bluuebunny/crossref_metadata_2025 huggingface dataset:
The function of DcR3.Fc and its derivatives in macrophage differentiation and skeletal muscle repair
Authors: Weng Chao-Yuan , Sung Pei-Shan , Hsieh Shie-Liang
Published: November 2024

Macrophages play a crucial role in regulating inflammation and tissue repair, influencing whether the tissue will regenerate or undergo fibrosis and dysfunction. However, the specific factors that can induce tissue repair/resolving macrophages remain unclear. Decoy receptor 3 (DcR3) is a soluble receptor without a transmembrane domain that can neutralize the effects of the tumor necrosis factor superfamily members FasL, LIGHT, and TL1A. In our previous study, we discovered that recombinant DcR3.Fc not only possesses neutralizing capabilities but also promotes M2-like macrophage differentiation. To delve deeper into its mechanisms, we created GRC2.Fc, which lacks the ligand-binding domain, thereby excluding the involvement of DcR3 ligands. Here, we observed that DcR3.Fc and GRC2.Fc induces differentiation of human CD14+ monocyte-derived macrophages and mouse bone marrow-derived macrophages toward a tissue repair/resolving phenotype in vitro. Furthermore, in a cardiotoxin-induced muscle injury model, intramuscular injection of GRC2.Fc protein promotes skeletal muscle repair and induce the expression of M2 macrophage markers in vivo. Our findings indicate that GRC2.Fc can induce macrophages toward a tissue repair/resolving phenotype and promote skeletal muscle repair.
Molecular Pixelation - dissecting the spatial organization of 80 surface proteins in single immune cells
Authors: van Ooijen Hanna , Karlsson Max , van Hoef Vincent , Schiller AnnaLotta , Thiagarajan Divya , Leijonancker Louise , Schweitzer Maud , Sathe Prajakta , Branting Annika , Martinez Barrio Alvaro , Karlsson Filip , Fredriksson Simon
Published: November 2024

Immune cell function is dependent on the activity of membrane proteins. Most widely-used cell analysis methods, flow cytometry and scRNAseq, directly or indirectly assess membrane protein abundance, yet provide no information about their spatial distribution. The gold standard technology for spatial protein analysis, fluorescence microscopy, has revealed that the spatial organization of membrane proteins is in fact of importance for their function, as exemplified by the immune synapse and uropods in migrating cells. Limited multiplexing and throughput scalability of microscopy, reduces its potential for forefront spatial proteomics discoveries, highlighting the need for new innovative methods.

Here, we present Molecular Pixelation (MPX), an advanced DNA barcode-based method for high-multiplex spatial analysis of proteins in single cells. Combining an NGS-based readout with our dedicated software, Pixelator, MPX enables the reconstruction of 3D cell-surface maps of 80 proteins on over 1000 individual cells, simultaneously. We applied MPX to compare the surface proteome of single resting and in-vitro activated T cells, highlighting differences in protein abundance, as well as revealing dramatic spatial protein re-organization, both in terms of supramolecular clustering and protein-protein co-localization. These results demonstrate that MPX has the potential to pave the way for expanding our understanding of cell surface protein architecture and its role in T cell activation.
GenSci120, a humanized PD-1 agonistic monoclonal antibody, enhances the binding of both PD-L1 and PD-L2 to PD-1, and mitigates the symptoms of GVHD in a mouse model
Authors: Jiang Wenbo , Chu Xuebin , Song Lei , Li Xue , Xue Weili , Li Lingyun , Zhao Ranran , Gu Fei , Xu John
Published: November 2024

Background: Autoimmune diseases impose tremendous health and economic burden to the individuals and families they affect. The fundamental mechanism of autoimmune diseases is a break in immune tolerance and homeostasis. PD-1 plays an important role in maintaining immune tolerance by antagonizing CD28/TCR signaling in autoreactive T cells. Stimulating the PD-1 co-inhibitory pathway by an agonistic antibody would suppress pathogenic immune responses and restore immune homeostasis. Methods: A PD-1 agonistic monoclonal antibody, GenSci120, was generated by Beacon single plasma cell cloning and murine antibody humanization technologies. In vitro T cell inhibition, ADCC and ligand blocking activities of GenSci120 were characterized. In vivo efficacy in a mouse GVHD model and PK in the human FcRn transgenic mice were evaluated. Results: GenSci120 exhibited potent T cell inhibition activity in a luciferase reporter assay and a primary T cell proliferation assay. GenSci120 also showed ADCC activity in an LDH release assay. Remarkably, GenSci120 enhanced the binding of both PD-L1 and PD-L2 to PD-1 in a flow cytometry assay. In a mouse model, GenSci120 significantly alleviated the symptoms of GVHD. Furthermore, GenSci120 demonstrated longer half-life than competitors’ antibodies in the human FcRn transgenic mice. Conclusion: These data suggest the potential of GenSci120 as a novel therapy to treat autoimmune disorders and support the evaluation of this molecule in clinical studies.
Phenotypic and Therapeutic Evaluation of Humanized C3 Transgenic Mice as a Model for C3 glomerulopathy-Like Autoimmune Disease
Authors: Liang Juan , Zi Kaiyuan , Xu Qiuping , Xu Shuxin
Published: November 2024

C3 glomerulopathy (C3G) encompasses a range of kidney disorders with autoimmune and genetic origins, where dysregulated alternative pathway (AP) complement activation stands as the underlying cause. This study develops a transgenic mouse strain that overexpresses human C3, thereby spontaneously generating a rapidly progressing and severe murine model of C3G.

