bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence-based learning modules and culturally responsive mentoring to engage underserved undergraduate students
Authors: Mishra Manoj K
Published: January 2023

The number of underrepresented minorities in the biomedical workforce has quadrupled over the past three decades, yet doctoral-level minority scientists still make up less than 5% of the total workforce. Thus, it is imperative to implement strategies to maintain the degree and diversity of scientific research and education expertise in biomedical disciplines. In this study, we develop, deploy, and assess the effectiveness of immunology learning by integrating evidence-based learning (EBL) modules to engage and motivate underserved students. Additionally, we provided robust culturally responsive mentoring to engage students in immunology education and research. Our initial finding demonstrates that EBL modules enhanced the collaborative learning and research activities and broadened their understanding of immunological problems. Therefore, our study suggests that a) implementing EBL modules in the immunology curriculum and b) engaging students through in-person mentoring during classroom engagement, attract and sustain underrepresented minority students to the immunology courses, improve undergraduate research training, and enhance educational appetite in biomedical disciplines.

Supported by grants from NIH UO1GM132769 and NSF 1912322.
Thymic Interferon Impacts T cell Selection
Authors: Ashby Katherine Maude , Martinez Ryan J , Salgado Oscar C , Hogquist Kristin Ann
Published: January 2023

Autoimmune diseases are triggered by the loss of immunological tolerance to self-antigens. Although previous work has characterized mechanisms driving immune tolerance to tissue restricted self-antigens, less is understood about how tolerance is established for inflammation-associated self-antigens that are expressed transiently during acute infection. This is particularly important with the discovery that autoantibodies to type I interferons (IFN) are commonly found in elderly individuals, and predispose to severe COVID19. Recent work from several groups has found that Type I and Type III IFN are expressed in the thymus at steady state (in the absence of infection) and drive expression of interferon stimulated genes in antigen presenting cells and developing thymocytes. We hypothesize that IFN is present in the thymus in order to tolerize developing T cells to self-antigens presented during acute infection (such as those derived from interferon and interferon stimulated genes.) In line with this, T cell receptor (TCR) repertoire analysis showed that mice lacking the type I IFN receptor (Ifnar KO) have distinct T conventional (Tconv) and T regulatory (Treg) TCR repertoires from wild type (WT) mice. We have identified two groups of interesting receptors from this TCR repertoire analysis; 1) IFN-deleted receptors(~50 TCRs) that are present in the Ifnar KO Tconv repertoire and absent from the WT repertoire, and 2) IFN-dependent Treg receptors(~750 TCRs) that are present in the WT Treg repertoire but lost from the Ifnar KO Treg repertoire. We have expressed TCRs from each group in hybridomas and retrogenic mice in order to determine the specificity of these TCRs and characterize the development of thymocytes expressing them.

Supported by grants from NIH (T32 AI007313)
Single-cell proteogenomics reveals that SLAM/SAP signaling regulates the development of innate-like γδ T cell subsets with distinct TCR repertoires
Authors: Boyson Jonathan E , Andretta Emma , Hampel Kenneth J , Sidiropoulos Nikoletta , Dienz Oliver , Majumdar Devdoot , Mistri Somen
Published: January 2023

γδ T cells are non-conventional T cells that are highly enriched in the mucosal tissues where they play critical roles in immunity. We recently demonstrated that the SLAM/SAP signaling pathway regulates the thymic development of innate-like γδT17 and γδTIFN subsets, in addition to γδNKT cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. This analysis not only confirmed our previous finding that SLAMF1 and SLAMF6 expression marks γδT17 and γδTIFN subsets, respectively, it also identified SLAMF7 as a novel marker of the SAP-dependent innate-like CD44+ CD45RB+ γδTIFN cells in both the thymus and periphery. Next, our data indicated that disruption of SAP-dependent signaling impaired γδT17 development at a very early (CD24high CD73−) stage, and was associated with the decreased expression of critical regulators of γδT17 development such as Blk and c-Maf. In contrast, while SAP also impaired γδTIFN development at an early (CD24high CD73+) stage, SAP-deficient γδTIFN cells exhibited increased expression of genes associated with TCR signaling and thymic export such as Prkch, Dgka, Klf2, and S1p1r. Finally, our analysis revealed significant alterations in the γδT17 TCR repertoire in both embryonic/neonatal thymus and the lung, as well as the presence of a SAP-dependent IFN-γ-producing lung Vγ4 population that preferentially utilized TRDV7. Altogether, these data suggest that SLAM/SAP signaling acts during the very early stages of γδ T cell development where it regulates critical pathways in both γδT17 and γδTIFN development, and influences the development of the innate-like γδ TCR repertoire.

Supported by NIH (R03AI153902, P30GM118228), AAI Careers in Immunology Fellowship
Collaboration between Osteopontin and Macrophage Migration Inhibitory Factor in Autoimmune Myocarditis
Authors: Medrano Megan T , Nalawade Saisha , Huizar Carol Chase , Yuhara Alec , Forsthuber Thomas G
Published: January 2023

