked inflammatory in Psoriasis. Primary Psoriasis CD4+ T cells exposed to NR reduced IL-17 secretion and RNA-seq/pathway analysis implicated NR in modulating sequestosome 1 (SQSTM1/p62)-coupled oxidative stress. In parallel, NR reduced CD4+ T cell reactive oxygen species (ROS) levels and activated the NRF2 transcription factor. Depletion of NRF2 or SQSTM1 diminished NR effects on ROS and TH17 activation. Metabolomic analysis implicated NR in arginine biosynthesis and L-arginine replicated NR CD4+ T cell effects. Genetic disruption of the argininosuccinate lyase synthesis enzyme ameliorated NR antiinflammatory effects, supporting that NR, via arginine biosynthesis orchestrates NRF2 activation, CD4+ T cell antioxidant defenses and blunted TH17 responsiveness. A pilot in-vivo human study similarly showed that NR increased arginine, antioxidant enzyme gene expression and blunted TH17 regulation, implicating NAD metabolism in CD4+ T cell-linked inflammation.

NHLBI Division of Intramural Research (MNS – ZIA-HL005102), NIH Bench to Bedside award (MNS and RT, HL-129510-04S1) and the NIH Office of Dietary Supplementation (JT) and the UK MRC (JLG – MR/P011705/2; UKDRI-5002; MAP UK).
Enteric helminth infection confers protection against lethal respiratory influenza virus infection
Authors: Tai Chi , Xie Jia-Xun , Lin Jr-Shiuan
Published: August 2023

Chronic helminth infection has been considered a serious public health issue. Due to the widespread of helminthiasis, people co-infected with helminth and other pathogens, such as virus, may be common. Studies suggest that helminth infection exert immunomodulatory effects that may alter host susceptibility to subsequent unrelated pulmonary pathogens, such as Mycobacterium tuberculosis. However, the outcome of helminth-respiratory viral co-infection remains controversial.

Here, we established an enteric helminth Heligomosomoides polygyrus bakeri (Hpb)-influenza virus co-infection mouse model, then characterized the immune responses modulated by Hpb infection in the lungs of the helminth chronically infected mice. Our results showed that enteric helminth-infected mice exhibited increased survival and reduced weight loss after lethal influenza virus infection compared with helminth-inexperienced mice. Further analysis revealed that enteric helminth infection altered immune landscape in the lungs by increasing effector CD4+ and CD8+ T cells infiltrated into the airway, as well as upregulating type I interferons and pro-inflammatory cytokines in the lung tissues before influenza virus infection. After influenza virus infection, viral titers were significantly decreased, whereas the viral-specific antibody responses and cellular responses were increased in the lungs of helminth-infected mice. Moreover, histological examination of the lungs revealed that cell infiltration in lung parenchyma and airway lumen was reduced in helminth-infected mice. Taken together, these results suggested that enteric helminth infection confers “bystander” protection against pulmonary influenza virus infection.

Supported by grants from MOST (109-2320-B-002-074-MY3), NHRI (EX110/111-11033SI) and NTU (111L7829), Taiwan
Lung pathogenic, P2RX7-dependent CD4+ T cell immune responses to influenza are exacerbated by co-exposure with lung allergens
Authors: de Gois Macedo Bruna , Santiago-Carvalho Igor , Jacobsen Elizabeth A , da Silva Henrique Borges
Published: August 2023

