bluuebunny/crossref_metadata_2025 huggingface dataset:
Let’s talk about sex, baby: sex influences age-related changes in natural antibodies and natural antibody producing B-1a cells
Authors: Webster Sarah E , Ryali Brinda , Tsuji Naomi E , Clemente Michael J , Holodick Nichol E
Published: January 2023

Natural antibodies are primarily produced by B-1a cells and are essential for protection against Streptococcus pneumoniae. The incidence and mortality rate for pneumococcal infection increases dramatically after age 65, disproportionately affecting males in both human and murine systems. To date, there is a significant gap in our understanding of the relationship among sex, aging, natural IgM efficacy, and IgM repertoire. Our investigation demonstrates the protective capacity of serum IgM against pneumococcal infection is maintained in IgM obtained from aged female mice but absent in IgM from aged male mice. To understand this difference in protective capacity, we examined serum immunoglobulin, discovering that the protective change was not associated with shifts in levels of phosphorylcholine (PC) nor PPS3-specific IgM. Interestingly, we observed aged females have an increase in the total number of B-1a cells, higher serum IL-5 levels, and a larger percentage of aged female B-1a cells expressed CD86 as compared to aged males. Furthermore, single-cell IgM repertoire analysis from peritoneal and splenic PC+ B-1a cell subsets demonstrated greater diversity with age and a higher level of germline status in female mice than previously observed in studies of aged male mice. Aged female B-1a cells also expressed higher levels of transcripts associated with cell activity and self-renewal, such as Nanog and Hmga2. Together, these data indicate females maintain a more diverse and active B-1a cell pool and natural IgM repertoire, which has implications for sex-related susceptibility to infection and disease.

Supported by grants from NIH NIAID (R01AI154539)
Single-cell TCR and mRNA sequencing of antigen-specific T cells reveal spatial trajectories from single time points with HLA and VDJ bias
Authors: Andersen Anna Halling Folkmar , Schleimann Mariane Høgsbjerg , Rosas-Umbert Miriam , Olesen Rikke , Gunst Jesper Damsgaard , Krogsgaard Michelle , Tolstrup Martin
Published: January 2023

Yellow fever (YF) vaccination induces massive CD8 expansion, comprising up to 10% of all circulating CD8 T cells. We hypothesize that HLA type and VDJ gene usage implicate the highly biased response towards certain immunodominant epitopes. PBMCs were collected from healthy human donors 3 weeks after vaccination with the yellow fever vaccine, Stamaril, and enriched for YF-CD8 T cells using HLA class I tetramers. We focused on the immunodominant epitopes NS4B and NS2A, and the less dominant epitope NS3. Cells were analyzed by single-cell T cell receptor (TCR) and mRNA sequencing, yielding detailed gene phenotypes for around 2000–3000 quality cells from each donor. Data was analyzed using SeqGeq, VDJ explorer, Seurat and Monocle2. From three donors, we performed in depth analyses on both TCR gene use bias across epitope specificity, and established pseudotime cell trajectories from a single sampling time point. We found that within the CD8 population from the same donor, VDJ usage was highly biased for the target antigen. Based on mRNA expression of a panel of 259 T-cell relevant genes, we performed unsupervised clustering, that shows cells with different epitope targets cluster interchangeably, indicating similar functional patterns. However, cells specific for the immunodominant epitopes had lower TCR clonal divergence between naïve-like and activated clusters, indicating a higher degree of clonal expansion compared to less immunodominant clones. Taken together, we show antigen-driven T cell responses are biased based on donor HLA-types, which could implicate development and success of T-cell driven immunotherapies.