The C3G phenotype observed in humanized C3 transgenic mice may arise from a dysregulated interaction between human C3 protein and various mouse complement proteins, resulting in uncontrolled C3 activation via the AP. Male humanized C3 transgenic mice exhibit increased morbidity early in life. Proteinuria initiates at 4 weeks of age, accompanied by an elevation in the albumin/creatinine ratio. Significantly increased serum levels of ALT, AST, ALP, LDH, and CK in humanized C3 transgenic male mice compared to wild-type mice indicate liver and heart injuries. Moreover, serum T-CHOL and HDL levels are significantly reduced, suggesting anemia and malnutrition. Pathohistological analysis of the kidney reveals various renal lesions. Oral administration of Lnp023, a small-molecule inhibitor of complement factor B, effectively increases body weight and serum hC3 levels, improves anemia, and preserves liver, heart, and renal function.

In conclusion, this novel model contributes significantly to unraveling the mechanisms underlying C3G and exploring potential therapeutic interventions.
The combined use of statin and rapamycin treatments synergistically inhibit the proliferation of B cell Lymphoma
Authors: Zhu Zhenhan
Published: November 2024

Sterol Regulatory Element-Binding Protein (SREBP) signaling plays a crucial role in maintaining sterol homeostasis during B cell activation and the proliferation of germinal center B cells. It is unclear whether this pathway can be targeted to effectively treat B cell lymphoma. We discovered that inhibiting SREBP signaling or its downstream target 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) using Fatostatin or Simvastatin effectively restrains the proliferation of B cell lymphoma cells. However, B cell lymphoma cells activate the mTORC1-pS6 pathway in response to statin treatment, suggesting a possible mechanism to counteract statin-induced cell cycle arrest. Combining statin treatment with the mTOR inhibitor rapamycin demonstrates a synergistic effect in inhibiting B cell lymphoma proliferation, impeding cell cycle progression, and regulating sterol production. These findings emphasize the potential of a combined therapy approach targeting both SREBP and mTORC1 as a treatment strategy for B cell lymphoma.
The tumor immune environment in advanced breast cancer following treatment with pembrolizumab and nab-paclitaxel
Authors: Tyagi Apoorvi , Voloshyna Iryna , Klar Natalie , Darvishian Farbod , Hao Yuan , Zhurova Anastasia , Cynthia Loomis , Marks Douglas K , Adams Sylvia , Krogsgaard Michelle
Published: November 2024

Metastatic breast cancer poses significant clinical challenge, necessitating improved treatment options. Immune checkpoint blockade approval for PD-L1+ triple-negative breast cancer highlights potential for durable response. However, PD-L1 diagnostics can be discordant and responses in PD-L1 low/negative patients emphasize the need for deeper understanding of tumor microenvironment (TME). In a single center phase II trial, specimens were obtained from 70 breast cancer patients at baseline, treatment cycle 1 featuring monotherapy using nab-paclitaxel (ER+/HER2- BC, cTNBC) or pembrolizumab (iTNBC), followed by combination therapy in cycle 2. We used PhenoImagerHT and NanoString for spatial phenotyping and gene expression analysis of ER+ cohort. Preliminary multiplex imaging (qMIF) showed correlation in cell phenotype, activation state, and spatial location. NanoString revealed transcriptional differences between responders and non-responders at baseline. Moreover, a positive relationship (r=0.8) was found between CTLs and macrophage count, validating qMIF targets. Our research extends to study tumor immune infiltrates and gene expression in cTNBC (n= 30) and iTNBC (n= 20) cohorts. The final results will determine intrinsic subtype signatures of each cohort and study crosstalk between different cell types in the TME. In conclusion, our approach aims to enhance the understanding of treatment response and TME's role in metastatic breast cancer for improved therapeutic strategies.
The EGFR inhibitor exerts a suppressive influence on the response of CD8+ T cells in Non-Small Cell Lung Cancer
Authors: Ko Chun-Jung , Chi Hao-Chen
Published: November 2024

Lung cancer is the leading cause of death worldwide, predominantly characterized by non-small cell lung cancer (NSCLC). The foremost treatment for NSCLC patients with EGFR mutations involves EGFR-tyrosine kinase inhibitors (EGFR-TKIs), demonstrating notable enhancements in patient survival and the preservation of quality of life. Immune checkpoint blockade (ICB) that reinvigorates T cell response is a promising immunotherapeutic approach to treat NSCLC patients with an efficacy of approximately 20%. Thus, comprehending the intricate interplay between EGFR-TKIs and immune regulation is pivotal, laying the groundwork for strategic combination therapies. Despite its critical importance, the relationship between EGFR-TKIs and immune regulation remains elusive, necessitating further investigation into the immunomodulatory role of EGFR-TKIs. This study unveils that EGFR-TKIs contribute to the reduced proliferation and effector cytokine production of CD8+ T cells. Treatment with EGFR-TKIs suppresses the response of tumor-infiltrating T cells in two syngeneic tumor mouse models. Furthermore, employing EGFR-TKI-specific antibodies and mass spectrum analysis, we identified the target profile of EGFR-TKIs. Remarkably, NSCLC patients undergoing EGFR-TKI therapy exhibited a diminished frequency of IFNγ-producing CD8+ T cells. These findings significantly inform the future development of therapeutic strategies and the optimization of drug administration timing for NSCLC patients.
The functional role of KLRG1+ Tregs in Non-small Cell Lung Cancer.
Authors: Oluwatola Oluwaseyi , Bag Arup , Oseni Saheed , Pilon-Thomas Shari , Adeegbe Dennis
Published: November 2024

Killer cell lectin-like receptor G1 (KLRG1) is a co-inhibitory receptor and upon engagement with its ligand, E-, N-, or R-cadherins, Akt phosphorylation is inhibited leading to proliferative dysfunction in T and NK cells. This study aims to investigate how E-cadherin engagement affects KLRG1 function.