Extracellular ATP (eATP) is a danger signal released by inflamed tissues, and can be sensed by immune cells via P2RX7. P2RX7 promotes lung pathology induced by influenza infection, by inducing exacerbated numbers of lung CD4+ T cells. In response to influenza, lung CD4+ T cells are comprised of a type 1 (Th1)-like population, and a subset resembling follicular helper (Tfh) CD4+ T cells; this subset is called tissue-resident helper (Trh). Trh cells amplify lung effector immune responses. Trh cells express high P2RX7 levels, and current research in our laboratory shows that P2RX7 is crucial for Trh cell establishment and subsequent lung damage. We have also found that the intensity of lung damage caused by influenza is positively correlated with P2RX7+ lung Trh cells. It is not clear, however, how concomitant exposure to influenza and type 2 immunity-inducing lung allergens (a common clinical occurrence in the United States) affect P2RX7+ lung Trh cells and their resultant tissue damage. We investigated this, using House Dust Mite (HDM) allergen exposure together with Influenza (PR8) infection. HDM/PR8 co-exposure induced worsening of lung inflammation, compared to single exposure to PR8. This inflammation had type 1 characteristics (i.e. parenchymal immune cell accumulation and absence of mucus). The increased lung damage generated by HDM/PR8 exacerbated the generation of influenza-specific P2RX7+ lung Trh cells. The accumulation of lung Trh cells (and subsequent tissue damage) caused by HDM/PR8 was abolished in T cell-specific P2RX7-knockout mice. Our results suggest that local inflammation, eATP release and sensing, and lung Trh cells together dictate the intensity of lung damage, regardless of the nature of the lung disease agent.

R01AI170649
The Upper Respiratory Tract Exhibits Reduced Interferon Response that Contributes to Limited CD8+ T Cell Recruitment and Delayed Clearance of Human Metapneumovirus
Authors: Sojati Jorna , Parks Olivia Bartlett , Williams John V
Published: August 2023

Human metapneumovirus (HMPV) is a leading cause of acute upper and lower respiratory infections. Although nearly everyone is infected during early childhood, re-infections occur often, highlighting difficulty in building long-term immunity. We employed a mouse model of HMPV infection to elucidate differences in upper and lower tract immune responses, and address why upper airway infection does not establish durable protective immunity. We found HMPV burden was higher in nasal airways and exhibited delayed clearance compared to lung. Additionally, there was low nasal expression of antiviral interferons (IFN) and inflammatory cytokines. Key innate immune cells, including macrophages, monocytes, and dendritic cells, were present in the nose at baseline but were not upregulated by infection. We previously showed HMPV-specific lung CD8+T cells (TCD8) are impaired, exhibiting sustained high PD-1 levels and reduced functionality. Despite higher HMPV viral load, there were few HMPV-specific nasal TCD8 and they did not exhibit impairment. To test whether low nasal IFN led to this phenotype, we administered type I IFN in an upper airway-restricted fashion early post-infection. This led to lower HMPV nose titers, increased cell number, higher PD-1 expression, and reduced functionality in HMPV-specific nasal TCD8. Despite the subdued nasal immune response, intranasal vaccination with a small-volume HMPV dose elicited modest reduction of lung and nose titers on challenge but no improvement in clinical disease. Our findings reveal a quiescent nasal immune landscape despite high HMPV burden that contributed to slow clearance and was modulated, in part, by low IFN expression.

Supported by NIH R01 AI085062-06 (JVW), T32 AI138954-4 (JS), and UPMC Children’s Hospital of Pittsburgh Research Advisory Council Graduate Fellowship (JS).
The Immunomodulatory Effects of E. coli Heat-Labile Enterotoxins on the Respiratory Immune
Authors: Ryan Madison , Stroud Margaret , Buchanan Zachary , Rojas Andrea , Knapp Mary-Peyton , Okinaga Adam , Connell Terry , Fiester Steven , Arce Sergio , Grier Jennifer Theresa
Published: August 2023

Heat-Labile Enterotoxins (HLTs) are bacterial secreted proteins with unique immunomodulatory properties. Type II HLTs are produced by E. coli and have been found to possess mucosal and systemic adjuvant properties, but they have never been investigated in respiratory-specific responses. The goal of this study was to determine the impact of three Type II HLTs (LT-IIa, LT-IIb, LT-IIc) on the respiratory-specific immune response. Cell cultures of the human lung epithelial cell line, A549, were stimulated with LT-IIa, LT-IIb, or LT-IIc, in the presence or absence of lipopolysaccharide (LPS), a known immunostimulant. Cell viability was measured by flow cytometry, with cell morphology observations via light microscopy. Immune responses were evaluated via quantitative polymerase chain reaction (qPCR) to quantify RNA expression or enzyme-linked immunosorbent assay (ELISA) for cytokine secretion at 6 and 18 hours post treatment. HLT treatment resulted in no significant changes to cell viability, although cytoplasmic inclusions were consistently visible in LT-IIc-treated cells. IL-6, a marker of inflammation, showed reduced RNA expression in HLT-treated cells at 6 hrs with a return to baseline by 18 hrs. IL-6 cytokine secretion in HLT-treated cells was also decreased, with the greatest reduction at 18 hrs of treatment. Thus, immune responses triggered by HLTs are potentially less inflammatory than those induced by LPS, although a more in-depth characterization of the expression of other cytokines is needed. These results demonstrate that Type II HLTs drive a modified, and potentially less inflammatory, respiratory immune response compared to LPS, which could have significant impacts in the treatment of inflammatory lung conditions.