Supported by grants from Independent Research Fund Denmark (1029-00004B)
Enhanced T cell effector function upon differentiation of CD8+ and CD4+ T cells in the presence of BioE-1197 through potential off target ARK family inhibition
Authors: Helms Rachel Shannon , Patel Chirag H , Leone Robert D , Duvall Bridget , Gao Run-Duo , Tsukamoto Takashi , Slusher Barbara S , Pomerantz Joel L , Powell Jonathan D
Published: January 2023

We sought to evaluate PAS domain containing kinase (PASK) inhibition during T cell differentiation using the previously described PASK inhibitor BioE-1197. Notably, CD8+, Th1, and Th2 T cells activated and expanded in the presence of BioE-1197 display elevated lineage specific cytokine production upon restimulation compared to control differentiated T cells, suggesting BioE-1197 may prove to be a powerful means to enhance T cell function. Curiously however, BioE-1197 mediated these effects even in PASK KO T cells suggesting its mechanism of action was independent of PASK. BLAST alignment of the PASK kinase domain revealed members of the AMPK related kinases (ARK) family as potential targets of BioE-1197. ARKs phosphorylate class IIa histone deacetylases (HDACs) in response to activation. To assess whether BioE-1197 inhibits ARK family member activity, we activated and expanded CD8+ T cells with or without BioE-1197. Control differentiated CD8s show elevated phosphorylated and total HDAC7 over time after activation. However, BioE-1197 differentiated cells do not exhibit elevated phosphorylation or total expression of HDAC7 after activation. Concurrent reduction of phosphorylation and total HDAC7 is a phenotype previously reported in cells treated with ARK family kinase inhibitors, suggesting BioE-1197 may be functioning to inhibit an ARK family member(s). Ongoing work aims to definitively demonstrate that the ability of BioE-1197 to enhance effector function is via inhibition of ARK family members and the subsequent regulation of HDAC7. Overall these studies identify BioE-1197 as a novel agent to enhance effector T cell function and suggests potential utility in tumor immunotherapy and enhancing vaccine efficacy.

Supported by grants from NIH (P41EB028239-03, R01AI077610-10) and the Bloomberg-Kimmel Institute for Cancer Immunotherapy.
Immune requirements for protection during secondary Plasmodium infection
Authors: Fontana Mary F , Pepper Marion
Published: January 2023

Malaria, caused by Plasmodium parasites, is one of the top infectious killers of children worldwide. Decades-long efforts to develop a vaccine have so far resulted in a single licensed vaccine with modest efficacy; a better understanding of naturally acquired protective mechanisms could lead to improved vaccine design. Previous work in humans and animal models has identified roles for B cells, CD4+ T cells, and phagocytes in clearance of a primary or established infection, but we still lack a clear understanding of which immune factors are required to restrict Plasmodium from replicating in previously exposed, immune hosts. In this study, we tested whether various immune effectors were required to limit parasitemia in immune mice experiencing a second infection with the rodent-adapted strain P. chabaudi. We found that phagocytes and circulating antibody were critical for homologous secondary protection, whereas B cells were dispensable. CD4+ T cells were also required for full protection, indicating that they continue to play an important role even after antibody responses are established. We speculate that memory B cells may be important for protection from heterologous parasite strains, a hypothesis that remains to be tested. This study underlines the need for a vaccine to induce multiple immune axes in order to confer protective immunity from malaria.
Gut-derived vaginal microbiota protects female reproductive organ from viral infection through pseudonormoxia of immune cells
Authors: Park Jang Hyun , Lee Heung Kyu
Published: January 2023

The detrimental role of obesity in various diseases, including metabolic, cardiovascular, autoimmune, infectious, and cancerous diseases, has been known for many years. Conversely, a phenomenon wherein obesity exerts a positive effect in certain conditions has recently been discovered, and this is known as the obesity paradox. However, the obesity paradox remains controversial, and the underlying mechanisms are still not clear. In particular, a possible role for obesity in response to viral infection has not been fully elucidated. In this study, we investigated a case of the obesity paradox in vaginal herpes simplex virus 2 (HSV-2) infection and uncovered the mechanisms whereby obesity protects from vaginal HSV-2 infection by inducing vaginal dysbiosis. Critically, these obesity-related vaginal microbiota produce L-arginine, which promotes γδ T cell immunity that enhances early protection against HSV-2 infection. This study reveals that obesity-induced translocation of gut microbiota to vagina potentiates anti-HSV-2 immunity via reprogramming of γδ T cell metabolism which provides novel insight for development of anti-HSV-2 drugs and understanding the role of microbiota, antiviral γδ T cell immunity, and mechanisms of obesity paradox.