KLRG1 fl/fl and cKO mice were orthotopically implanted with KP tumor cell line. T cells were isolated and stimulated for intracellular staining. KLRG1+ and KLRG1- Tregs sorted from lung suspensions of KPM tumors were stimulated with α-CD3+α-CD28 in the presence or absence of soluble E-cadherin for 24hours. CD4+CD25- effector T cells were labelled with cell-trace violet (CTV) and then co- cultured with graded numbers of each population of Tregs in the presence/absence of soluble E-cadherin. Finally, KLRG1+ and KLRG1- Tregs were sorted from lung tumors of KP mice for single-cell RNA sequencing.

We show that GZB is upregulated in KLRG1+ Tregs upon engagement with E-cadherin as confirmed by RNA sequencing, and this GZB expression by KLRG1+ Tregs is associated with enhanced death of effector T cells. Also,Treg-specific deletion of KLRG1 in the cKO mice does not impact Treg development or peripheral homeostasis; instead it permits enhanced anti-tumor responses in lung adenocarcinoma-bearing mice.

NSCLC-associated KLRG1+ Tregs are armed with a functional program that drives their enhanced immunosuppressive activity and attenuates effector function compared to KLRG1- Tregs.
A microbiota derived metabolite and pediatric celiac disease pathogenesis
Authors: Kelley Kristina , Girdhar Khyati , Dogru Yusuf , Yang Yi , Tolstikov Vladimir , Kiebish Michael , Palm Noah , Ludvigsson Johnny , Soto Genesis , Zhang Michelle , Altindis Emrah
Published: November 2024

Celiac Disease (CD) is an autoimmune condition triggered by gluten ingestion and defined by small intestine inflammation leading to villous atrophy in genetically predisposed individuals. 20-40% of the general population possess the predisposing HLA-DQ2 or HLA-DQ8 alleles, however only 1-2% of the population develops CD, indicating that there are additional environmental factors at play in the pathogenesis of the disease.

In a prospective cohort study using fecal and plasma samples from children at ages 2.5 and 5 years, before the onset of celiac disease (CD progressors), we found that CD progressors have an altered gut microbiome, plasma metabolome and cytokine profile years before diagnosis. IgA sequencing revealed an increased IgA response and unique microbial targets of IgA. One metabolite increased in CD progressors is taurodeoxycholic acid (TDCA), a secondary bile acid produced by gut microbes. Treating C57BL/B6 mice with TDCA caused villous atrophy as well as upregulated CD4+T-cells in intraepithelial lymphocytes and natural killer cells in the lamina propria (LP). TDCA increased the expression of Qa-1 and NKG2D on T-cells.

The DQ8-Dd-villin-IL-15tg mouse model of CD possesses the predisposing HLA-DQ8 molecule and overexpresses IL-15 in the LP. Treating these mice with TDCA caused the villi/crypt ratio to decrease in the treatment group of pooled villi. Our results indicate that TDCA and TDCA-producing bacteria could potentially contribute to the pathogenesis of CD.
Infection of the human lymphoid organ on-a-chip with Borrelia burgdorferi reveals novel mechanisms of immunosuppression and autoimmunity in Lyme disease
Authors: Goyal Girija , Ku Min Wen , Zhai Yunhao , Menon Kavya , Laforest Demi , Gould Russell , Gulati Aakanksha , Feigenson Marina , Feitor Jessica , Morton Shad , Shanmugam Nandhinee , Singh Rohini , Patil Aditya , Prabhala Pranav , Gharpure Supriya , Horvath Viktor , Isaacs Abdul
Published: November 2024

In mice and one human case study, Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD), disrupts germinal center architecture and function. Bb proteins also mimic self-antigens. Thus, the antibody response may be of poor quality and target self antigens. To understand this pathology and its relevance to human infection-induced autoimmune diseases, we infected human lymphoid organ chips (Goyal et al., Adv Sci., 2022) created from healthy volunteers with Bb. We discovered unexpected and severe suppression of the adaptive immune system by Bb. T cell and B cell activation were absent in all of the donors (n>10) activated by Bb. Further, T cell proliferation was suppressed by Bb. Yet, some donors displayed hypergammaglobulinemia (hyperIg), seen in many antibody-mediated autoimmune diseases.

We collected the exosomes secreted from the lymphoid organ chips and performed mass spectrometry to identify proteins associated with Bb infection and hyperIg. A heat-killed, fixed suspension of Staph. aureus Cowan I (SAC), commonly used to benchmark patient immune responses, was used as a control. We identified complement and coagulation factors, chaperone proteins, and immunological synapse proteins as key players in discriminating between successful activation, immunosuppression, and hyperIg (Bb). Our studies establish an innovative way to study human infectious diseases and their role in autoimmunity and reveal novel therapeutic targets to treat post-treatment Lyme disease.
COMPOUND 1 is a first-in-class small molecule cGAMP competitive oral STING antagonist that reduces lung inflammation and fibrosis in chronic bleomycin and silica mouse models
Authors: Banerjee Monali , Middya Sandip , Shrivastava Ritesh , Basu Sourav , Ghosh Rajib , Middya Anindita , Mane Nagaswamy , Sharma Priti , Rawat Nidhi , Soram Thanilsana , Mansuri Rubeena , Yadav Dharmendra , Gautam Anuj , Shinde Vaibhav , Padaliya Manisha , Bano Shahnaj , Roy Sneha
Published: November 2024

