bluuebunny/crossref_metadata_2025_split huggingface dataset:
Trehalose-based lipid improves mRNA vaccine safety
Authors: Bae Seo-Hyeon , Bang Eun-Kyoung , Lim Byung Kwan , Lee Jisun , Yoo Soyeon , Park Hyo-Jung , Park Sang-In , Lee Yu-Sun , Roh Gahyun , Lee Seonghyun , Youn Sue Bean , Keum Gyochang , Nam Jae-Hwan 
Published: August 2023

Abstract
The rapid development of lipid nanoparticle (LNP)-formulated mRNA vaccines strongly contributed to overcoming the COVID-19 pandemic. However, in parallel with this success, the toxicity of ionizable cationic lipids has emerged as a potential issue in clinical safety because it might cause mRNA vaccine side effects, including myocarditis, anaphylaxis, and pericarditis. In this study, to reduce the risk posed by ionizable cationic lipids, we employed LNPs containing trehalose-based lipid (LNP-126), which increased the stability of the LNP-mRNA molecules. Trehalose has been shown to improve heart function and increase mRNA stability through hydrogen bond interactions. The LNP-126 was formulated with 50% fewer ionizable cationic lipids than a commercial LNP (LNP-128). Furthermore, the mRNA encoding the reporter gene was encapsulated by LNP-126 so that gene expression (Renilla luciferase) and organ toxicity could be examined. We also assessed immune efficiency immunizing mice that had been treated with an LNP-126-formulated mRNA vaccine expressing influenza hemagglutinin (HA). The results showed that LNP-126 reduced organ toxicity in the liver and heart compared to LNP-128, while maintaining gene expression and immune efficiency in vivo. In conclusion, the LNP-126, trehalose-based lipid will facilitate the development of safer mRNA vaccines beyond the current barriers.
A grant (22213MFDS421) from the Ministry of Food and Drug Safety in 2022 supported this research
Selection for disease resistance in Oncorhynchus mykiss increases the myeloid to lymphoid balance and is affected by rapamycin as measured by flow cytometry and scRNAseq
Authors: Reid Ross M , Osbourn Keira , Leeds Timothy , Wiens Gregory D 
Published: August 2023

Abstract
Heritable variation in disease resistance has been described in many species, but the effects of selection on innate immunity are unclear. Here, we compare the cellular response of two lines of rainbow trout that differ in survival due to five generations of selection for resistance to Flavobacterium psychrophilum (Fp), an important fish pathogen. We examined bacterial load, plasma biomarkers, whole blood cellular composition, and blood chemistry at days 1 and 5 after bacterial or PBS injection. We also examined the effect of 0.8 mg/kg rapamycin pretreatment, which results in immunosuppression and a ~100-fold reduction in the challenge LD50. On day 1, bacterial load did not differ between genetic lines and treatment, but by day 5, the R-line exhibited no increase in Fp load, while rapamycin increased R- and S-line loads 33- and 506-fold, respectively. Biomarkers: cathelicidin 2 and C1q-LP3 significantly differed between genetic lines and by time, corresponding to Fp load. Analysis of whole blood by flow cytometry identified a response to infection and an ~8 percentage point higher frequency of myeloid-gated cells in R-line fish that was consistent in both naïve and challenged animals. S-line fish exhibited a higher percentage of IgM+ B cells. Single-cell RNAseq was conducted on day 1 purified blood and identified 23 cell clusters including multiple B cell, neutrophil and macrophage subpopulations. In naïve animals, S-line fish harbored a cxcr4 and mych expressing B cell subpopulation absent in R-line fish. Rapamycin pretreatment caused profound loss of multiple B cell subpopulations in R- and S-line fish. In summary, selection of rainbow trout for resistance to Fp is correlated with a cellular shift in peripheral blood and is mTOR dependent.
US Department of Agriculture, Agricultural Research Service; Project number 1930-32000-007
The role of bystander activation among CD8αα T cells in controlling Mycobacterium tuberculosis infection
Authors: Winter Cassandra R , Zhou Angela , Tappen Tori , Ou Henry , Seshadri Chetan 
Published: August 2023