Supported by the National Research Foundation of Korea grant NRF-2019R1A2C208749013
The microbiome enhances transcriptional inflammatory signatures and delays clinical resolution in cutaneous leishmaniasis
Authors: Amorim Camila Farias , Lovins Victoria , Novais Fernanda O , Harris Jordan , Carvalho Lucas , Carvalho Edgar M , Beiting Daniel P , Grice Elizabeth , Scott Phillip A
Published: January 2023

Cutaneous leishmaniasis (CL) is a protozoal infection in which the immune response plays a major role in lesion development. The activation of local responses such as cytolysis by CD8+ T cells and release of IL-1β by myeloid cells is associated with poor clinical outcome in patients. We found that lesions from cutaneous leishmaniasis patients also exhibit a bacterial dysbiosis, but whether this dysbiosis affects immune responses and consequently impacts chemotherapy to heal the patients is unknown. We carried out an integrative multi-omics analysis of 64 patients, including RNA-seq and 16S-seq from lesion biopsies and clinical metadata. Staphylococcus was the dominant overrepresented genus in lesions from most patients, so we generated an in-house S. aureus pangenome to quantify transcript abundances through dual RNA-seq mapping analysis. Increased overall bacterial burden assessed by qPCR and the presence of S. aureus assessed by dual RNA-seq indicated enrichment for cytokines (e.g., IL1B), inflammatory responses (e.g., PTGS2), and chemotaxis (e.g., CXCL8) by Gene Ontology analysis. Of particular importance, we found that lesions with increased S. aureus transcript abundances exhibited high expression of inflammatory-related genes that have been associated with treatment failure, such as PRF1, GNLY, and GZMB. Correspondingly, we found that patients with a Staphylococcus spp. dysbiosis exhibited a delay in resolving their lesions following treatment. These results indicate that the microbiome influences the local immune responses in CL, affecting how patients respond to therapy by delaying the healing time. These results suggest that the use of antibiotics combined with chemotherapy may improve treatment outcome for CL.

Supported by grant from NIH (R01 AI149456)
Contribution of PU.1 and STAT6 to fibrosis-mediated growth suppression of the Diphyllobothriidae family cestode, Schistocephalus solidus.
Authors: Steinel Natalie C , Weber Jesse , Peng Foen , Shim Kum Chuan , Lohman Brian , Fuess Lauren , De Lise Stephen , Bolnick Daniel I
Published: January 2023

Threespine stickleback (Gasterosteus aculeatus) fish are naturally infected with the Diphyllobothriidae family tapeworm, Schistocephalus solidus. The anti-parasite response to this body cavity infection varies greatly among wild stickleback populations. Some populations generate extensive peritoneal fibrosis which suppresses cestode grown, thereby preventing the parasite from reaching sexual maturity and blocking its ability to infect its terminal host. In extreme cases this response can encapsulate the cestode. On the other hand, other fish populations tolerate S. solidus, carry heavy parasite burdens, grow parasites 34-fold larger than resistant fish, and do not produce fibrosis. Combined genetic mapping and population genomics identified regions of the genome associated with this resistance-tolerance axis. Quantitative trait loci (QTL) mapping of the fibrosis phenotype led us to a 78bp deletion in SPI1b which encodes the transcription factor PU.1 which is associated with the initiation of tissue fibrosis. This deletion was fixed in tolerant fish, but not resistant fish. SPI1b expression increases with infection and is higher in fibrotic fish compared to non-fibrotic controls. QTL mapping of cestode mass detected STAT6 on Ch 12, which is associated with alternatively activated macrophages and fibrosis. In tolerant fish, but not resistant, a deletion in stat6 exon 5 is nearly fixed. Several additional QTL were also found containing genes associated with fibrosis initiation or regulation, evidence for strong selection on this anti-parasite mechanism and identifying pathways involved in the regulation of a pathologic fibrotic response against a Diphyllobothriidae cestode.
Malaria antigens are presented to CD8 T cells via the non-classical HLA-E
Authors: Wilder Brandon Keith , de Lacerda Luna , Barbosa Camila R R , Aleshnick Maya , Martinson Thomas , Morrow David , Zhao Zeshou , Gaiha Gaurav , Junqueira Caroline
Published: January 2023