Background: STING activation by cGAMP leads to canonical Type I IFN responses and non-canonical signaling that increase inflammation, pyroptosis, autophagy, senescence and fibrosis in conditions such as Idiopathic Pulmonary Fibrosis. COMPOUND 1 is a novel cGAMP competitive synthetic small molecule STING antagonist discovered at Curadev. Materials and Methods: Competitive binding was established using radioligand binding assays. Anti-fibrotic activity was established in Bleomycin and Silica induced lung fibrosis models in mice. PK and safety studies were evaluated in rodents. Results: COMPOUND 1 inhibits the binding of cGAMP to STING and causes reductions in the level of Type I interferons and pro-inflammatory cytokines from immune cells as well as in mice treated with a STING agonist. Once a day oral administration of COMPOUND 1 that commenced one week after intratracheal injury with bleomycin or silica in C57/Bl6 mice ameliorated lung inflammation and fibrosis with concomitant decreases in collagen load and inflammatory cytokines. Orally administration of the compound was well tolerated at high doses in a 14-day non-GLP rat safety study. Conclusions: COMPOUND 1 is an orally administered novel first-in-class cGAMP competitive STING antagonist with promising single agent anti-inflammatory and anti-fibrotic activity in chronic lung injury models making it is well suited for IND directed studies.
Highly Motile Neutrophil Cytoplast ameliorate Bacterial Pneumonia
Authors: Tsukasaki Yoshikazu , Prasad Nithish , Vellingiri Vigneshwaran , Mehta Dolly , Malik Asrar
Published: November 2024

Polymorphonuclear neutrophils (PMN) function as innate immune cell through rapid mobilization at site of infection and prompt phagocytosis and bactericidal activity. We addressed in vivo in mouse lungs the function of cytoplasts (PMNcyt), the PMN generated following release of extracellular traps (NETs). We found ~50% of PMN lost their nucleus post-NETosis following pneumonia induced by Pseudomonas aeruginosa (PA), and become PMNcyt. Interestingly, , PMNcyt in lung microvessels showed >2 greater velocity and > 3 greater deformability as compared to PMN. PMNcyt were also 9x more efficient in killing airway PA as compared to PMN. Using the protein arginine deiminase 4 (PAD4) inhibitor (Cl-amidine) to prevent NET formation in mice, we prevented the generation of PMNcyt demonstrating that PMNcyt were derived post-vital NETosis. We also showed PMNcyt retained the ability to phagocytose PA. Moreover, the inhibition of PMNcyt generation by PAD4 blocker induced severe lung injury in the pneumonia model, indicating an important anti-bacterial protective role of PMNcyt. Adoptive transfer of PMNcyt in PA infected mice also significantly reduced bacterial load and prevented lung tissue injury in mice. The results suggest a crucial host-defense function of PMNcyt generated post vital NETosis and their usefulness as cell-based therapy in inflammatory diseases such as pneumonia.
Identify new Foxp3 regulators in human primary regulatory T cells using a whole-genome CRISPR screening
Authors: he huan , chen mingkuan , chanda sumit
Published: November 2024

Regulatory T (Treg) cells are crucial in maintaining immune homeostasis. However, they can also promote tumor progress by suppressing anti-tumor immunity. Conversely, Treg instability, marked by the loss of the key transcription factor Foxp3, can facilitate effective tumor immunity. Therefore, gaining a deeper understanding of the pathways that regulate Foxp3 could offer promising therapeutic opportunities for cancer treatment. In this study, we performed a genome-wide CRISPR screening on human in vitro induced Treg cells to identify Foxp3 regulators. Our preliminary results have identified 191 potential positive and 75 potential negative regulators of Foxp3 in human Treg cells. Gene ontology analysis revealed several important functional clusters, including chromatin organization, histone modification, and transcription regulator complex. In particular, we identified many genes encoding components of the INO80 complex and the Sin3-type complex. Next, we aim to conduct further validation and analysis of the INO-80 and Sin3 complexes to gain insight into their role in regulating Foxp3 expression and the functioning of Treg cells. Given that Treg cells can promote tumor growth by inhibiting anti-tumor immunity and Foxp3 is the master regulator for Treg cells, obtaining a comprehensive understanding of the regulation of Foxp3 can reveal novel therapeutic targets to modify the number and function of Treg cells, leading to potential cancer treatment strategies.
Gamma-delta T cells suppress microbial metabolites that activate striatal neurons and induce repetitive/compulsive behavior in mice
Authors: Rezende Rafael , Cox Laura , LeServe Danielle , Mayrink Julia , Oliveira Marilia , Fonseca Roberta , Lanser Toby , Rosa Ana , Schwerdtfeger Luke , Moreira Thais , Weiner Howard
Published: November 2024

Intestinal gamma-delta (gd) T cells play an important role in shaping the gut microbiota, which is critical not only for maintaining intestinal homeostasis but also for controlling brain function and behavior. Here, we found that mice deficient for gd T cells (gd-/-) developed an abnormal pattern of repetitive/compulsive (R/C) behavior, which was dependent on the gut microbiota. Colonization of WT mice with gd-/- microbiota induced R/C behavior whereas colonization of gd-/- mice with WT microbiota abolished the R/C behavior. Moreover, gd-/- mice had elevated levels of the microbial metabolite 3-phenylpropanoic acid in their cecum, which is a precursor to hippurate (HIP), a metabolite we found to be elevated in the CSF. HIP reaches the striatum and activates dopamine type 1 (D1R)-expressing neurons, leading to R/C behavior. Altogether, these data suggest that intestinal gd T cells shape the gut microbiota and their metabolites and prevent dysfunctions of the striatum associated with behavior modulation.
Fc-Optimized Agonistic Anti-Human CD40 mAb Demonstrates Strong Antitumor Activity in hCD40/hFcγRIIB Dual Humanized Mice
Authors: Hong Juan , Hong Jane , Han Boang , Li Tao , Feng Dongxiao , Sun Ruilin , Qing Shuang , He Daniel
Published: November 2024