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine elevated in multiple conditions. MIF is also elevated in autoimmune myocarditis (AM), a condition in which the immune system aberrantly attacks the heart muscle. AM can be induced by antigenic mimicry, autoimmune stimulation, genetic defects, issues with regulatory T cells, dysregulation of immune effector cells. AM also has the possibility to progress to the fatal disease dilated cardiomyopathy (DCM) through chronic inflammation, the deposition and crosslinking of collagen, proliferation by fibroblasts, and differentiation into myofibroblasts. Our lab has previously shown that MIF and OPN synergize to enhance the progression of disease to DCM, by affecting cardiac fibrosis and the deposition of collagen. Glucocorticoids (GCs) are given to suppress the inflammation caused by immune reactions and therefore are prescribed as treatment for autoimmune myocarditis. However, GCs are unable prevent the progression of AM to DCM. Our lab investigated the role of GCs for promoting the actions of MIF by enhancing the OPN-dependent deposition of collagen in the myocardium by gene expression of extracellular matrix proteins affected by AM through qPCR and their protein concentration by ELISA. Establishing a pathway for collagen deposition in AM will allow for the development of more effective treatment and prevention of the progression to DCM.
Archived antigen boosts CD8 T cell memory responses during an unrelated infection.
Authors: Doan Thu A , Tamburini Beth
Published: January 2023

Numerous studies have shown viral antigens can persist in lymph nodes after the resolution of the infection. We showed that lymphatic endothelial cells (LECs), which comprise the lymphatic vasculature, are the predominant cell type required for the persistence of antigen within the lymph node. We termed this process antigen archiving due to the ability of LECs to actively acquire and store antigens to which an active immune response occurred. This process involves antigen acquisition, retention, and exchange of the antigen between LECs and dendritic cells, resulting in the presentation of the archived antigens to CD8 memory T cells and improving effector function. In more recent data, we demonstrated that LEC death causes the release of archived antigens during LN contraction. We found that archived antigens could be released during an unrelated viral infection, during the time frame of LEC death. This release of antigen was visualized by a significant increase in archived antigen-specific endogenous or transferred memory CD8 T cells, but not memory T cells of an unrelated specificity. The increase in archived antigen-specific CD8 T cells maintained a more effector-like phenotype and performed significantly better upon antigenic challenge with Listeria monocytogenes (LM) expressing ovalbumin. Mice that received an unrelated infection 2–3 weeks prior to antigenic challenge demonstrated improved protection against LM-ova. In conclusion, archived antigen release during a secondary unrelated infection boosts the archived antigen-specific memory CD8 T cell population. These findings bring into question whether some previous data demonstrating heterologous immunity may be a result of archived antigen release.

Supported by R01 AI121209 R21 AI155929
The anti-inflammatic activity of a sulfated polysaccharide Fucoidan in innate immune cells
Authors: Miyazaki Yoshiyuki , Satoyama Toshiya , Nakano Hayato , Takeuchi Shugo , Takeuchi Hideaki , Tachikawa Daisuke
Published: January 2023

Fucoidan is a series of natural sulfated polysaccharides derived from brown seaweeds and have reported to possess various biological activities such as anti-tumor, anti-virus, and immuno-modulatory effects. In previous study, we revealed that fucoidan derived from Cladosiphon okamuranus activated murine macrophage-like cell line RAW264 cooperatively with Zymosan, a Saccharomyces cerevisiae-derived β-glucan. On the other hand, several reports demonstrated that fucoidan inhibited lipopolysaccharide (LPS)-induced production of nitrogen oxide (NO) and inflammatory cytokines by RAW264 cells. Therefore, in this study, we investigated the potential of fucoidan derived from Undaria pinnatifida to regulate the excessive activation of RAW264 cells in response to overstimulation with pathogen components. RAW264 cells were inoculated at 20000 cells/well in 96-well culture plates, and after a recovery culture for 24 hr, the cells were treated with fucoidan with or without appropriate concentrations of each ligand for pattern recognition receptors (PRRs) including Toll-like receptors (TLRs). Whereas the production of NO was significantly enhanced with fucoidan alone, it was dose-dependently inhibited by fucoidan under stimulation with Pam3CSK4 (TLR1/TLR2 ligand), heat killed Listeria monocytogenes (HKLM, TLR2 ligand), LPS (TLR4 ligand), Pam2CGDPKHPKSF (FSL-1, TLR2/TLR6 ligand). These results suggested that fucoidan have beneficial ability to alleviate excessive inflammatory reaction during pathogenic infection and maintain suitable immune balance.

Supported by Laboratory of Analytical Food Immunoscience belonging YM is a donated fund laboratory established in Faculty of Agriculture, Kyushu University by Ventuno Co., Ltd.
THC decreases bone marrow-derived macrophage activation in a dose-dependent manner
Authors: Carter Taylor H , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Macrophage activation is a major factor in progressive illnesses such as atherosclerosis, arthritis, and HIV-associated Neurological Disorder (HAND). Δ9-Tetrahydrocannabinol (THC), the main psychoactive compound found in cannabis, has been shown to have beneficial effects in treating these diseases, although the mechanism of action is not clear. Our lab and others have demonstrated the role of THC as an anti-inflammatory agent. In the current study, we attempted to investigate the effect of THC on macrophage activation. For this purpose, bone marrow was collected from tibia and femurs of C57BL/6 mice (10–12 weeks) and cells cultured in presence of macrophage colony stimulating factor (MCSF) for 4 days, in order to induce macrophage differentiation. On day 4, Lipopolysaccharide (LPS), gp120 of HIV or vehicle was added to the cultures and flow cytometry performed. Our results showed that THC treatment decreased F4/80+ macrophages in both LPS and gp120 groups in a dose-dependent manner when compared to vehicle-treated controls. MicroRNA-sequencing analysis showed a large number of miRs were dysregulated several fold. Importantly, there was an increase in the THC group of various miRNA such as miR-2137 and miR-486-5p that are involved in cell development and proliferation. Together, the role of THC in decreasing bone marrow-derived activated macrophages through miR dysregulation could provide new treatment strategies for diseases such as atherosclerosis, arthritis, and HAND.