Acknowledgement: This work was funded in part by a Transformative Seed Grant from the Health Sciences Center at Prisma Health and a University of South Carolina School of Medicine Greenville Summer Research Scholars Fellowship awarded to MR.
Naïve T cell activation in the draining lymph nodes is accelerated by airway resident CD4 T cell activation
Authors: Finn Caroline M , Prokop Emily , McKinstry Kai
Published: August 2023

Tissue-resident memory CD4 T cells (TRM) protect against pathogens through the early shaping of inflammation at sites of infection. The extent to which their activation can impact subsequent systemic T cell responses is not clear. To ask if lung CD4 TRM recognizing antigen can impact systemic T cell responses against the same antigen presented in secondary lymphoid organs, we generated OVA-specific lung TRM primed by infection with an influenza virus expressing OVA peptide. We isolated the OVA-specific TRM 30 days after infection and transferred one million to naive mice intranasally resulting in an airway-resident TRM cohort similar in magnitude to TRM generated in virus-primed mice. We then recalled the transferred TRM by intranasal administration of FITC-labelled OVA protein to focus on TRM recall in the absence of virus-induced inflammation. One day after OVA challenge more FITC+ APC were present in the draining lymph nodes (dLN) of mice with TRM, suggesting antigen presentation to systemic T cells may be accelerated in the presence of airway TRM. We confirmed this by tracking naive OVA-specific CD4 T cells (OT-II) and CD8 T cell (OT-I) cohorts transferred i.v. to mice harboring OVA-specific TRM or not: more responding naive cells, expressing higher levels of activation markers, were detected 3 days after OVA challenge in mice with TRM. By 4 days, more responding cells were present in the lungs of TRM recipients, indicating that TRM and systemic T cell responses form a circuit via enhanced antigen presentation in the lymph nodes. Our results suggest that enhanced kinetics of systemic T cell activation induced by TRM activation in the airways may represent a novel mechanism of their protection against influenza and other respiratory viruses.
EFFECT OF ARTIFICIAL MITOCHONDRIAL TRANSFER FROM LINE CELLULAR HSC-3 TO LYMPHOCYTE TCD4+, AS A MECHANISM INDUCTOR OF EXHAUSTED PHENOTYPE
Authors: Cáceres Bárbara Evelyn Antilef , Cisterna Solange Estefania , Quiroga Romina Andrea , Novoa Sergio Andrés Sanhueza , Cabrera-Garcia Camilo Daniel , Grez Camila Paz Muñoz , Zuñiga Felipe , Ferrada Luciano , Gonzáles Wilfredo , Luz Patricia , Caicedo Andrés , Labarca Gonzalo , Perez Andy , Nova-Lamperti Estefania
Published: August 2023

Background

Oral squamous cell carcinoma (OSCC) is the most frequent type of oral cancer. The tumor microenvironment of OSCC induces an alteration of the T lymphocyte, promoting an exhausted phenotype. The main organelle of metabolism is the mitochondrion and in recent years it has been indicated that several cells have the capacity to transfer mitochondria, including cancer cells. However, it has not been evaluated whether mitochondria transfer from cancer cells to T lymphocytes induces an exhausted phenotype in T lymphocyte. The aim of this work was to analyze the exhausted phenotype in TCD4+ lymphocytes after artificial transfer of mitochondria (MitoCeption) obtained from the oral cancer cell line HSC-3.