As a promising antitumor immunotherapy, the agonistic anti-CD40 antibody is limited by its clinical response and toxicity. The selective binding of anti-CD40 antibodies to FcγRIIb has been reported to improve the anti-tumor efficacy. Therefore, we developed the CD40 single humanized and CD40/FcγRIIB dual humanized mouse model, and characterized the expression and pathway activation of chimeric h/m CD40 and FcγRIIB by FACS. Moreover, to evaluate the efficacy and toxicity of anti-hCD40 in vivo, the hCD40 and hCD40/hFcγRIIB mice were engrafted with MC38 syngeneic tumors and treated with anti-human CD40 antibody (MIL97) with mouse or human Fc domain. The binding epitope of MIL97 has a strong potential to rely on cross-linking CD40 signaling activation, and the functional modification of Fc enhances the binding to FcγRllB, resulting in stronger cross-linking and agonistic effects on CD40 signaling. Notably, in comparison with MIL97-mFc, the MIL97 with human Fc domain failed to inhibit the tumor growth in hCD40 mice, but led to ~50% tumor growth inhibition in MC38 tumors in hCD40/hFcγRIIB dual knockin mice. Meanwhile, we observed the toxicity indicated by the body weight change and liver function damage of the MIL97-hFc in hCD40/hFcγRIIB mice, mirror clinical toxicities. Collectively, the hCD40/hFcγRIIB mice provide a powerful preclinical model to predict the activity and safety of anti-hCD40 antibodies in vivo, especially of human-specific Fc-engineered antibodies.
Harnessing a potent combination to address a pandemic virus: SARS-CoV-2-specific antiviral and anti-inflammatory activity of BromAc
Authors: Coelho-dos-Reis Jordana , Marques Ferreira Geovane , Reis Erik , Clarindo Felipe , Lopes-Ribeiro Ágata , Gomes-de-Pontes Letícia , Carvalho Luciana , Martins-Filho Olindo , Guimaraes da Fonseca Flavio , Teixeira Mauro , Sabino Adriano , Eapen Mathew , Morris David , Valle Sarah
Published: November 2024

Herein, the impact of BromAc on the SARS-CoV-2-specific inflammatory response was evaluated. A reduction of the cytokine storm, composed of chemokines, growth factors, proinflammatory and regulatory cytokines produced after short-term in vitro culture with the inactivated virus (iSARS-CoV-2). The immunophenotypic analysis showed that BromAc was able to downmodulate CD16+ neutrophils and CD14+ monocytes observed after stimulation with iSARS-CoV-2. Conversely, BromAc treatment increased steady-state HLA-DR expression in CD14+ monocytes and preserved this activation marker in this subset upon iSARS-CoV-2 stimuli, indicating improved monocyte activation upon BromAc treatment. Additionally, BromAc downmodulated the iSARS-CoV-2-induced production of TNF-α by the CD19+ B-cells. System biology approaches, utilizing comprehensive correlation matrices and networks show distinct patterns of connectivity in groups treated with BromAc, suggesting loss of connections promoted by the compound and by iSARS-CoV-2 stimuli. In addition, BromAc seems to disengage proinflammatory responses and their interactions with other soluble factors and axis orchestrated by SARS-CoV-2. These results give new insights on the mechanisms for the robust anti-inflammatory effect of BromAc in the steady state as well SARS-CoV-2-specific immune leukocyte responses, indicating its potential as a therapeutic strategy for COVID-19.
Escalating Dose Immunization Regimen in Non-Human Primates to Improve Immunogen-Specific Responses
Authors: Carson Audrey , Cheng Cheng , Biju Andrea , Changela Anita , Duan Hongying , Foulds Kathryn , Irvine Darrell , Kwong Peter , Koup Richard , Pierson Theodore
Published: November 2024

HIV-1 remains a major health threat, highlighting the need for a vaccine that induces broadly neutralizing antibodies. Epitope-based vaccine designs that focus humoral responses to vulnerable sites on the HIV-1 Env trimer protein are promising approaches, including those targeting the HIV-1 fusion peptide (FP). Escalating dose (ED) immunizations are a strategy to increase immunogen uptake into germinal centers and boost affinity maturation of the antiviral antibody response. In this study, we tested the impact of an ED immunization scheme on enhancing the FP-directed humoral response. Five groups of non-human primates were immunized: four received an ED immunization of either FP or a cocktail of FP+trimer, and one control group was dosed with a bolus FP+trimer cocktail. Two ED groups were adjuvanted with SMNP. The remaining groups used 3M052/alum. Fourteen weeks post-immunization, all ED groups had higher anti-FP IgG titers compared to the control, with the cocktail ED group adjuvanted with 3M052/alum characterized by 14.5-fold higher titers than the bolus control. While the ED cocktail and bolus groups had similar anti-trimer IgG titers, the ED groups had a higher non-base trimer response, shown by an 8.6-fold IgG increase between the 3M052/alum cocktail ED group and its bolus control. ED prime seems promising in raising anti-immunogen titers and lowering responses to the artificial trimer base, and further boosts will be administered to elicit serum neutralizing activity.
Validation of a Microbiome Dependent Colitis Model via Fecal Microbial Transplant in Germ-Free IL-10 Deficient Mice
Authors: Parello Caitlin , Van Dam Brett , McKim Andrew , Knights Mathew , Durrant Jessica , Panwell Kristin , Van Engelenburg Alex , Dube Philip , Lyng Gregory , Sonis Stephen
Published: November 2024