Supported by NIH grants P01AT003961, P20GM103641, R01ES030144, R01AI129788, R01AI123947 and R01AI160896
Inhibition of Glycolysis Modifies Distinctive Metabolic and Immune Pathways Across Multiple Tissue Compartments Associated with B and T Follicular Helper Cells
Authors: Wells Ann , Wilson John , Roopenian Derry C , Chang Chih-Hao , Carter Gregory
Published: January 2023

Systemic lupus erythematosus (SLE) is the most common form of lupus; however, only one new drug was approved for SLE in the last 60 years. Inhibition of glycolysis via 2-deoxyglucose (2DG), a competitive inhibitor of glucose-6-phosphate, has recently shown efficacy in attenuating tumor growth and reducing autoimmune pathology; however, the mechanism of this efficacy is somewhat unclear. To elucidate how 2DG longitudinally modulates pathways within the context of systemic lupus erythematosus (SLE), BXSB.YaaIL15−/−,CD8−/− mice, an SLE model, were treated orally with 2DG for 96-hours or 4-weeks, after which the transcriptional profiles of 9 tissues (heart, hippocampus, hypothalamus, kidney, liver, prefrontal cortex, skeletal muscle, small intestine, and spleen) were analyzed using unsupervised clustering and weighted gene correlation network analysis. Principal component analysis showed mice clustered by tissue. Tissues correlated to B cells and T follicular helper cells grouped together according to their summary gene expression for modules treatment or treatment, in a time dependent manner, and three pathway groups, transcription, immune, and glucose, emerged. However, within each tissue cluster, pathways for each module were uniquely altered by treatment or treatment, in a time dependent manner, according to ANOVA. Furthermore, while most pathways identified were grouped into either immune or metabolic, there was little overlap of specific pathways identified between tissues. These results show that 2DG has a systemic impact altering immune and metabolic pathways, but in a tissue-specific manner. Ongoing work includes identifying genes that are potentially central to pathway alterations for follow-up analysis.

Supported by the AAI Intersect Fellowship
An immunomodulatory human commensal coordinately regulates inflammatory and metabolic disease
Authors: Tan Chin Yee , Surana Neil
Published: January 2023

Specific members of the microbiota are known to modulate the immune system, which also plays an important role in regulating metabolism. Indeed, there are emerging data detailing specific bacterial species that impact multiple physiological outcomes, but the mechanisms underlying these pleiotropic effects are unclear. We previously discovered a new human commensal bacteria species, Clostridium immunis, that protects against colitis. Here, we define the host and bacterial mechanisms of action for C. immunis-mediated disease regulation. We found that C. immunis treatment decreases ILC3 numbers and function in mice and humans. Consistent with this finding, C. immunis regulates the severity of intestinal and extra-intestinal inflammatory diseases, as well as body composition in an ILC3-dependent manner. Interestingly, C. immunis influences metabolism by decreasing expression of lipid processing genes, thereby inhibiting dietary fat processing. The bioactive component of C. immunis is a secreted product and comparative genomics identified 8 C. immunis genes associated with ILC3 regulation. Collectively, we have demonstrated how a single bacterial species impacts different physiological pathways via its suppression of ILC3s. More broadly, our work reveals a microbiome-based therapy for diseases driven by pathogenic ILC3s.

Supported by a Development Award from the National University of Singapore and a Graduate Student Award from the Triangle Center for Evolutionary Medicine
Rab27A-dependent transfer of CD11c+ cell exosomes regulate gut immunity
Authors: Bauer Kaylyn , Round June , O’Connell Ryan M
Published: January 2023

Intercellular communication is critical for maintaining homeostasis in mammalian organ systems, including the gastrointestinal (GI) tract. Exosomes are small nanoscale lipid extracellular vesicles that mediate communication between a wide variety of cell types. Notably, the roles played by immune cell exosomes in regulating homeostasis and inflammation in the GI tract are largely uncharacterized. Here, we describe an unknown mechanism of exosome-mediated immune regulation within the GI tract. By generating novel mouse strains, we demonstrate that disruption of exosome signaling via the deletion of Rab27a in CD11c+ myeloid cells exacerbates colitis, which is reversed through administration of dendritic cell (DC)-derived exosomes. Profiling small noncoding RNAs within exosomes from colonic tissues revealed a distinct subset of microRNAs (miRNAs) carried by both gut- and DC-derived exosomes. Among the DC-enriched exosomal miRNAs, miR-146a was transferred from gut CD11c+ cells to myeloid and T cells through a Rab27-dependent mechanism to decrease macrophage-mediated inflammation during colitis. Clinically, RAB27A is also expressed at lower levels in a cohort of ulcerative colitis patients versus healthy controls. Together, this work reveals an exosome-mediated modulatory mechanism of gut inflammation, paving the way for exosomes containing miRNA cargo as a potential therapeutic for inflammatory bowel disease.

