bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multimodal single-cell analysis of human TILs across multiple tumor types reveals heterogeneity and potential opportunities for personalized immunotherapy
Authors: Redmond William L , Koguchi Yoshinobu , Miller William L , Christie Tanisha , Kaufmann Johanna , Seestaller-Wehr Laura , Yanamandra Niranjan , Griffin Sue , Smothers James 
Published: January 2023

Abstract
Immune checkpoint blockade (ICB) efficacy varies among tumor types likely due to differences in tumor infiltrating lymphocyte (TIL) composition and function within the tumor microenvironment (TME). To help understand these differences, we conducted multimodal single-cell analysis of TILs including single-cell RNA sequencing (scRNA-seq), CITE-seq (oligo-tagged antibodies), and scTCR-seq (10× Genomics) in (non-small cell lung cancer: NSCLC; head and neck squamous cell carcinoma: HNSCC; renal cell carcinoma: RCC; and breast cancer: BrCa; n=48). We found that regulatory T cell (Treg) frequency was higher in HNSCC, whereas exhausted T cells (Tex) were higher in NSCLC and RCC. In contrast to other tumor types, Tex in RCC lacked the expression of CD103, a hallmark of tissue-resident T cells. On the other hand, expression of PD-1, TIM-3, and LAG-3 were more prominent in Tex in RCC. Interestingly, Tex in HNSCC showed higher expression of TIGIT than other tumor types. Previous work has demonstrated an increased presence of CD4+CD8+ double-positive T cells (DPT) in RCC, which was associated with better overall survival. Therefore, we used CITE-seq to identify DPT and then compared the composition of DPT among different tumor types. DPT were CD39+, a marker for tumor-reactive T cells, and the vast majority were transcriptionally categorized as CD8+ T cells in RCC, whereas DPT in other tumor types are mixture of CD4+ or CD8+ T cell subsets. We also found overlap of TCR profiles between DPT and CD8+ T cell subsets (Tex, ZNF683-CD8, and GZMK-CD8) in RCC. Together, multimodal single-cell analysis of TILs highlighted heterogeneity among tumor types that may provide insight into novel strategies to treat cancer.
Supported by a research grant from GlaxoSmithKline and the Providence Portland Medical Foundation.
Supported by a research grant from GlaxoSmithKline and the Providence Portland Medical Foundation.
Precise spatial multiplexing of immune cell diversity in clinical FFPE tumor samples with ChipCytometry™
Authors: Schwarz Spencer , Christians Arne , Brooks Jennifer , Northcutt Adam , Kwarta Karen , Winkeler Crystal 
Published: January 2023

Abstract
Highly multiplexed spatial biomarker analysis has demonstrated the potential to advance our current understanding of the immune system and its role in cancer – from tumor initiation to metastatic progression. Previously, a trade-off between plex and spatial context meant that our understanding of immune cell involvement in cancer was limited by either single-plex technologies with spatial context (i.e., immunohistochemistry) or highly multiplex technologies without spatial context (i.e., single-cell RNA sequencing). ChipCytometryTM is a novel, highly multiplexed technology that preserves both plex and spatial context to deeply profile immune cell diversity at single-cell resolution. ChipCytometry uses commercially available antibodies and combines iterative immuno-fluorescent staining with high-dynamic range imaging to profile dozens of protein biomarkers in a single tissue specimen. Cellular phenotypes are identified with via flow cytometry-like hierarchical gating from standard multichannel OME-TIFF images, compatible with a variety of computational tools being developed for multiplexed analysis and visualization. Here, we use ChipCytometry to identify and quantify key immune cell subtypes in both normal (i.e., appendix) and tumor tissues (i.e., melanoma and non-small cell lung cancer) FFPE samples. The results show precise expression levels for each biomarker in the assay in each individual cell in the sample, while maintaining spatial positioning of each cell. Spatial analysis reveals quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for the in-depth immune profiling in clinical FFPE samples.
Defining the cooperation between T cell responses to MHC-I and MHC-II melanoma neoantigens towards developing effective personalized cancer immunotherapies.
Authors: Williams Charmelle , Shavkunov Alexander , Keshari Sunita , Salmon Avery , Highsmith Anna , Pineda Josue , Gubin Matthew 
Published: January 2023