Inflammatory Bowel Diseases (IBD) are immune-mediated intestinal tract diseases with undefined etiologies. Proposed pathogenic mechanisms include abnormal inflammatory response to the intestinal microbiome. Mice deficient in IL-10, a critical cytokine for mucosal immune homeostasis, spontaneously develop enterocolitis; phenotypes are microbiome-sensitive. To establish a microbiome-dependent colitis model with improved construct validity and translational clinical relevance, we assessed colitis development in Germ-Free IL-10 knockout mice (GF IL-10 KO; Taconic Biosciences) following inoculation with fecal microbial transplant (FMT) from wild-type C57BL/6NTac mice at the Murine Pathogen Free™ (MPF™) health standard and assessed clinical responses to anti-IL-12/23p40 (anti-p40). Compared to control GF IL-10 KO mice, FMT-inoculated mice demonstrated reduced weight gain, elevated levels of fecal lipocalin 2 and calprotectin, and increased colon weight:length ratio at 8 weeks following FMT. FMT-inoculated mice also showed elevated histopathology scores, increased colonic CD3+ T cells and CD4+ TH cells, and epithelial hyperplasia by Ki-67 immunolabeling. Several colon cytokines were elevated in FMT-inoculated mice. Anti-p40 treatment significantly reduced all of these disease indicators with the exception of colon weight:length. These data provide a validated colitis model with relevant mechanisms for assessing the role of the microbiome and response to therapeutics in IBD.
Next-generation B cell ImmunoSpot™ assays permit in-depth assessment of the B cell response elicited by SARS-CoV-2 infection and/or COVID-19 mRNA vaccination
Authors: Kirchenbaum Greg , Lehmann Paul , Becza Noemi , Chepke Jack , Yao Lingling , Valente Alexis , Liu Zhigang
Published: November 2024

The affinity distribution of antigen-specific Bmem in the body is a critical variable that defines an individual’s ability to rapidly generate protective antibody specificities. Moreover, defining the Ig class/subclass usage of such antigen-specific Bmem is also critical since it forecasts the anamnestic response to be engaged upon antigen re-encounter. Here, we leveraged multiplexed ImmunoSpot to comprehensively define the magnitude and Ig class/subclass usage of the SARS-CoV-2 S-specific Bmem following infection and/or multiple COVID-19 mRNA vaccinations. Beyond evidencing past SARS-CoV-2 infection more reliably than assessment of plasma IgG reactivity against the nucleocapsid protein, direct assessment of Bmem provided insights into their affinity distribution and cross-reactivity profiles against emerging variants such as Delta and Omicron. Following stimulation of PBMC to transition resting Bmem into antibody-secreting cells, our studies entailed tracking of the S-specific Bmem compartment in subjects as their immune histories became increasingly more complex. Of note, our next-generation ImmunoSpot assay approaches evidenced improved functional affinity at single-cell resolution. Collectively, such B cell ImmunoSpot assays offer tremendous value for future B cell immune monitoring efforts owing to their ease of implementation, applicability to essentially any antigenic system, economy of PBMC utilization and suitability for regulated testing.
Regulation of NK cell effector function by Acly
Authors: Sohn Stacey , Cooper Megan , Kolicheski Ana , Laurent Jennifer , Saucier Nermina , Trammel Kelsey , Tobin Joshua
Published: November 2024

Natural Killer (NK) cells, crucial for viral immunity and tumor clearance, are influenced by metabolism. A prior study revealed that cytokine stimulation boosts the citrate-malate shuttle and cytosolic acetyl-CoA through ATP citrate lyase (ACLY) in NK cells. Acetyl-CoA is vital for fatty acid synthesis and protein acetylation, including histones. To explore the role of ACLY in NK cell function, we generated an inducible NK-specific Acly knockout mouse model. ACLY loss in NKp46+ NK cells did not alter maturation or IFN-γ production in naïve NK cells. However, ACLY-deficient NK cells exhibited notable proliferation defects in homeostatic and IL-15-stimulated conditions, associated with impaired glycolysis. The stimulation and priming of NK cells through IL-15 is an important mechanism for enhancing effector and anti-tumor functions. In addition to proliferation defect, IL-15-primed ACLY-deficient NK cells showed reduced IFN-γ responses to DAP12-associated activating receptors, specifically NKG2D, Ly49H, and Ly49D. RNA-seq revealed ACLY-deficient NK cells failed to upregulate DAP12 expression with IL-15 stimulation, confirmed at the protein level. These findings suggest the importance of ACLY in NK cell proliferation and potentially DAP12-driven effector functions. Ongoing experiments are focused on investigating how ACLY and cytosolic acetyl-CoA are linked to DAP12 expression, and the effects of this during in vivo effector functions.
Impaired Early Interferon Responses to Influenza Virus Infection in Aged Mice are Associated With Increased Pulmonary Inflammation
Authors: Metcalf Jordan , Alexander Jeremy , Booth J Leland , Miller Craig , Drevets Douglas , Wu Wenxin
Published: November 2024

Background: Seventy to ninety percent of seasonal influenza deaths occur in those aged 65 or older. Methods: To determine the innate immune responses to influenza A virus (IAV) infection, young (12-week) and old (70-week) C57BL/6J mice were infected intranasally (i.n.) with IAV PR8. Immune responses and viral replication were determined by qRT-PCR. Lung inflammation and injury were evaluated by histology and bronchoalveolar lavage (BAL) and lung to body weight ratio respectively.

Results: Comparing young and old mice at the times indicated there were no differences in weight loss percentages, lung-to-body weight ratios and total immune cells in BALF after IAV infection. However, aged mice had significantly higher viral loads and lower type I interferon (IFN) expression in the lung at day 3 p.i. In contrast, at this time point, aged mice had significantly higher amounts of type III IFN expression, which correlated with higher viral loads. Histopathology revealed that IAV infection in aged mice resulted in lower pathological scores early (day 5) and higher lung pathological scores later (day 7) of disease than in young mice.