Supported by National Institutes of Health Grants T32 AI 138945-1 (K.M.B.) and AI123106-01A1 (R.M.O.)
Mechanosensory channel Piezo1 is essential in pathogenic T cell-mediated intestinal inflammation
Authors: Choi Sung Hee , Aguilar Alicia , Myers Jay , Kim Byung-Gyu , Eid Saada , Tomchuck Suzanne , Kingsley Daniel , Huang Alex
Published: January 2023

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the gastrointestinal tract. CD4+ T cells are especially known to be the main drivers of IBD when they show an elevated level of activation. Elevation of intracellular Ca2+ is one of the key triggering signals for T cell activation. Piezo1 is a mechanosensitive nonselective Ca2+-permeable cation channel, which is broadly expressed in mammalian cells. However, the role of Piezo1 in the pathogenesis of T cell-mediated colitis remains unknown. We have generated T cell-specific Piezo1 knockout (Piezo1fl/flxCD4-cre) mice and observed that loss of Piezo1 in CD4+ T cell increased Th1 and Th17 cell polarization. RNA-sequence analysis of Piezo1fl/flxCD4-cre T cells identified elevated pathogenic Th17 cell pathway, IFN-γ signaling pathways and inflammatory response gene signature compared to that of wild type. These results suggest that Piezo1 controls the inflammatory response of pathogenic T cells. Next, we examined the function of Piezo1 on intestinal inflammation in vivo using acute and chronic colitis mouse model. For the acute colitis mouse model, we used a chemically induced mouse model of colitis using DSS in drinking water. Piezo1fl/flxCD4-cre mice with DSS developed severe colitis compared to Piezo1fl/fl mice with DSS. However, in chronic colitis mouse model, which is the adaptive transfer of naïve CD4+ T cells (CD4+ CD45RBhigh) from Piezo1fl/fl or Piezo1fl/flxCD4cre into Rag1−/− mice, T cells from Piezo1fl/flxCD4cre mice failed to induce colon inflammation, while mice that received T cells from Piezo1fl/fl mice developed severe intestinal inflammation. Thus, our data demonstrate a critical role of Piezo1 in CD4+ T cell-mediated intestinal inflammation.

Supported by R03 CA230840, P30 CA043703, St. Baldrick’s Foundation, Hyundai Hope-on-Wheels Scholar Hope Grant, Andrew McDonough B+ Foundation, Curing Kids Cancer, Center for Pediatric Immunotherapy at Rainbow
Vitamin A regulates phagocytosis by resident macrophages of the small intestine
Authors: Dende Chaitanya , Pendse Mihir , Propheter Daniel , Quinn Gabriella , Hooper Lora V
Published: January 2023

Intestinal Tim4+ CD4+ macrophages are a distinctive macrophage subset that express Tim4, a receptor for phosphatidylserine on dying apoptotic cells, Unlike other macrophage subsets, they do not depend on blood monocytes for their turnover, instead self-maintained in the small intestine. The signal(s) responsible for the self-maintenance and function of Tim4+ CD4+ macrophages is not known. We have discovered that maintenance of the gut resident Tim4+ CD4+ macrophage population depends on dietary vitamin A and its derivative retinoic acid (RA). Retinoic acid receptors, which direct RA-dependent transcription, were required for maintenance of Tim4+ CD4+ macrophages. Chemical blockade of retinoic acid receptor (RAR) signaling and macrophage-specific genetic inactivation of RARs in mice further revealed that macrophage-intrinsic RARα signaling was required for Timd4 expression and maintenance of Tim4+ CD4+ macrophages. Macrophage RARα signaling was furthermore essential for phagocytosis by Tim4+ CD4+ macrophages. Ongoing studies are examining the role of Tim4+ CD4+ macrophages and vitamin A in the clearance of apoptotic intestinal epithelial cells. Our findings reveal that vitamin A provides an essential dietary signal for the maintenance and function of a gut resident macrophage subset.

Supported by Welch foundation grant I-1874
Targeting tonsillar B cells with IL-9 and IL-33 enhances the rhesus macaque humoral response to DNA/protein-based HIV envelope vaccine
Authors: Kobie James , Spencer David , Barnette Philip , Pandey Shilpi , Basu Madhubanti , Sutton William , Rangel-Moreno Javier , Haigwood Nancy Logan , Hessell Ann Jones , Sarkar Sanghita
Published: January 2023

Development of an effective HIV vaccine is dependent on the quantity and quality of HIV Envelope specific antibody at the mucosal sites. Priming of the oral mucosa is a promising approach to generate mucosal antibody at HIV entry sites. Our recent work in mice has demonstrated the ability of IL-9 and IL-33, known regulators of mucosal immunity, to promote robust development of HIV Env-specific IgG; in addition they increase the breadth, magnitude, and durability of the Env-specific IgG response when combined with the DNA/protein HIV Env immunogen platform VC10014, which elicits Tier 2 neutralizing antibody in rhesus macaques. We hypothesized that conditioning the tonsil microenvironment with IL-9 or IL-33 would enable the rapid induction of durable and effective mucosal humoral immunity by the VC10014 DNA/protein HIV vaccine platform. Rhesus macaques were primed with VC10014 gp160 DNA plasmids in the absence or presence of IL-9 or IL-33 intra-tonsillar (IT), followed by intramuscular (IM) boosting with the DNA/protein HIV vaccine regimen. IT immunization with IL-9 or IL33 induced plasma Env-specific IgG that was present as early as week 6, mucosal Env-specific IgG and heterologous plasma neutralizing antibody. IL-33 treated animals exhibited the greatest number of germinal centers (GCs) and largest GCs following the first IM boost whereas IL-9 treated animals showed the greatest number of GCs following the second IM boost in the draining lymph nodes. The difference in kinetics, with IL-33 promoting an earlier B cell response, and IL-9 promoting greater magnitude later, may suggest that IL-9 and IL-33 promote the development of qualitatively different B cells subsets and may have synergistic potential in HIV vaccine development.