Abstract
Classical cytotoxic T cells have a CD8 coreceptor with an αβ heterodimer and recognize peptide antigens via major histocompatibility (MHC) molecules. CD8 T cells with an αα homodimer structure are nonclassical and can recognize non-MHC molecules independently of antigen. CD8αα T cells have a higher propensity to undergo T cell receptor (TCR) independent bystander activation via pro-inflammatory cytokines. Bystander activation of CD8 T cells has shown a protective effect during Mycobacterium tuberculosis (M.tb) infection, but the distinct roles of CD8αα and CD8αβ T cells have not been elucidated. To begin assessing this, we compared the levels of CD8αα and CD8αβ T cell proliferation in response to TCR dependent stimulation using anti-CD3/CD28 beads. We observed that CD8αα T cells had lower levels of proliferation compared to CD8αβ T cells after three days of stimulation. After seven days, a population of CD8αα T cells did not proliferate while nearly all CD8αβ T cells showed high proliferative capacity. We next stimulated healthy peripheral blood mononuclear cells with IL-12, IL-15, and IL-18 to induce bystander activation and found 52.8% of CD8αα T cells secreted granzyme B compared to 25.1% of CD8αβ T cells. Interestingly, CD8αα T cells also had higher levels of CD69 expression than CD8αβ T cells (62.6% and 38.2%, respectively). Finally, we present plans for defining the role of bystander activated CD8αα and CD8αβ T cells during M.tb infection using tonsil organoids. These data reveal that CD8αα T cells have a distinct bimodal response to TCR mediated activation as well as an increased cytotoxic and activated phenotype after bystander activation. We further establish a foundation for future bystander activation studies in M.tb infection.
Supported by grants from the NIH (R01 AI146072)
What did the T cell see? A deep-learning model of CD8+ T cell activation reveals sharp antigen discrimination at the single cell level
Authors: Wahlsten Madison , Akhshi Amin , Achar Sooraj , Krishnan Anagha Yogam , François Paul , Altan-Bonnet Grégoire 
Published: August 2023

Abstract
Immunotherapies such as checkpoint blockade antibodies to block T cell exhaustion have been successful in several cancers such as non-small cell lung cancer and melanoma, but limited in others (e.g., pancreatic or prostate carcinomas) owing to differences in tumor antigenicity. Therefore, quantifying tumor antigenicity is critical for successful immunotherapies. Our lab has shown that antigenicity can be encoded in a single parameter derived from bulk cytokine dynamics in ex vivo co-cultures between antigen presenting cells (APCs) and T cells. Here we built a model that can capture the antigenicity seen by individual cells. Using a custom robotic platform, we generated high-throughput kinetics of T cell activation in co-culture with APCs by analyzing cells at various timepoints across a large set of activation conditions. We performed spectral flow cytometry to measure the expression of up to 30 surface markers and intracellular signals per cell. To analyze our content-rich datasets, we designed a machine learning-based model that can classify the antigen seen by an individual cell using expression values from flow cytometry. The model performs well not only at classifying T cells (ROC-AUC &gt; 0.91), but also APCs (ROC-AUC &gt; 0.88), suggesting that each individual leukocyte may register the quality of antigen being presented. Blocking cytokine signaling disrupted this antigen classification. Our study demonstrates that every individual lymphocyte can bridge local and global response to achieve high discriminatory power of antigens.
Phenotypic and transcriptional heterogeneity of memory CXCR5+ CD8+ T cells in human blood and lymphoid tissue
Authors: Rivera Olga , Buggert Marcus 
Published: August 2023

Abstract
CXCR5+ CD8+ T cells are involved in antiviral and -tumor immunosurveillance. Animal models have demonstrated that CXCR5+ CD8+ T cells have multiple roles, including cytotoxic, stem-like, and regulatory functions. Many of these functions might be disease-dependent, where human studies are lacking to understand if CXCR5+ CD8+ T cells show signs of significant heterogeneity. To this end, we performed simultaneous single-cell (sc) RNA−, TCR−, and CITE-seq on CXCR5+ and CXCR5− memory CD8+ T cells from matched tonsils and blood to unravel if CXCR5+ CD8+ T cells show potential differences between human lymphoid tissue and blood. Our analysis indicates that a high proportion of CXCR5+ CD8+ T cells in tonsils show tissue residency-like (TRM) features, including CD69 and CD103 expression. Instead, blood CXCR5+ CD8+ T cells showed increased signs of cytotoxic functions in blood compared to their counterparts in tonsils. GSEA analysis identified groups of genes differentially expressed, such as the p53 pathway enriched in tonsils compared to ribosome-related pathways in blood CXCR5+ memory CD8+ T cells. Additionally, with CITE-seq markers, we identified the protein landscape of sub-populations and found increased PD-1 and CD45RO expression in tonsils compared to blood CXCR5+ memory CD8+ T cells. Overall, these findings indicate that CXCR5+ CD8+ T cells are heterogeneous with potentially distinct functional subsets that patrol blood and lymphoid tissues.
PD-1 signaling serves a dual role in suppressing T cell activation but also in protecting from activation-induced cell death
Authors: Vick Logan V , Khuat Lam Tan , Le Catherine , Dunai Cordelia , Collins Craig , Canter Robert , Monjazeb Arta , Murphy William J 
Published: August 2023