Abstract
Immune checkpoint therapy (ICT) (e.g. anti-CTLA-4, anti-PD-1) enables durable T-cell dependent anti-tumor immunity in patients with solid tumors. Since not all patients respond to ICT, this work aims at developing a more in-depth understanding of T-cell responses to MHC class I (MHC-I) and MHC class II (MHC-II) tumor antigens that occur as a consequence of aberrant expression of non-mutant antigens or driver and passenger mutations that form tumor neoantigens. We used a poorly immunogenic Brafv600e Pten−/− Cdkn2a−/− YUMM1.7 (Y1.7) murine melanoma line that with a paucity of endogenous neoantigens that is unresponsive to ICT, and introduced model neoantigens previously identified in a murine sarcoma tumor line expressing the same MHC haplotype as the Y1.7 line. The MHC-I neoantigen used was formed by a G1254V point mutation in the Laminin alpha subunit-4 (Lama4) and the MHC-II neoantigen was formed by an A710T point mutation in Integrin beta-1 (Itgb1). Anti-CTLA-4-treated mice bearing the parental Y1.7 melanoma displayed progressive tumor growth, whereas the modified Y1.7 line expressing mLama4 and mItgb1 (Y1.7.mLama4.mItgb1) was rendered sensitive to ICT. Preliminary data showed that anti-CTLA-4 treated Y1.7.mLama4.mItgb1 tumors reject in mice in a CD4+ T cell and CD8+ T cell-dependent manner. Additionally, intratumoral mLama4-specific CD8+ T cells were detected in anti-CTLA-4-treated Y1.7.mLama4.mItgb1 melanoma bearing mice. Overall this model will allow us to better understand T cell mediated anti-tumor responses to both MHC-I and MHC-II neoantigens, as well as shared, non-mutant melanoma antigens (e.g. Pmel/GP100 and Trp2), which may facilitate development of improved neoantigen-based personalized immunotherapies.
Differential Immune Response to Klebsiella pneumoniae O-antigen subtypes O2v1 and O2v2
Authors: Wantuch Paeton L , Harding Christian M , Rosen David A 
Published: January 2023

Abstract
Klebsiella pneumoniae (Kp) infections are sharply on the rise among at-risk populations. We have previously shown that use of bioconjugate vaccines targeting Kp are beneficial in preventing disease and seek to increase our vaccine breadth of coverage. Kp has nine serogroups of O-antigens. Recently, additional O-antigen subtypes within these serogroups have been identified; yet, the pathogenic fitness, immunogenicity, functional antibody response, and cross-protection between subtypes is unknown. We investigated how the addition of the single branched galactose in O-antigen subtype O2v2 compared to O2v1 changes its bacterial fitness and host immune susceptibility. We knocked out the gmlABC region of an O2v2 strain of Kp, converting it to O2v1. Complementation of this mutant allowed us to identify the specific gene responsible for the addition of the single branched galactose of O2v2. Further, experiments using the O2v1 mutant and its parent O2v2 strain confirmed similar phenotypic expression of virulence factors beyond the O-antigen. Well-established murine models of pneumonia were used to determine the pulmonary fitness of the pathogens and assess the host innate immune responses. Neutrophil and complement-mediated killing assays suggested differences in susceptibility to innate immune defenses. Further, differences in IgG subclass were observed 4 weeks post respiratory infection with the O2v2 parent and O2v1 mutant strains. Lastly, using novel bioconjugate vaccines against these specific O-antigen subtypes, we determined only partial cross-protection is elicited. These studies advance our understanding of the immune response to Kp O-antigens and aid in vaccine design to combat this drug-resistant pathogen.
Supported by the W.M. Keck Postdoctoral Fellowship in Molecular Medicine Dr. Wantuch is a W.M. Keck Fellow
Effect of E-cigarette use on lung immunity to influenza infection
Authors: Cannon Judy L , Sciesczka David , Mrass Paulus , Garland Devon , Major Katharine , Hunter Russell , Begay Jessica , Lucas Selita , Campen Matthew J 
Published: January 2023