Supported by a grant from NIH (R01DE027245).
Microbiota regulation of intestinal inflammation influences colorectal cancer
Authors: Ruiz Daniel Fernando Zegarra , Kim Dasom V , Saldana-Morales Fatima B , Ng Charles , Callaghan Ryann , Uddin Maisha , Chang Lin-Chun , Longman Randy S , Diehl Gretchen E
Published: January 2023

Inflammatory bowel disease (IBD) patients with poorly controlled intestinal inflammation are at an elevated risk for colorectal cancer (CRC). IBD patients exhibit intestinal dysbiosis with expanded proteobacteria such as E. coli. Here, we find that colonization with an E. coli isolated from the intestine of an IBD patient (E. coli 541-15) prevents tumorigenesis in an inflammation-related model of CRC. Colonization increased tumor infiltration of T helper 1 (Th1) cells, cytotoxic T lymphocytes (CTLs), and type 1 innate lymphoid cells (ILC1s) and decreased myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Prevention of tumorigenesis occurs if colonization takes place before induction of inflammation. Intestinal inflammation in colitis models was ameliorated by E. coli 541-15 and this protection depended on IL-10 production by macrophages and IL-10 signaling to the intestinal epithelium. Colonization with E. coli 541-15 also promotes these IL-10 pathways if colonization occurs after tumorigenesis is established. However, this leads to worse CRC outcome, with increased tumor burden alongside decreased tumor infiltration of Th1 cells, CTLs, and ILC1s and increased MDSCs and Tregs. These results identify activation of an IL-10 signaling loop between immune cells and the intestinal epithelium after E. coli colonization that modulates intestinal inflammation and CRC. Importantly, these pathways can be protective or pathogenic depending on timing of activation.

Supported by 2021 Ludwig Center Basic and Translational Research Award
Migratory type 2 dendritic cells mediate mucosal Th17 response to gut commensal bacteria
Authors: Ngoi Soo M , Yang Yi , Iwanowycz Stephen , Gutierrez Jennifer , Hill Megan , Allen Carter , Chung Dongjun , Liu Bei
Published: January 2023

Dendritic cells (DCs) are professional antigen-presenting cells equipped with MHC-restricted antigens, co-stimulations, and cytokines that effectively prime and differentiate naïve T cells into distinct functional subsets. The immune signals that DCs carry reflect the route of antigen uptake and the innate stimuli they received. In the mucosal tissues, owing to the great variety of foreign antigens and inflammatory cues, DCs are predominantly activated and migratory. The gut commensal bacteria contribute to immune health by educating the immune system at this unique location interfacing with the outside environment. In the small intestine, CD4 Th17 cells are abundant and play a critical role in mucosal defense against bacterial and fungal infections. We identified that the early priming step of commensal-driven Th17 cells is controlled by bona fide Zbtb46-expressing DCs. CCR7-dependent migration of DC2s from the small intestine to the mesenteric lymph nodes is essential for the activation of naïve CD4 T cells. The migratory DC2 population in the MLN are almost exclusively Esam+ cells. Single-cell RNA sequencing highlighted the abundance of co-stimulatory markers (CD40 and OX40) and chemokines (Ccl22 and Cxcl16) on MLN migratory DCs. Further resolution of MLN migratory DC2s revealed that the Th17-polarizing cytokine IL-6 colocalizes with DC2s expressing CD40, Ccl17, and Ccl22. Our study indicates that the mucosal Th17 cell response is regulated by a restricted subset of migratory DC2s in the gut draining lymph nodes.

Supported by NIAID U01AI125859
Interactions Between Aryl Hydrocarbon Receptor and the Microbiome Regulate Intestinal Macrophages in Non Obese Diabetic Mice
Authors: Ehrlich Allison , Malany Keegan , Sandoval Simone , Shulzhenko Natalia , Morgun Andrey
Published: January 2023

Type 1 diabetes (T1D) arises from a complex interaction between genetic and environmental factors. Multiple environmental signals predicted to impact T1D development occur at the intestinal surface, as a result of exposures through ingestion, dietary antigens, and microbial colonization. It is hypothesized that an inappropriate immune response to these exposures precipitates overt T1D. We discovered a novel mechanism by which environmental surveillance interacts with the immune system: immunosuppressive lamina propria (LP) macrophage regulation via the aryl hydrocarbon receptor (AhR). In NOD mice, AhR negatively regulates a macrophage subset (CD11b+ CX3CR1+ MHCIIhi; AhR-MFs) that have a strong positive correlation CD4+ IL10R+ Foxp3+ Tregs in the small intestine. To identify the potential source of AhR ligands that regulate these resident-like macrophages, mice were fed an AhR ligand-deficient diet. These cells were not further regulated by dietary ligands, suggesting that AhR activation originates from ligands produced by the intestinal microbiome. Therefore, germ-free NOD AhR knockout mice were derived and the AhR-MF population was measured. In the absence of the microbiome, both AhR knockout and wild type mice had high levels of AhR-MFs, demonstrating that the microbiome is required for AhR-mediated regulation of LP macrophages. RNAseq analysis of sorted AhR-MFs demonstrate that these cells are antigen presenting but lack an inflammatory gene signature. Currently, single cell sequencing of CD45+ LP cells from AhR knockout NOD mice is being performed to identify how AhR alters macrophage differentiation and predict mechanisms of crosstalk with intestinal CD4+ T cell populations.

Supported by R00 DK117509-03, T32 ES007059
Modeling Inflammatory Immune Cell Recruitment and Response on Human Colon Intestine-Chip
Authors: Carman Christopher , Kanellias Marianne , Ramos Dennis , Maniar Kairav , Saud John , Lucchesi Carolina , Kulkarni Gauri , Apostulou Athanasia , Kujala Ville J , Ewart Lorna
Published: January 2023

Objective

Immune cell recruitment into tissues is an essential step in inflammatory responses. This occurs in a highly tissue- and stimulus-specific manner, which presents a significant challenge to modeling disease and testing therapeutics ex vivo. We previously developed an advanced primary human vascularized Colon Intestine-Chip model and showed that it recapitulates physiologic cell composition, morphology and barrier function. The goal of this study was to test the ability of this system to model inflammatory bowel disease (IBD)-like immune cell reactions ex vivo.