CD8 T cells have long been known to target infected hepatocytes during the intracellular liver stages of Plasmodium—the causative agent of malaria. Current T cell vaccine strategies are limited by the inability to obtain sufficient amounts of Plasmodium-infected hepatocytes to identify peptides presented on MHC-I (“immunopeptidomics”). Recently, we demonstrated that the blood stages of Plasmodium vivax are also susceptible to CD8 T cell killing and performed immunopeptidomics on Pv-infected reticulocytes. Peptides mapped to proteins conserved across Plasmodium spp. and were common across volunteers. Furthermore, ~50% of the peptides detected are not predicted to bind classical HLA-I molecules and thus could be presented by non-classical HLA. In support of this, blocking HLA-E during in vitro stimulation of PBMCs with a pool of newly-identified peptides substantially reduced the CD8 T cell response. Mechanistic studies conducted in the rhesus macaque/P. cynomolgi malaria model demonstrated that CD8 T cell killing of infected reticulocytes is conserved in this model as are CD8 responses to the newly identified peptides. In vitrostimulation assays showed that CD8 T cell responses to at least 3 of 9 peptides tested are completely restricted to MHC-E. This is supported by in vitro binding assays showing peptide binding to HLA-E. In summary, we demonstrate for the first time that malaria infection induces HLA/MHC-E-restricted CD8 T cell responses to newly identified peptides that are conserved across host and parasite species. Given the extreme conservation of MHC-E, these results pave the way to explore a universal, species-transcending vaccine for malaria based on MHC-E presentation of novel antigens.
Hypoxia promotes cytolytic activity of CD8 T cells and pathogenesis in cutaneous leishmaniasis
Authors: Fowler Erin , Novais Fernanda O
Published: January 2023

Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and the most common form of the disease is cutaneous leishmaniasis (CL). A fundamental question in CL is what regulates the development of severe disease, information that is critical to develop therapies to ameliorate disease. In a series of studies, we demonstrated CD8 T cell-dependent cytotoxicity as the main inducer of immunopathology in CL. This result was unexpected since IFN-γ production by CD8 T cells plays a protective role by promoting pathogen elimination. To resolve this paradox, we studied the CD8 T cells in different anatomic sites and found that the effector function of CD8 T cells in CL depends on their location: while CD8 T cells are cytotoxic (GzmB+) and produce little IFN-γ in leishmania lesions, CD8 T cells in the draining lymph nodes (dLN) have the opposite profile. Importantly, GzmB− CD8 T cells from dLN quickly upregulate GzmB after injection into CL lesions. By transcriptional profiling, we found that CD8 T cells in lesions and not dLN have a hypoxic signature. In vivo, we observed that leishmania lesions are hypoxic using the Oxyphor G4 oxygen probe and pimonidazole staining. In vitro, we found that induction of hypoxia was sufficient to convert GzmB− into GzmB+ CD8 T cells. In vivo, blocking the dimerization of HIF-α, a master regulator of hypoxia, with acriflavine decreased lesion development in mice. Transcriptional profiling in patients showed that hypoxia is a signature of human CL and correlates with GZMB expression. Together, our results suggest that the hypoxic microenvironment of leishmania lesions alter the function of CD8 T cells and convert protective CD8 T cells into pathogenic cytotoxic T cells.

Supported by Host Defense and Microbial Biology (OSU)
Small Intestine Epithelial CD4 Cytotoxic T Lymphocytes Provide Innate-Like Protection against Enteric Pathogens without Risk of Immunopathology
Authors: Chen Angeline , Thiault Nicolas , Iwaya Hitoshi , Cheroutre Hilde
Published: January 2023

CD4 T helper (Th) cells reprogram to cytotoxic T lymphocytes (CTLs) in the small intestinal epithelium. In the present study, CD4 CTLs were evaluated for their potential to promote tolerance during steady state as well as their ability to fend off enteric pathogens during infection.