Conclusion: Our models demonstrated that aging dysregulates early IFN responses to influenza infection resulting in enhanced viral replication. These altered IFN responses in aged mice also result in enhanced lung inflammation late after infection and may also be a cause of the increased incidence of secondary bacterial infections seen in the aged.
Single cell transcriptomes from fetuses exposed to HCMV during gestation reveals a distinct subset of memory-like NKG2C+ fetal NK cells
Authors: Khalil Mohamed , Terhune Scott , Malarkannan Subramaniam
Published: November 2024

Congenital cytomegalovirus (cCMV) infection is the most common cause of intrauterine infection in the USA, impacting 1 in 100 live births. Although most infected newborns are asymptomatic, 10% display severe congenital anomalies, including microcephaly, sensorineural hearing loss, cerebral palsy, growth restriction, and perinatal mortality. Natural killer (NK) cells are the first lymphocytes to develop during gestation and are required to manage CMV infections. The role of fetal NK cells during cCMV is limited and their development and functions within the umbilical cord has not yet been explored. To investigate this, four sets of umbilical cord blood (UCB) and matching umbilical cord tissues were collected from 2 CMV seropositive (CMV+) and 2 CMV seronegative (CMV-) fetuses that did not experience any complications during gestation. We were able to successfully sort and capture over 5,000 fetal NK cells and perform single-cell RNA-sequencing (scRNA-seq). Using scRNA-seq, we observed and characterized 5 unique fetal NK cell subsets in the UCB and 4 subsets in the corresponding tissue. Interestingly, we identified a distinct subset of NKG2C+ memory fetal NK cells displaying elevated expression levels of NKG2C, CD52, CD2, CD16, and CD3E. Here, we demonstrate that cCMV can induce the formation of memory-like NKG2C+ NK cells that display a unique transcriptional profile. These findings have the potential to influence the future application of fetal memory NK cells.
The role of CX3CR1+ FALC-associated macrophages in health and inflammation
Authors: Dominguez Joseph , Kim Jiseon , Kim Seung Hyeon , Adams Hannah , Ludewig Burkhard , Kim Ki-wook
Published: November 2024

The omentum is a visceral adipose tissue that contains fat-associated lymphoid clusters (FALC) to aid in peritoneal immunity. These clusters consist of large aggregates of lymphocytes, dendritic cells, macrophages, and innate lymphoid cells that participate in the clearance of pathogens. Tissue-resident macrophages (TRM) are immune cells found in every tissue and can be defined by their origins, consisting of subsets maintained either by embryonic precursors or by bone-marrow progenitors. A recent study found embryonically derived TRM express at least one marker amongst Tim4, LYVE1, FOLR2 while bone marrow derived macrophages maintain CCR2 expression. Previous publications have shown the existence of LYVE1+ macrophages surrounding each cluster, here we study an unexamined subset of FALC-associated macrophages that are imbedded within the lymphoid structures. Using confocal imaging, flow cytometry analysis with reporter/ conditional knock-out mice, we have characterized this TRM subset as CX3CR1+ Tim4- LYVE1lo/- and CCR2 dependent. In addition, we examined the interaction between CX3CR1+ macrophages and CCL19+ fibroblastic reticular cells by generating CCL19-driven Csf1 depleted mice and found CX3CR1+ macrophages were partially depleted. Lastly, we looked at the role of tissue-resident macrophages in the omentum in early ovarian tumor burden and found omental/peritoneal TRM macrophage depletion by Wt1-driven Csf1 depletion led to increased tumor burden.
Single-cell transcriptomics of CD8 T cells in autoimmune diabetes after anti-CD3 monoclonal antibody treatment
Authors: Wu Ying , Spurrell Maxwell , Deng Songyan , Herold Kevan
Published: November 2024

The FcR non-binding anti-CD3 mAb, teplizumab is the first drug approved by FDA to delay Type 1 diabetes (T1D) by modulating the immune mediated destruction of insulin-producing β cells. However, we and others have observed that after remission is induced in diabetic NOD mice that are treated with F(ab’)2 fragments of mAb 145-2C11, insulitis remains. To understand how the antibody induces long-term tolerance, we performed single-cell RNA-seq and TCR-seq on T cells from the islets and pancreas-draining lymph nodes (pLN) of long-term remitted NOD mice treated by anti-CD3 mAb or pre-diabetic NOD mice with insulitis. In the islets, CD8 T cells of remitted NOD had higher expressions of TCF-1 and EOMES, than their counterparts from pre-diabetic control. Notably, CD8 T cells from islets of remitted NOD had increased expressions of TOX and EOMES while with no difference in TCF-1, compared to their counterparts from paired pLN. In contrast, islet-infiltrating CD8 T cells of pre-diabetic NOD had decreased expressions of EOMES and TCF-1 but with no difference in TOX, compared to their pLN counterparts. Moreover, islet-infiltrating CD8 T cells of remitted NOD had higher median TCR entropy indicating a more diverse TCR repertoire than their counterparts from pre-diabetic NOD. These results suggest that anti-CD3 modulates the terminal differentiation of islet-infiltrating CD8 T cells. Tolerance is maintained by cells with stem-like and exhaustion features and a more diverse TCR repertoire.
Hyaluronidase 2 induces pathogenicity in Th17 cells
Authors: Adhikari Laxmi Prasad , Woodman Kaitlyn , Reynolds Joseph
Published: November 2024