Methods

We perfused primary human peripheral blood mononuclear cells (PBMC) across the vascular channel in untreated ‘resting’ or TNFα/chemokine-treated ‘inflamed’ Colon Intestine-Chips. We analyzed total cell recruitment, inflammatory cytokine secretion and barrier function in the following 24–72 hours.

Results

We show that the perfused PBMC efficiently adhered and transmigrated to the epithelial channel in an inflammation-specific manner. This was followed by an accumulation of proinflammatory cytokines characteristic of IBD (e.g., INFγ, IL1β, IL18), as well as loss of barrier function, the hallmark feature of IBD. We further showed that 1) the recruited cells were strongly enriched in the ‘gut trophic’ α4β7+/CCR9+ PBMC subsets and 2) this recruitment could be blocked with the IBD therapeutic Entyvio, which targets the α4β7-MAdCAM-1 interaction.

Conclusion

Our findings indicate that our Colon Intestine-Chip can model inflammatory immune cell recruitment and in situ immune reactions that reflect key clinical correlates of IBD. This model may prove effective for development of new anti-inflammatory therapeutics for human intestinal diseases.

Supported by Emulate Bio
High-precision micro-wells in polymer for single cell cloning and monitoring
Authors: Schimke Magdalena M
Published: January 2023

STRATEC Consumables is focusing on collaborations in development of smart consumables that enable precise clinical diagnostics, single cell handling and detection of rare proteins out of complex samples. Creation of micro-and nano-features in polymers that allow high-volume replication with great accuracy to ensure reliable results in clincis and laboratories. Micro-wells are supportive to novel approaches in single cell handling, cloning, sequencing and proteomics. STRATEC’s partnership with ALS GmbH from Jena, led to the development and production of nanowell plates for automated screening and recovery of single cells, under the name “CellCelector™”. This unique system combines high-resolution fast fluorescence imaging, sensitive cell detection technology as well as patented robotics for fast recovery of pure and viable cells and is compatible with various upstream cell enrichment approaches.

In order to realize the nanowell plates STRATEC Consumables made use of mastering of high aspect ratio features up to 1:10, followed by injection molding with a high pattern fidelity, assembly to a standard micro-titer plate and surface functionalization to enable the application of ALS. Together, this project shows outstanding results though tight collaboration combining capabilities to innovate in the vibrant field of single cell understanding and applications.
Incorporation of Id3 enhances CAR T cell therapeutic efficacy against solid tumors in an immunocompetent murine model
Authors: Reed Steven James , Tower Samantha J , Dunga Meenakshi , Toumi Ryma , Kalia Vandana , Sarkar Surojit
Published: January 2023

T cell exhaustion in the tumor microenvironment (TME) has hindered the success of T cell-based immunotherapy against multiple types of solid tumors. To combat this exhaustion, we have engineered a CD19-targeted fourth generation CAR construct to express the pro-memory transcription factor Inhibitor of DNA Binding 3 (Id3). Id3 critical for the formation of long-lived memory T cells in viral infection models, and retrovirally-induced expression of Id3 in T cells has been shown to enhance the survival of effector T cells in both acute infection and tumor models.Using an immunocompetent murine model of syngeneic solid tumors expressing truncated human-CD19 (hCD19t), we demonstrate that ectopic expression of Id3 via 4th generation CARs containing Id3 (Id3-CAR) augments CAR T cell therapeutic efficacy against solid tumors. Amongst cells recovered from established solid tumors, Id3-CAR T cells retained a memory-precursor effector cell (MPEC) like phenotype including elevated expression of TCF-1, while the 3rd generation CAR T cells largely displayed a terminally exhausted (TEx) phenotype. Importantly, the inclusion of Id3 did not compromise CAR T cell proliferation or expression of the effector cytokines IFN-γ and TNFα following restimulation. In addition to superior control of primary tumors, the Id3-CAR T cells instilled greater long-term protection from tumor relapse in mice, which cleared primary tumors (6/7) compared to 3rd generation CAR T cells (3/7). Highlighting Id3 as a candidate, these data present incorporation of pro-memory transcription factors as an attractive engineering strategy to intrinsically combat CAR T cell exhaustion in the TME and promote long-term CAR T cell memory and protection from tumor relapse.

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number F32CA265056, and Pediatric Cancer Research Foundation.
The effect of alloferon on chemosensitivity of pancreatic cancer through the regulation of SLC6A14 expression
Authors: Jo HyeJung , Lee DaHae , Go Cheolhyeon , Kim Yejin , Kang Jae Seung
Published: January 2023

Pancreatic cancer is one of the most malignant solid tumors with a high mortality rate. Although there have been many trials for effective therapies using chemotherapeutic drugs, such as gemcitabine, pancreatic cancer still shows significantly higher mortality than other types of cancer. Therefore, it is needed to examine the effective therapeutic ways to improve conventional therapy for pancreatic cancer. It has been previously reported that cancer cells possess a high rate of glutamine uptake needed for their rapid proliferation. In addition, it has recently been reported that amino acid transporter SLC6A14 is up-regulated in some cancers in close relation to glutamine metabolism. Alloferon is a peptide isolated from the insect immune system and shows anti-viral and anti-inflammatory effects based on its immune modulating function. In addition, its anti-tumor effect was recently discovered, but the related mechanisms are largely unknown. For this reason, I investigated the effect of alloferon on pancreatic cancer cell lines Panc-1 and AsPC-1. When Panc-1 and AsPC-1 were exposed to alloferon for 3 weeks, I found down-regulation of SLC6A14 expression and decreased glutamine uptake. Given that SLC6A14 is involved in tumor progression and survival by promoting glutamine uptake into cancer cells, alloferon could be an effective way with conventional chemotherapeutic drug, gemcitabine. Therefore, alloferon could be a useful adjuvant for an effective therapeutic way with conventional chemotherapeutic drug, gemcitabine.