The CD4 T cell adoptive transfer model of colitis was used to determine if CTLs can promote tolerance. Naïve CD4 T cells were differentiated towards a CTL, Treg, or inflammatory Th (Th1 or Th17) fate and transferred into RagKO recipients. Strikingly, the recipients that received CTLs, like the Treg condition, had minimal disease; in contrast, the Th conditions all exhibited pathology. To determine if the forced presence of Th cells exacerbates colitis disease, CD4 T cell adoptive transfers were performed using cells that were forced to stay Th or predisposed to becoming CTLs. Altogether, the conversion of Th to CTL mitigated disease pathology and promoted tolerogenic conditions.

The protective role of CD4 CTLs to rapidly respond to enteric pathogens and contain infections was also examined. Using Salmonella as a bacterial model, the data show that pre-existing CD4 CTLs have the capacity to kill infected cells, thereby preserving the integrity of the barrier and preventing bacterial dissemination. Functionally, CD4 CTLs represent a strategy of protective tolerance by the immune system to fortify the epithelium with quiescent but primed CTLs that can provide rapid immunity during enteric infection.

Supported by grants from the NIH (U01 AI125957, F31 DK124078)
Gene signatures for intestinal and peripheral innate lymphoid cells in pigs reveal tissue-specific imprinting and similarities to human cells via single-cell RNA sequencing
Authors: Wiarda Jayne E , Trachsel Julian M , Sivasankaran Sathesh K , Tuggle Christopher K , Loving Crystal L
Published: January 2023

Intestinal innate lymphoid cells (iILCs) impact intestinal health outcomes, but study of iILCs in humans is limited. Human peripheral ILCs (pILCs) are easily obtained but may vary substantially from iILCs, thus requiring comparison of iILCs and pILCs to determine applicability of pILCs as surrogates to study iILC function. Pigs have anatomic, physiological, nutritional, and immune similarities to humans that are lacking in rodent models, making pigs a relevant biomedical model; however, ILCs are poorly defined in pigs. Single-cell RNA sequencing was performed to compare porcine iILCs to pILCs and determine potential similarities of porcine to human ILCs. Porcine pILCs matched porcine natural killer (NK) cell descriptions, while iILCs were annotated as group 1 and group 3 ILCs. Gene modules obtained independent of cell annotations were distilled to core signatures of genes most highly specific to pILCs, pan-iILCs, group 1 iILCs, and group 3 iILCs. The pILC signature included conventional NK cell genes, while the group 3 iILC signature included genes associated with type 3 immunity. Pan-iILC and group 1 iILC signatures included genes associated with tissue residency, cell activation, and metabolism, indicating tissue-specific, activation-associated imprinting. Gene profiles were used to develop in situ iILC detection methods, establishing group 1 iILCs were intraepithelial, while group 3 iILCs resided in lamina propria/gut-associated lymphoid tissue. Findings indicated iILCs were ILC subsets distinct from pILCs, thus opposing pILCs as surrogates to study iILC dynamics. Moreover, gene profiles and in situ locations of iILCs drew close parallels to human iILCs functions, supporting pigs as a biomedical model for iILC research.

Research was supported by appropriated funds from USDA-ARS CRIS 5030-31320-004-00D, an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE. This research used resources provided by the SCINet project of the USDA ARS project number 0500-00093-001-00-D.
Microbially-induced exosomes in dendritic cells promote paracrine immunosenescence: Novel mechanism of inflammatory alveolar bone loss in mice
Authors: Elsayed Ranya M , Elashiry Mahmoud , Cutler Christopher W
Published: January 2023