Abstract
PD1/PDL1 interactions exert potent inhibitory effects on T cell responses. Inhibition of this pathway results in greater T cell responses and is increasingly used in cancer immunotherapy but the long-term effects of PD1 inhibition on T cell activation responses remain unclear. We hypothesized that these gains in proliferation and activation on T cells over time may result in greater T cell loss due to apoptosis via activation induced cell death (AICD). We investigated this using both mouse and human T cell models of activation. T cells from wild-type or PD1−/− mice were activated in vitro by a variety of stimuli. We observed significantly increased total T cell numbers, as well as expression of Ki67 and CD69 in the PD-1−/− T cells compared to controls. However, we identified that this increase was accompanied by increased cell death and apoptosis at later time points. Using in vivo adoptive transfer models (allogeneic & xenogeneic) in which PD-1−/− murine cells, or human T cells given in tandem with PD-1 checkpoint blockade we assessed PD-1 inhibition in vivo on T cell activation, proliferation, and apoptosis. We observed early expansion and activation of murine PD-1−/− cells in the allogeneic environment compared to controls. Furthermore, human T cells transplanted into immunodeficient mice given anti-PD1 also had greater activation and proliferation. Yet, this was followed by a sharper contraction and increased apoptosis identified through decreased cell numbers and increased Annexin V staining. These data suggest PD-1 regulates T cell activation as a means of protection, but when this balance is manipulated, T cells experience a “short term gain” for a “long term loss” in which cells undergo greater early activation at the cost of AICD.
Supported by National Institute of Health grant numbers NIH RO1 HL140921 (WJM), National Institute of Health grant numbers NIH RO1 CA214048 (WJM).
Asymmetrical Expression of PD1 and CD25 in T-cells Post-Initial Activation
Authors: Sheng Michael K , Vick Logan , Collins Craig , Yoon Daniel J , Murphy William J 
Published: August 2023

Abstract
After TCR engagement, T cells induce expression of CD25, which forms the IL2 high affinity receptor complex, concurrently with PD1, which plays a down-regulatory role in activation. Cessation of TCR stimulation leads to eventual contraction of T cell responses. Chimeric antigen receptor (CAR) T cell therapy has seen increased use in the past decade. CAR T-cells are activated and transduced ex vivo and expanded with low amounts of cytokines prior to administration. The expression of CD25 and PD1 on CAR T cells at different stages of the process has been incompletely characterized. Purified T cells were activated in vitro for 3 days followed by expansion using low dose IL2. We demonstrate that both CD25 and PD-1 are strongly upregulated during initial activation, but by day 8, under expansion conditions, PD1 is downregulated to negligible levels. Despite absence of TCR signaling, CD25 expression remains moderate-to-high explaining the ability to maintain proliferation. This discrepancy was higher in transduced CAR T-cells compared to activated non-transduced (NT) T cells indicating the possibility of CAR tonic signaling. Furthermore, restimulation of both NT and CAR-T cells after this expansion phase resulted in further increased CD25 expression whereas PD-1 expression remained low. Our studies suggest that during initial activation, TCR engagement is necessary to maintain PD-1, but not CD25 expression. However, upon restimulation, TCR engagement is capable of further upregulating CD25, but not PD-1 expression. These results indicate that TCR signaling induces asymmetrical expression of PD-1 and CD25, a dynamic interplay that may regulate T-cell function and memory response and that PD1 may not be a limiting factor in the process.
Metabolic changes induced by T cell-mediated cutaneous vaccinia virus clearance
Authors: Pei Luxin , Jarmusch Alan K , Hickman Heather D 
Published: August 2023