Abstract
Use of E-cigarettes, also called vaping, has been significantly increasing, with over 10 million adults and 3 million adolescents reported to actively vape. Of particular concern recently is the association of vaping with increased risk of respiratory infection, including influenza and COVID-19. Despite the claims of safety, lung immunological development appears to be impacted by vaping, with associated pulmonary toxicity and potential to impair immunity against respiratory infections.
Immunity against respiratory pathogens, particularly viral pathogens is dependent on a robust CD4 and CD8 T cell responses responsible for viral clearance. CD4 and CD8 T cells then develop memory responses to protect against reinfection. T cell memory also provides protection against infection after vaccination. We asked whether vaping exposure affects T cell responses to influenza infection. We exposed animals for 8 weeks to base components of vape devices, including propylene glycol (PG), vegetable glycerin (VG), and a novel cutting agent, phytol, then assessed immune responses to influenza. We find that PGVG and phytol exposure alters pulmonary function, promotes proteomic changes to the lung, and modify immune cell subsets in the lung. We also find that PGVG and phytol exposure alters T cell responses to influenza infection, particularly T cell memory protection against heterologous infection. These results suggest that vaping affects immune responses to respiratory influenza infection, including T cell memory responses. Effects of vaping on T cell memory may have wide ranging consequences for T cell mediated protection against other respiratory disease including SARS-CoV-2 and protection after vaccination.
Supported by NIH (P20 GM121176; P20GM130422)
Human lung tissue-resident memory CD8+ T cells are transcriptionally, epigenetically, and phenotypically diverse
Authors: Elliott Jennifer L , Kost Kirsten N , Lacetti Kristiana H , Thomas Jeronay K , Williams M Elliott , Mattingly Cameron L , Lobby Jenna L , Lawrence Laurel A , Kohlmeier Jacob E 
Published: January 2023

Abstract
Tissue-resident memory CD8+ T cells (TRM) are strategically located in peripheral tissues, especially at mucosal surfaces, where they can provide protection against invading pathogenic microbes. In the lungs, TRM play a critical role in limiting disease and transmission of respiratory viruses; however, the heterogeneity present in TRM populations in the human lung remains largely unexplored. Here we show that human lungs harbor transcriptionally, epigenetically, and phenotypically distinct populations of memory CD8+ T cells expressing the tissue residency-associated markers CD69 and CD103. High-dimensional flow cytometry and single-cell RNA sequencing of memory CD8+ T cells isolated from human lungs shows that heterogeneity exists both between and within CD69+ CD103− and CD69+ CD103+ subsets, with transcriptional diversity among the CD69+ CD103− subset being most prominent. Single-cell ATACseq demonstrates that similar levels of epigenetic diversity exist between and within these different TRM subsets. However, flow cytometry and single-cell RNAseq of influenza- and SARS-CoV-2-specific lung TRM demonstrates that heterogeneity between CD69+ CD103− and CD69+ CD103+ subsets is largely eliminated when the cells are specific for a common antigen. Together, these data illuminate underappreciated and unexplored aspects of heterogeneity in TRM populations in humans.
Supported by grants from NIH/NIAID (75N93019R00028) and NIH/NHLBI (R35 HL150803)
CD4 T cell deficiency results in impaired neutralizing IgG against polyomavirus carrying a frequent PML-associated capsid mutation
Authors: Ayers Katelyn Nicole , Lauver Matthew D , Lukacher Aron E 
Published: January 2023