Methods

Isolated MitoTracker-labeled mitochondria from oral cancer tumor cells were Mitocepted into CD4+ T lymphocytes. Then, surface molecule expression, proliferation and cytokine secretion mediated by tumor mitochondrial transfer were analyzed by flow cytometry.

Results

The results showed that TCD4+ lymphocytes that acquired mitochondria had increased expression of 2 inhibitory proteins (TIGIT and CTLA4) and 3 proteins associated with exhausted phenotype (PD-1, PLD-1 and LAG3), compared to the control group. In addition, the mitocepted lymphocytes exhibited a significant decrease in proliferation compared to the control. For cytokine analysis, a significant decrease was observed in the mitocepted group in the secretion of IFN-gamma, TNF-alpha, IL-10 and IL-4, but not IL-17, compared to the control.

Conclusions

Therefore, we concluded that the acquisition of isolated mitochondria from HSC-3 cancer cells by CD4+ T lymphocyte induces an exhausted phenotype in the TCD4+ lymphocyte.

Regular Fondecyt Project 1211480
Role of the E3 ubiquitin ligase Peli1 in regulating antitumor T cell responses
Authors: Ko Chun-Jung , Sun Shao-Cong
Published: August 2023

Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor signal (TCR) and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased effector function and the number of tumor-infiltrating CD4 and CD8 T cells. Moreover, Peli1 deficiency inhibited the expression of PD-1 and TIM-3 and reduced the exhaustion of tumor-infiltrating CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of mTOR signaling, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through the regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting the stabilization of the TSC complex. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity.
Pre-operative exercise therapy suppresses colorectal liver metastases by triggering anti-tumor trained immunity of Kupffer cells
Authors: Huang Hai , Zhang Hongji , Tsung Allan
Published: August 2023

Hepatic recurrence after surgical resection of colorectal liver metastasis is the major cause of treatment failure. Pre-operative exercise therapy (PEx) has been shown to improve outcomes from major abdominal surgeries. We demonstrate that PEx significantly attenuates liver ischemia/reperfusion (I/R) injury by inducing an anti-inflammatory phenotype in Kupffer cells (KCs). However, the mechanisms by which PEx prevents surgery-induced hepatic recurrence remain unclear.

Results

Four weeks of treadmill running significantly reduces I/R-induced hepatic metastatic tumor growth in mice compared to sedentary controls. Depletion of KCs substantially decreases I/R-induced tumor progression, indicating a pro-tumor role of KCs. Our scRNA-seq data reveal that PEx causes dramatic transcriptomic changes in KCs. A clear induction of trained immunity with noticeably higher levels of anti-tumor cytokines IFN-γ and TNF-α is observed in PEx-KCs. With scATAC-seq, we find that PEx triggers the chromatin accessibilities profile changes in KCs, which is associated with the metabolic reprogramming of itaconate/IRG1 pathway. Furthermore, PEx significantly changes the transcriptomic profile of polymorphonuclear myeloid-derived suppressive cells (PMN-MDSCs) in the tumor microenvironment (TME) and decreases them from forming neutrophil extracellular traps (NETs), which promote tumor progression. We identify the cell-cell interaction between KCs and PMN-MDSCs in the TME is significantly enriched after PEx, suggesting that PEx may facilitate KCs to modulate PMN-MDSCs. In summary, PEx ameliorates surgical stress-induced pro-tumorigenic inflammatory responses and sustains an anti-tumor immune microenvironment in the liver.

Supported by grants from NIH (R01GM137203 for Hai Huang, R01CA214865 for Allan Tsung)
Immunometabolic modulation by targeting UCP2 and IL-17A boost anti-tumor immunity against pancreatic cancer
Authors: Liu Chuan-Teng , Yen Hung-Rong
Published: August 2023