Abstract
Viral infections induce prominent alterations to the host metabolism. Vaccinia virus (VACV) has been used as a live vaccine preventing smallpox and monkeypox. However, much remains unknown about the metabolic regulations of the adaptive immune response mounted to VACV infection in the skin.
To survey the metabolic changes during VACV clearance, we performed a longitudinal metabolomics study based on our cutaneous VACV infection murine model. We adoptively transferred OT-I CD8+ T cells into Rag−/− mice, then infected one ear with VACV expressing SIINFEKL and VACV lacking SIINFEKL in the contralateral ear. Ears were harvested on 0,5,6,7,8,10 days post infection (dpi), which were homogenized and methanol extracted prior to untargeted mass spectrometry analyses.
Metabolomics profiling revealed that VACV infection induced significant metabolic changes over time in the ear tissue. Interestingly, the metabolic profile after viral clearance was still distinct from uninfected control group. The levels of phenylalanine (adj. p=0.032), oleic acid (adj. p=0.011), S-lactoylglutathione (adj. p=0.022), and hypoxanthine (adj. p=0.039) were significantly altered between samples undergoing T cells-mediated killing and those without. Furthermore, glutamic acid level was significantly different (adj. p=0.036) between groups with and without T cells, suggesting T cell utilization during VACV clearance.
Overall, our results indicated that VACV infection caused significant and persistent metabolic perturbation in the skin tissue environment. Antiviral T cells that mediate VACV clearance may require specific metabolites or metabolic pathways to regulate cytotoxic functions.
Quantitation of Immune Synapse Duration Supports the Contribution of Perforin Haploinsufficiency to Hyperinflammation
Authors: Frank-Kamenetskii Anastasia M , Klinghoffer Hannah , Dang Vinh , Varghese Jemy , Canna Scott 
Published: August 2023

Abstract
Hyperinflammation is an acute state that occurs in a variety of infectious, inflammatory, and genetic contexts. Familial Hemophagocytic Lymphohistiocytosis (FHL) is a rare Inborn Error of Immunity resulting from biallelic disruption of perforin or other genes necessary for granule-mediated cytotoxicity. Some reports suggest that heterozygous defects in FHL genes contribute to hyperinflammation in concert with infection or rheumatic diseases. Most known causes of hyperinflammation arise from abnormal regulation of the Immune Synapse (IS) between cytotoxic CD8 T-cells and target cells. Using live imaging, we sought to assess the effects of Perforin and other putative contributors to hyperinflammation on IS duration and magnitude.
We evaluated the IS between CD8 T cells from mice with different T-cell receptor signal strengths (OT1 vs P14) and various target cell types, as well as that between Prf1+/+, +/−, and −/− P14 cells and BMDCs. IS duration was calculated based on time between killer cell calcium flux and target cell Propidium Iodide uptake, and cytokine ELISA of supernatants was performed.
We found that OT1, but not P14, CD8 T-cells could kill MC57 fibrosarcoma cells, but both were able to kill BMDCs. Moreover, both complete Prf1 deficiency and Prf1 haploinsufficiency prolonged the IS and increased cytokine production. Excess IL18, a hyperinflammatory susceptibility factor that synergizes with Prf1 deficiency in vivo to drive spontaneous hyperinflammation, shortened IS duration but dramatically increased IFN-γ production. Thus, showing that our in vitro IS quantitation model is sensitive to small changes that may be important contributors to pathogenic T-cell activation and life-threating hyperinflammation.
Supported by grants from NIH (R01 HD098428-03-04), Children’s Hospital of Philadelphia (IDF FY21-FY24), and from Histiocytosis Association (FP00039723_A1)
A commensal-derived exopolysaccharide regulates ILC3-mediated diseases
Authors: Tan Chin Yee , Theriot Barbara S , Rao Meghana V , Surana Neeraj K 
Published: August 2023