Abstract
Deficiencies in the CD4 T cell compartment, such as HIV/AIDS, idiopathic CD4 lymphopenia, predispose patients to progressive multifocal leukoencephalopathy (PML), an oft-fatal demyelinating brain disease caused by the JC polyomavirus (JCPyV). PML is associated with immunomodulatory therapies that preferentially diminish CD4 T cells in the CSF (natalizumab) or B cells (rituximab). The connection between CD4 T cells and JCPyV control is unclear. Work by others show that PML patient sera have “blind spots” in neutralizing JCPyV variants with mutations in the VP1 capsid protein. We hypothesized that defects in the humoral response after CD4 T cell depletion led to the emergence of antibody (Ab)-escape PyV variants. Using mouse PyV (MuPyV) to model JCPyV CNS disease, we found immune cell aggregates consisting of B cells, CD4 T cells, and CD8 T cells along the ventricles of PyV-infected WT mice but only a few infected cells. In contrast, CD4 T cell-depleted mice had more infected ependyma but lacked lymphocytic aggregates. There were fewer anti-PyV Ab-secreting B cells in the CNS despite similar B cell numbers in MuPyV-infected WT and CD4 T cell deficient mice brains. CD4 T cells prevent emergence of VP1 mutant PyVs that escape neutralizing Abs. MuPyV infection elicits a CD4 T cell-independent anti-VP1 IgG response that neutralizes WT virus equivalently to IgG from CD4 T cell-sufficient mice. Serum from MHC-II KO and CD4 mAb depleted mice had significantly lower neutralization activity toward a MuPyV with a frequent PML-equivalent VP1 mutation than to WT MuPyV. This data demonstrate that a narrowed antiviral IgG response caused by CD4 T cell deficiency is a critical antecedent for outgrowth of Ab-escape PyV VP1 mutant viruses.
Supported by 1. Finkelstein Memorial Student Research Award, Pennsylvania State University College of Medicine, Hershey, PA 2. T32 CA060395 Training Grant (Meyers), NIH/NCI 3. R01 NS088367, NIH/NCI
IFN-γ promotes TH1 at the expense of TFH differentiation during viral infections
Authors: Sala Eleonora , Mangione Marta , Laura Chiara , Consolo Eleonora , Beccaria Cristian Gabriel , Iannacone Matteo , Kuka Mirela 
Published: January 2023

Abstract
Although humoral and cellular immunity upon viral infections usually co-exist, sometimes one of the two responses emerges and is responsible for most of the antiviral activity. For example, vescicular stomatitis virus (VSV) infection induces early and potent neutralizing antibody (nAb) responses, whereas lymphocytic choriomeningitis virus (LCMV) infection induces strong cellular responses, but weak nAb responses. Recent work from our laboratory has shown that unbalance is observed also at the level of CD4 T cells responses, with VSV inducing strong TFH polarization that support nAb responses, and LCMV in contrast promoting TH1 differentiation. Analysis of the VSV and LCMV priming niches led to identification of the spatiotemporal regulation of type I IFN expression as a critical regulator of antiviral CD4+ T cell polarization, with early type I IFN sensing leading to TFH polarization. Notably, this mechanism did not explain the strong TH1 differentiation observed during LCMV infection. In this study we aimed to elucidate the role of known TH1-polarizing cytokines in CD4+ T cell differentiation upon LCMV infection. We found that IFN-γ, but not IL-12, plays a key role in early CD4+ T cell differentiation upon LCMV infection, inducing TH1 cell polarization and suppressing TFH cell development, thus resulting in a shift in the equilibrium towards TFH and humoral responses. Future studies will determine the cellular source for this TH1-polarizing cytokine and the mechanism by which IFN-γ exerts its function, possibly unveiling the mechanisms underlying the reduced humoral response in the context of viral infections like LCMV.
Immunogenetic determinants of HSV-2 infection and disease
Authors: Graham Jessica B , Swarts Jessica , Mooney Michael , Lund Jennifer M 
Published: January 2023