Autoreactive Th17 cells express high levels of inflammatory cytokine IL-17A promoting the development and progression of autoimmune diseases. Previous studies show that differentiation of Th17 cells with IL-1 and IL-23 or TLR2 activation drives their aggressive proliferation, making them pathogenic to induce experimental autoimmune encephalomyelitis (EAE). We aim to study a previously undefined role of hyaluronic acid (HA) and its modulation in influencing inflammatory potential and pathogenicity of Th17 cells in autoimmune diseases. Our in vitro differentiation experiments with HA and its modifying enzymes show that a baseline level of HA is essential for the normal differentiation and proliferation of Th17 cells. The overexpression of hyaluronidase 2 (Hyal2) that cleaves HA in Th17 cells decreases IL-17A production but increases cell proliferation in vitro, and adoptive transfer of these cells is sufficient to induce EAE in mice. Furthermore, flow cytometric analysis of infiltrated CD4+ T cells in the CNS of EAE mice indicates lower levels of inflammatory cytokines. Our findings suggest that Hyal2 itself exhibits a level of pathogenicity challenging the prevailing paradigm that these cells can induce the disease only when producing higher levels of encephalitogenic cytokines. Future experiments will delve into the regulation and mechanisms underlying the impact of HA on pathogenic Th17 cells, potentially identifying target molecules to treat autoimmune disorders.
Single-cell RNA sequencing reveals an immunomodulatory population of double negative T-cells in the diabetic NOD mouse
Authors: Robben Michael , Luber Jacob , Kostic Aleksander
Published: November 2024

NOD mice are a widely used model of type 1 diabetes, an inflammatory autoimmune condition characterized by autoreactive T-cells that target insulin producing beta cells of the pancreatic islets. While T cell biology in the context of this disease is important it is unclear why autoreactive T-cells develop in this model or what they do within the islets themselves. We have designed a single cell sequencing experiment where enriched T-cells taken from diabetic and non-diabetic NOD mice are sequenced for mRNA expression and TCR sequence concurrently. The resulting sequencing data shows abnormal representation of Double Negative (DN) T-cells in all tissues and conditions (~30%). We have observed that like CD8+ and CD4+ T-cells, this population displays normal maturation phenotypes, but experience reduced exhaustion of effector subsets after infiltration of the pancreatic islets. Looking at clonal subpopulations of T-cells we have discovered a subset of CD8+ T-cells that have high expression of GzmA even after loss of the survival signal marker, IL7r. Through investigation of these aberrant T-cell populations, we have found that the diabetic NOD mouse pancreatic is highly immunosuppresive and expression of T-cell co-receptors may have a significant impact on diabetic onset.
Unraveling the Role of CD137 in Treg-Mediated Suppression of Type 1 Diabetes: Insights from Treg-Specific CD137 Knockout Mice
Authors: Nabi Rabia , Ciecko Ashley , Lin Chien , Chen Yi-Guang
Published: November 2024

CD137, encoded by Tnfrsf9, acts as a costimulatory molecule on activated T cells. Conversely, soluble CD137 exerts immunosuppression by binding to CD137L on T cells, inhibiting activation. In the NOD mouse model of Type 1 Diabetes (T1D), CD137 expression in Foxp3+ regulatory CD4 T cells (Tregs) inhibits T1D, but the underlying mechanism remains elusive. To investigate the role of CD137 in Treg-mediated T1D suppression, we generated Treg-specific CD137 knockout mice (NOD.Treg-Tnfrsf9-/-). While no systemic inflammation was observed, these mice developed accelerated T1D compared to wildtype control. Reduced serum-soluble CD137 in these mice indicated Tregs are its main producers. scRNA-seq revealed phenotypic alterations of Tregs isolated from pancreatic islets of NOD.Treg-Tnfrsf9-/- mice, showing lower activation and clonal expansion than wildtype counterparts. Reduced Treg activity in these mice was associated with increased frequencies of islet-activated CD8 T cell subsets and their clonal expansion. MHC class I tetramer staining revealed a higher frequency of β-cell antigen IGRP206-214 specific CD8 T cells in NOD.Treg-Tnfrsf9-/- mice. No discernible differences in the initial expansion and activation of β-cell autoreactive CD8 T cells in pancreatic lymph nodes were observed. Our results suggest CD137 expression in Tregs is critical for restraining ongoing autoimmune inflammation in islets of NOD mice but dispensable for controlling autoreactive T cells in lymphoid tissues.
Chitinase 3-Like-1/BRP-39 regulates extracellular matrix remodeling during allergic fungal asthma
Authors: Ellis Diandra , Makullah Mgayya , Jones Mary Jane , Godfried Jarrett , Rokicki Ellie , Cheung Suki , Steele Chad
Published: November 2024

We previously reported that fungal-sensitized human asthmatics had higher levels of the chitinase-like protein YKL-40 in sputum than non-fungal sensitized asthmatics. Unexpectedly, we found that mice deficient in BRP-39 (Chi3l1-/- mice; the murine ortholog of human YKL-40) had increased airway hyperreactivity (AHR) during experimental allergic fungal asthma, despite no correlation with classic drivers of AHR (type 2 and eosinophilic responses). BRP-39 has been reported to bind lectins and complex carbohydrates and is involved in non-immune processes such as cell proliferation, angiogenesis and tissue remodeling. The extracellular matrix (ECM) of the lung is mainly comprised of collagen and elastin that provides elasticity and resilience to compressive and expanding forces. We found that Chi3l1-/- mice subjected to allergic fungal asthma demonstrated restrictions in lung mechanics due to increased elastic stiffness in the conducting airways and the alveolar/parenchymal compartment. This correlated with increased levels of collagen and elastin in mutant mice. ECM enzymes involved in matrix remodeling of collagen and elastin, such as matrix metalloproteinases (MMPs) 2, 3, 9, and 12, were reduced in mutant mice. We further identified BRP-39 dependent differences in MMP secretion by stimulating primary lung fibroblasts. Ultimately, these findings highlight BRP-39 as a crucial regulator of MMPs needed to turnover lung ECM proteins during the progression of allergic fungal asthma.