This study was funded by the NRF of Korea (No.:2017R1A2B2010948, 2017R1A6A3A11032576, 2020R1C1C1009334)
Coupling the immunomodulatory properties of the HDAC6 inhibitor ACY241 with Oxaliplatin promotes robust anti-tumor response in Non-small cell lung cancer
Authors: Adeegbe Dennis O , Bag Arup , Schultz Andrew , Bhimani Saloni , Dominguez William , Cen Ling
Published: January 2023

Although immunotherapy provides durable therapeutic response in a subset of Non-small cell lung cancer (NSCLC) patients, there is still a need for broadly applicable therapeutic strategies. HDAC inhibitors are a promising class of drugs whose immunomodulatory properties are now being appreciated. In the present study, we evaluated the immunomodulatory properties of the HDAC6 inhibitor, Citarinostat (ACY241) on lung tumor immune compartment and its therapeutic potential in combination with Oxaliplatin. Lung adenocarcinoma-bearing mice were treated with ACY241 or vehicle after which immune profiling and RNA-sequencing were conducted in tumor-associated T cells and macrophages. Ex vivo T cell functional studies, tumor growth assessment and survival of tumor-bearing mice treated with ACY241 and/or Oxaliplatin were also conducted. ACY241 treatment promoted increased presence of T and NK cells as well as activation, proliferation, and effector profile of T cells in the lung tumors of treated mice. Furthermore, tumor-associated macrophages exhibited increased expression of MHC and co-stimulatory molecules while expression of inhibitory ligands were reduced. RNA-sequencing of tumor-associated T cells and macrophages revealed significant genomic changes that is consistent with ACY241-mediated enhancement of immune priming. Finally, ACY241 treatment led to significantly enhanced tumor-associated T cell effector functionality in lung cancer-bearing mice and in patient-derived tumors when combined with the chemotherapy drug Oxaliplatin. Collectively, our studies support ACY241 as a promising HDAC6 inhibitor which coupled with Oxaliplatin promotes robust therapeutic outcomes in a pre-clinical model of NSCLC.
High-dimensional single-cell analysis of the immune response in multiple myeloma and profiling of the T cell repertoire in response to immunomodulatory treatment
Authors: Prazich Jack
Published: January 2023

Multiple myeloma is a largely incurable malignancy of plasma cells in the bone marrow. The standard of care for this disease has increasingly included the use of immunomodulatory drugs such as thalidomide and its derivatives. However, high-dimensional analysis of the immune response in multiple myeloma tumors and the resultant response to immunomodulatory treatment is lacking. Here, we present a single-cell multi-omic analysis of the immune response in refractory multiple myeloma patient tumors including profiling of CD8+ T cell antigen specificities. We also analyze changes in the T cell repertoire and gene expression following treatment of pomalidomide. We observe a contraction of the repertoire that includes the expansion of select clones within the repertoire. These results provide a clearer map of the immune response in multiple myeloma and how patients respond to immunomodulatory treatment.

Supported by grants from CRI Lloyd J. Old STAR Program and NIH R33 IMAT 2018
Evaluation of tumor derived Serpina3n in experimental glioblastoma
Authors: Ayasoufi Katayoun , Wolf Delaney , Zheng Jiaying , Tritz Zachariah Peter , Jin Fang , Gulbicki Lauren , Hansen Michael , Johnson Aaron J
Published: January 2023

Glioblastoma (GBM) is an incurable brain cancer which is associated with severe peripheral immune suppression. Using experimental GBM, we recently determined that peripheral immunosuppression involves thymic atrophy, stunted T cell proliferation, and release of circulating factors that inhibit immune responses. RNA sequencing and proteomic studies have recently determined that transcripts and protein levels of Serpina3n are upregulated in the brain and serum of GBM patients and correlates with poor prognosis. We determined Serpina3n is a highly upregulated gene in the brain and the thymus of glioma-bearing mice using RNA sequencing technologies. We therefore sought to determine the role of Serpina3n in peripheral immunosuppression in GL261 glioma harboring wild type and Serpina3n knockout mice. Glioma implantation in Serpina3n knockout mice resulted in similar survival and peripheral immunosuppression compared to controls. Western blotting experiments confirmed that Serpina3n is expressed and secreted by GL261 gliomas isolated from WT and Serpina3n knockout mouse brains. Together this data suggests that tumor-derived, but not host derived, Serpina3n needs to be evaluated as a putative factor associated with peripheral immunosuppression.