Abstract
Herpes simplex virus-2 (HSV-2) is one of the most prevalent sexually transmitted infections, and can result in life-long, chronic disease. Disease severity, frequency of reactivation, and shedding rates vary between individuals, though little is known about how host genes regulate tissue-specific immune responses. We have previously used the Collaborative Cross (CC) mouse model system, which incorporates the extent of genetic variation found in the human genome, to better model the diversity of outcomes found in human viral infections, so we next probed the CC to identify host genetic regions that regulate viral shedding and disease following HSV-2 infection, as well as tissue-specific immune responses. We performed a screen of mice from different CC strains to assess viral titers and disease following vaginal HSV-2 infection, and then used this data to perform quantitative trait loci (QTL) mapping to identify chromosomal regions linked to vaginal viral shedding rates and levels, as well as virus-associated clinical disease. In parallel experiments, we assessed lymphoid, nervous system, and mucosal immune cell frequencies at innate, adaptive, and memory response timepoints. We observed a distinctive suppressive signature on vaginal Tregs compared to lymph node Tregs, at various times following HSV-2 infection. Additionally, these suppressive responses varied in mice with either higher viral titers, or more tissue inflammation, highlighting the interplay between host immune response and viral infection kinetics. Understanding host factors that contribute to HSV shedding, clinical disease, and immune responses may provide critical insights for developing new preventive strategies or interventions to HSV-2 infection.
Supported by grant R21 AI152559-01 from the NIH
EVALUATION OF Murine norovirus (MNV)-1 INFECTION IN PRIMARY MOUSE B CELLS.
Authors: Rodriguez Carlos Emilio Miguel , Lonches Clotilde Cancio , Escolano Ana Lorena Gutierrez , Argumedo Leopoldo Santos 
Published: January 2023

Abstract

Methods
Primary mouse spleen B cells were stimulated or not with LPS (10ug/ml) + IL-4 (10IU/ml) for 48 hours. Two million cells were infected 72 h with MNV-1 at a MOI of 5. The non-structural (NS7) viral protein expression was analyzed in cell lysates by Western blot with a specific anti-NS7 antibody. The supernatant-associated virus in Raw 264.7 cells was determined by paque assay.

Results
B cells can be infected by MNV-1, even though they do not express the functional receptor CD300LD and CD300LF present in macrophages and Tuft cells. Since MNV-1 can uses CD44 as a receptor in BMDCs, we wanted to investigate if this molecule has a role in MNV-1 entry into B cells. First, we corroborated MNV-1 infection of B cells by evaluating the presence of NS7 by western blot and viral particles production in the supernatant at 72 hpi. To evaluate the role of CD44 in MNV-1 entry into B cells, we blocked the CD44 receptor using two different anti-CD44 monoclonal antibodies, and we found a significant decrease in both the amount of NS7 protein and virus yield, indicating that CD44 plays a role during virus binding/entry into B cells. Since CD44 expression is increased in LPS + IL-4 activated B cells, we wanted to determine if MNV-1 infection could be favored in these conditions. A correlation between the activation of B cells for 48 h and a significant increase of twice of MNV-1 NS7 protein and virus yield production were detected after infection.

Conclusion
Blocking CD44 correlates with a reduction of MNV-1 protein synthesis and virus yield production, indicating a role of CD44 in MNV-1 binding/entry. The increase of MNV NS7 protein and virus yield production in C57BL/6J spleen B cells activated with LPS + IL-4 that overexpresses CD44 on its surface, also supports this conclusion.
CEMR is supported by a Conacyt fellowship, CVU fellowship number: 780860
The role of tumor necrosis factor receptor 2 in experimental autoimmune encephalomyelitis
Authors: Bartsch Brenden L , Negron Austin Joseph , Forsthuber Thomas 
Published: January 2023