Abstract
The gut microbiota plays a fundamental role in controlling many facets of human physiology, with impacts on the immune system being chief among them. Although there have been increasing successes in identifying disease-regulating commensal bacteria, the mechanisms by which they work remain poorly understood. We previously identified Clostridium immunis, a new human-derived commensal bacterial species that modulates colitis. Here we identify the immunological factors and specific bacterial determinant required for its disease modulating activity. We find that C. immunis directly regulates the effector function of group 3 innate lymphoid cells (ILC3s), with altered levels of IL-22 and GM-CSF in states of health and inflammation, respectively. This impact on ILC3s serves as a common immunological node for modulation of visceral adiposity and intestinal inflammation. Surprisingly, the two bacterial species most closely related to C. immunis lack this ILC3-modulating activity. Guided by comparative genomics, we purified an exopolysaccharide (EPS) that recapitulates the immunomodulatory activity of C. immunis in vitro and in vivo. Structural analysis revealed that phosphocholine on the EPS correlated with activity, and deletion of the C. immunis phosphocholine biosynthesis locus (LicABC) resulted in functionally inactive EPS. Our results reveal that a phosphocholine moiety present on a bacterial polysaccharide is critically required to orchestrate ILC3 effector function and regulate intestinal homeostasis. Considered together, we provide genetic and structural insight into how a human commensal bacterium provides a unique immunomodulatory function.
Supported by a predoctoral fellowship from the American Heart Association (897275), a postgraduate scholarship from the Tan Kah Kee Foundation, and a Development Grant from the National University of Singapore (to C.Y.T) Supported by grants from NIH (NIH R03 AI142341), The Hartwell Foundation, and as a Translating Duke Health Scholar and as a Whitehead Scholar (to N.K.S.).
Gut microbiome-derived isodeoxycholic acid promotes mucosal immune non-responsiveness
Authors: Ionescu Edward , Arnold Jack , Mimee Mark , Nagler Cathryn R 
Published: August 2023

Abstract
Perturbations to the commensal gut microbiota are associated with the increasing prevalence of food allergies. Our lab has reported that colonization of germ-free mice with feces from healthy, but not cow’s milk allergic, infants protects against anaphylaxis to a cow’s milk allergen. Protection was associated with higher levels of the secondary bile acid (SBA) isodeoxycholic acid (isoDCA) in the feces of healthy-colonized mice. IsoDCA has been reported to induce regulatory T cells (Tregs) which suppress inflammation. We hypothesize that isoDCA-driven induction of mucosal Tregs promotes non-responsiveness and protects against food allergy. To study this, we identified two Clostridia species, Peptacetobacter hiranonis and Ruminococcus gnavus, that together produce isoDCA from taurocholic acid (TCA), an abundant host bile acid. Using a Group II intron-based system, we engineered R. gnavus Δrumgna_00694, a functional knock-out of a 3β-hydroxysteroid dehydrogenase required to produce isoDCA. In our system, P. hiranonis converts TCA to deoxycholic acid (DCA) and R. gnavus WT, but not Δrumgna_00694, converts DCA to isoDCA. Ongoing experiments will compare isoDCA-dependent Treg induction in gut-associated lymphoid tissues of mice co-colonized with P. hiranonis and R. gnavus WT or Δrumgna_00694. Once optimized, this system can be used in our allergic sensitization model to investigate the role of SBA in protection against food allergy and maintenance of intestinal homeostasis.
Commensal-specific CD4 T cells can promote inflammation in the central nervous system via molecular mimicry
Authors: Sano Teruyuki , White Zachary , Cabrera Ivan 
Published: August 2023

Abstract
Commensal bacteria are critical regulators of both tissue homeostasis and the development and exacerbation of autoimmunity. However, it remains unclear how the intestinal microbiota contributes to inflammation in tissues such as the central nervous system (CNS) where these microbes are typically absent and whether T cell receptor (TCR) specificity for commensal-derived antigens is important to the development of tissue inflammation-related outcomes. Here, we found that ileum- and cecum-colonizing segmented filamentous bacteria (SFB)-specific T cells (clone TCR7B8) can infiltrate the CNS wherein they can be reactivated and produce high levels of inflammatory cytokines including IFNγ, IL-17A, TNFα, and GM-CSF in the absence of regulatory T cells. In contrast, other SFB-specific T cells (clone TCR1A2) recognizing an epitope in which 8/9 amino acids overlap with those recognized by TCR7B8 failed to induce such neuroinflammation. Despite their similar SFB-derived peptide antigen targets, TCR7B8 was found to recognize peptides derived from host proteins including receptor tyrosine-protein kinase ErbB2, trophinin 1, and anaphase-promoting complex subunit 2 in vitro, whereas TCR1A2 did not, indicating that TCR7B8 induces CNS inflammation via molecular mimicry. Immune checkpoint blockade accelerated TCR7B8-mediated CNS inflammation. Together, our findings reveal a potential mechanism whereby gut commensal-specific T cells are dysregulated, infiltrate into the CNS, and contribute to extraintestinal inflammation.
Supported by grants from UIC start-up funds and UIC COVID Relief Funding.