Supported by Brains Together for a Cure grant
Hydrogen sulfide signaling in promoting the anti-tumor T cell response
Authors: Oberholtzer Nathaniel , Mehrotra Shikhar
Published: January 2023

Adoptive cell therapy (ACT), a form of immunotherapy, continues to emerge as a novel therapeutic strategy for treating advanced malignancies. New approaches to improve ACT protocols are needed to enhance in vivo persistence of adoptively transferred tumor antigen-specific T cells and overcome tumor-induced immunosuppression and oxidative stress, which remain significant barriers to the clinical success of ACT. Here, we show that exogenous hydrogen sulfide (H2S), an important gaseous signaling molecule, promoted a central memory phenotype with enhanced antioxidant capacity in tumor antigen-specific T cells. In vitro, T cells treated with exogenous H2S upregulated key antioxidant enzymes and molecules associated with stemness while simultaneously downregulating immune checkpoint molecules. H2S-treated T cells also displayed an enhanced reactivity to tumor antigen, with an increase in production of pro-inflammatory cytokines as well as an increase in overall protein translation. H2S-treated T cells also displayed an enhanced ability to combat oxidative stress, with a reduced accumulation of reactive oxygen species and protection against hydrogen peroxide-induced cell death. In an in vivo murine model of melanoma, tumor-reactive T cells conditioned ex vivo with exogenous H2S demonstrated superior tumor control upon ACT. Even greater tumor control was observed when ACT-treated mice were injected systemically with an H2S donor compound. These data provide evidence that anti-tumor T cells exposed to H2S possess an enhanced metabolic and stem-like phenotype that allows them to persist in the immunosuppressive tumor microenvironment and exert greater tumor control upon adoptive transfer into tumor-bearing hosts.

Supported by NIH R01 CA236379
Targeting LSD1 rescues MHC class I antigen presentation and promotes immune checkpoint blockade response in small cell lung cancer
Authors: Nguyen Evelyn M , Taniguchi Hirokazu , Chow Andrew , Sen Triparna , Rudin Charles M
Published: January 2023

Purpose

Small cell lung cancer (SCLC) is an aggressive tumor with modest clinical response to immune checkpoint blockade (ICB). Repression of major histocompatibility class I (MHC-I) molecules represents a key mechanism driving ICB resistance in SCLC. Recent studies have kindled interests in the lysine-specific demethylase 1 (LSD1) as an immunomodulatory target. Our study investigated the potential functions of LSD1 in regulating MHC-I antigen presentation in SCLC.

Method

We employed the inhibitor ORY-1001 and RNA interference to assess changes in MHC-I expression in SCLC cell lines by flow cytometry. We then performed RNA-seq to characterize whole transcriptomic changes in SCLC cells following LSD1 inhibition. To explore effects of targeting LSD1 on T cell cytolysis, we co-cultured SCLCs engineered to present endogenous peptides with pre-activated cognate CD8+ T cells. Finally, we treated immunocompetent mice bearing syngeneic SCLC tumors with ORY-1001 and/or anti-PD-L1 to evaluate tumor growth and characterize intratumor immune activities.

Result

We first demonstrated that targeting LSD1 restores MHC-I cell surface expression, transcriptionally activates APP-regulatory genes, and activates tumor intrinsic immunity. We further show that LSD1 inhibition of SCLC cells enables for efficient MHC-I-restricted T cell-mediated cytolysis. Finally, the addition of LSD1 inhibitor to anti-PD-L1 therapy significantly augments intratumor CD8+ T cell infiltrates and sensitizes refractory SCLC model to ICB response.

Conclusion

Our data define a potent regulatory role of LSD1 in MHC-I antigen presentation and provide support for targeting LSD1 to overcome immune evasion in SCLC.

Supported by NIH NCI R01 CA197936 (CMR); U24 CA213274 (CMR); the Druckenmiller Center for Lung Cancer Research (CMR, TS); NIH/NCI R01 CA258784 (TS); Congressionally Directed Medical Research Programs (DOD-IITRA) LC190161 (TS); Parker Institute for Cancer Immunotherapy grant (TS); LCFA-BMS/ILC Foundation Young Investigator Research Awards in Translational Immuno-oncology (TS); the Geoffrey Beene Foundation (EMN); The Yasuda Medical Foundation (HT); NIH K08 CA-248723 (AC); and the Van Andel Institute – Stand Up to Cancer Epigenetics Dream Team grant (CMR). Stand Up to Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. We also acknowledge the use of the MSKCC Flow Cytometry Core Facility, the MSKCC Antitumor Assessment Core, and the Integrated Genomics Core, which are funded by MSKCC Support Grant/Core Grant (P30 CA008748).
Dietary low-fiber promotes resistance to immune checkpoint inhibitor immunotherapy in the LSL-KrasG12D Lung Cancer Model
Authors: Li Qingsheng , Goggin Kevin Edward , Seo Jamie , Egilmez Nejat
Published: January 2023

Dietary fiber interaction with the gut microbiota impact gut microbiota composition and is likely to play a large role in affecting the efficacy of immune checkpoint inhibitors (ICIs) therapy. Here we show that a diet with low-fiber promoted lung tumor development and reduced tumor-bearing animal long-term survival in both prophylactic early disease and established late disease of murine LSL-KrasG12D lung cancer model, which displays low neoantigen burden and is refractory to anti-PD-1 antibody. In this model, the administration of anti-PD-1 in the presence of IL-17 neutralization resulted in tumor suppression, indicating that limited intrinsic neoantigen alone was not the primary determinant of resistance to ICIs therapy. Interestingly, dietary low-fiber led to reduced anti-tumor effect of anti-PD-1 and anti-IL-17 partially in gut microbiota-dependent manner. Fecal microbiota transplantation from mice fed low-fiber diet also enhanced lung tumor burden. An altered immune response with significantly reduced gut lamina propria RORgt+IL-17A+ Th17 cells, lung Foxp3+Treg cells, enhanced lung Th17 cells, but no alteration of lung gdTCR+ cells were found in mice fed low-fiber diet. Furthermore, a low-fiber diet led to significant changes in metabolites including a high level of serine in the plasma of animals. Mice fed normal chow with drinking water containing serine increased lung tumorigenesis. Our study demonstrates a functional interconnection between dietary fiber and spontaneous lung tumor development.

Supported by grant from NIH/NIGMS(P20-GM135004)