Abstract
Multiple sclerosis (MS) is an autoimmune neuroinflammatory disease that affects over 2 million people globally. HLA-DR2b is a major histocompatibility complex (MHC) locus in humans responsible for antigen-specific immune responses. HLA-DR2b (DRB1*15:01) is a major genetic risk factor for MS. Experimental autoimmune encephalomyelitis (EAE) is the animal model for MS and can provide insights into the mechanisms behind MS pathogenicity. Tumor necrosis factor receptor 2 (TNFR2) was suspected to control EAE severity by altering regulatory T cell (Treg) function and impairing remyelination. In the absence of TNFR2, mice expressing the human DR2b allele and lacking mouse MHC II develop progressive EAE upon adoptive transfer of myelin oligodendrocyte glycoprotein (MOG)-reactive CD4 T cells. These mice show altered Treg phenotype, including decreased expression of Helios, a marker of thymic Tregs, by splenic Tregs from DR2bΔR2 mice compared with DR2b mice. However, it remains unclear how the presence or absence of TNFR2 alters the expression of specific protective genes in both astrocytes and Tregs in the context of EAE. Using the DR2bΔR2 and DR2b mouse models, we investigated the impact of TNFR2 on protective astrocyte subsets and regulatory T cells by single-cell RNA sequencing (scRNA-seq) in the context of both active and adoptive transfer EAE. Our results elucidate mechanisms underlying the protective function TNFR2 via modulating astrocyte and regulatory T cell function in EAE. Our results may aid in the development of new treatments for MS.
Supported by grant 1RO1NS117742 from the NIH/NINDS (T.G.F.) and by NIH/NIGMS RISE grant GM60655 (BB).
IL7Rα and TSLPR expression on Langerhans cells in response to inflammatory stimulation
Authors: Gonzalez-Rodriguez Martin Ignacio , Salomaa Tanja , Kummola Laura , Hiihtola Lotta , Junttila Ilkka 
Published: January 2023

Abstract
A key component of immune barriers are innate immune cells localized in the epithelium responsible for sensing local signals such as foreign antigens and cytokines. In the skin, specialized antigen presenting cells (APC) known as Langerhans cells (LC) can integrate these signals influencing downstream T cell activation and polarization. Thymic stromal lymphopoietin (TSLP) is a proinflammatory mediator released by keratinocytes that promotes LC to enhance type 2 immune response (Th2). When Th2 response becomes unbalanced, it induces type 2 inflammation as seen in atopic dermatitis. TSLP signaling is regulated by two cell surface receptor chains: TSLP receptor (TSLPR) and IL7Rα. Interestingly, IL7Rα is not commonly expressed in murine conventional APC such as dendritic cells (DC). Our previous findings have shown that IL7Rα expression regulates TSLP sensitivity of splenic DCs (sDC) ex vivo. However, the receptor expression dynamic in tissue resident LC and its biological implications has not yet been elucidated. In this study we enriched LC from healthy mice B6 mice using MACS magnetic beads. Cytokine surface receptors and pospho-STAT5 activation were assessed using flow cytometry. Moreover, topic administration of vitamin analog MC903 will be used to evaluate TSLPR and IL7Rα dynamic. Our current results indicate that IL7Rα is expressed on unstimulated LC contrary to sDC. Furthermore, TSLPR was not differently expressed between LC and sDC. Functional assessment of p-STAT5 ex vivo and the dynamic of these receptors in the context of MC903 treatment in vivo is yet to be measured. Our results suggest that tissue resident APC regulate differently cytokines receptors enabling spatial-temporal regulation of proinflammatory signals.
Academy of Finland (IJ: grants: 25013080481 and 25013142041), The Competitive State Research Financing of the Expert Responsibility Area of Fimlab Laboratories (grant X51409 to IJ), Tampere University Hospital Support Foundation (IJ) and Tampere Tuberculosis Foundation (IJ).