Metabolic reprograming of cytotoxic effectors by tumor microenvironment (TME) is one of the key mechanisms by which TME impairs the success of cancer immunotherapy. Therapeutic strategies that mitigate the immunosuppressive TME and selectively enhance bioenergetic fitness of cytotoxic effectors may enhance benefits of immunotherapy, particularly in TME-abundant pancreatic cancer. IL-17 is a key TME cytokine engaged in a feed-forward loop promoting the immunosuppressive milieu that facilitates cancer progression. Mitochondrial uncoupling protein 2 (UCP2) acts as an inhibitor of T cell metabolic fitness and directly promotes tumorigenesis. Therefore, we hypothesized that targeting IL-17 and UCP2 could act additionally to enhance antitumor immune response and directly inhibit pancreatic cancer growth. UCP2 deficiency regulated bioenergetic metabolism and mTOR signaling in cytotoxic T cells to enhance IFN-γ production and anti-tumor immune responses. UCP2 inhibition increased anti-tumor immunity in PBMCs and tumors of patients with PDAC. The dual inhibition of IL-17 and UCP2 boosted antitumor immunity and suppressed tumorigenesis in tumor-bearing mice. Combination of UCP2 and IL-17 blockade may offer additional complimentary and cooperative mechanisms of antitumor immunity against pancreatic cancer.

1. Ministry of Science and Technology (MOST 108-2320-B-039-024-MY3) 2. China Medical University Hospital (DMR-110-186) 3. Chinese Medicine Research Center, China Medical University from the Featured Areas Research 4. Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (CMRC-CHM-2)
Gut microbiota modulation by high glucose diet invigorates anti-tumor immune response to glioblastoma multiforme
Authors: Kim Jaeho , Kim Hyunjin , Kang Byeong Hoon , Kim Yumin , La Jeoungwoo , Ku Keun Bon , Lee Heung Kyu
Published: August 2023

Most studies about the relationship between high sugar diet and tumor progression state that the pro-tumoral effects of high sugar diet are induced by metabolic diseases such as obesity and diabetes caused by long-term, excessive sugar diet. However, there are still many unknowns about the effect of high sugar diet on tumors when it lasts for a relatively short period that does not cause metabolic diseases. In this background, we find that survival rate of glioblastoma (GBM) is increased through supplement of high glucose diet (HGD) for several weeks. We find that gut microbiota modulation by HGD plays a crucial role in enhancement of anti-tumor immune response. Through single cell RNA sequencing analysis, modulation of gut microbiota by HGD results in an increase in T cell-mediated anti-tumor immune response in GBM. In addition, we find the novel cytotoxic CD4+ T cell population in GBM is increased due to synergistic action with anti PD-1 and the change occurs only through the synergistic action of anti PD-1 treatment and HGD. As a result, we identify that HGD can enhance anti-tumor immune response in GBM through changes in gut microbiota and suggest that the role of glucose diet on GBM needs to be elucidated from a different perspective.
γ-aminobutyric acid drives glioblastoma in females in an immune-dependent manner
Authors: Bayik Defne , Chin Diana , Zhou Yadi , Cheng Feixiong , Lathia Justin
Published: August 2023

Interaction between tumor cells and neurons drives the progression of glioblastoma (GBM), which is characterized by enhanced immunosuppressive myeloid cell accumulation. Our previous results revealed that myeloid-derived suppressor cell subsets (MDSCs) play a critical role in tumorigenesis in a sex-specific manner, with monocytic MDSCs (mMDSCs) accumulating in male tumors and granulocytic MDSCs (gMDSCs) correlating with a worse outcome in females. However, host factors governing sex differences in MDSC subset activity remained unknown. Here, we report that gMDSCs express g-aminobutyric acid (GABA) receptor and female gMDSCs respond to GABA by upregulating the antiviral innate immune pathway. Female mice treated with the GABA analogue pregabalin succumbed to disease earlier, consistent with our previous observation that gMDSCs have a central role in GBM progression in females. In contrast, pregabalin treatment had no effect in male mice with tumors or immunocompromised female mice. Assessment of the antiviral response signature revealed that GABA upregulates nitric oxide synthase 2 (NOS2) expression in gMDSCs. Correspondingly, the tumor-accelerating effect of pregabalin was diminished in NOS2 KO female mice. Collectively, these results identify a neuron-immune-cancer communication axis and indicate that GABA alters anti-tumor immune response in a sex-specific manner. These results support the future assessment of GABA pathway inhibitors as potential immunotherapy agents.

VeloSano Philanthrophic Funds