Periodontitis (PD) is a chronic inflammatory disease of aging or inflammaging that affects 70.1% of Americans 65 years and older. Age related immunosenescence is implicated in chronic inflammation and impaired immune surveillance, but role in PD is unclear. The aim of this study was to determine cellular mechanisms of immunosenescence in PD in-vivo. Variables including age, bacterial infection with key stone oral pathogen, Porphyromonas gingivalis (Pg), and senolytic agent rapamycin (Rap) were assessed for influence on ligature accelerated PD in young (2–3 mo) and old (24 mo) B6 mice (n=30). Senescence profiling of gingiva and lymph node (LN) was performed by confocal microscopy, flow cytometry and qPCR. Increased SA-BGal, p16INK4A, p21Waf1/Clip1, CD57+ CD28− T cell, Th17/Treg ratio and IL6/TNFa/IL1b were observed in DCs and T cells in gingiva and/or LN as a function of advanced age, or exposure of young mice to Pg gavage. This was associated with a significant increase in bone loss shown by micro CT 3D analysis which was abrogated by Rap. In-vitro analysis revealed a~2-fold increase in secreted exosomes from DCs infected with Pg (PgDCexo). These exosomes were enriched in age-related, anti-apoptosis/anti-autophagy miRNAs, Pg fimbrial adhesin protein mfa1, and IL6/TNFa/IL1b. PgDCexo were injected intragingivally in young mice promoting a premature senescence profile in the gingiva and LN and inflammatory bone loss compared to exosomes from uninfected DCs. We conclude that advanced age and microbially-induced immunosenescence contribute to the pathogenesis of inflammatory bone loss in mice which can be inhibited by rapamycin. P. gingivalis-induced exosomes in dendritic cells promote paracrine senescence of normal bystander cells.
HIVE Single-Cell TCR Sequencing: Assay Principle and Applications
Authors: Whitney Irene E , Chen Li , Gierahn Todd , Flanigon Jim
Published: January 2023

Biological resolution at the level of individual cells is powering the next phase of precision health. Honeycomb Biotechnologies currently offers an assay for single-cell RNA sequencing with the HIVE™ scRNAseq Solution. The HIVE is a portable, handheld, single-use device that enables gentle capture, robust storage, and easy processing for the analysis of single-cell samples. The HIVE scRNAseq assay integrates sample storage and single-cell profiling into a single workflow, facilitating decentralized collection and centralized processing of samples. The design of the HIVE is ideal for the recovery of fragile cells, loading of large sample volumes, and efficient processing of numerous samples in parallel, particularly valuable for clinical applications.

Here, we extend the application of the HIVE by offering a single-cell T cell receptor enrichment and sequencing (scTCRseq) assay. Using the stable whole transcriptome amplification (WTA) product obtained from the scRNAseq assay as the sample input, the HIVE scTCRseq assay enriches TCR in a streamlined target-specific amplification workflow. The assay is compatible with all human tissues, and can accommodate a wide range of cell numbers. We typically achieve over 75% TCR molecule recovery from the WTA, and over 60% sequencing reads are mapped to TCR. Here we also present TCR profiling of several specimens, demonstrating its value in clinical applications.

Honeycomb aims to expand single-cell opportunities across basic, translational, and clinical research throughout the world.
Single-cell multi-omic analysis identify heterogeneity and distinct features in fate-mapped tissue-resident alternatively activated macrophages
Authors: Lin Jian-Da , Chou Tzu-Yin , Yang Chen-Hsuan , Chao Pei-An , Chen Yen-Ting , Loke P’ng
Published: January 2023

Peritoneal cavity cells play pivotal roles in inflammation, repair, and maintaining homeostasis in response to pathogenic infections and tissue injury. Two major tissue-resident macrophages (TRMs) are large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). These are the major macrophage subsets in the peritoneal cavity and originate from embryogenic (LPMs) or bone-marrow-derived myeloid precursors (SPMs). CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) provides simultaneous information for single cells in both cell-surface protein and gene expression levels. Here, we used CITE-seq to profile peritoneal cells by an oligonucleotide-labeled antibody panel designed to react with 189 unique mouse cell surface antigens. We identify 14 markers exclusively expressed on TRMs but not other immune cell types. These markers can classify phenotype differences between LPMs and SPMs during IL-4 stimulation. We further profile fate-mapped TRMs by scRNA-seq and identified more heterogenous phenotypes of TRMs that originated from embryogenic rather than bone-marrow-derived myeloid precursors. Notably, serum amyloid A-3 (Saa3) and platelet factor 4 (Pf4), can distinguish TRM clusters from alternative activation to IL-4 stimulation and Heligmosomoides polygyrus infection. Hence, we identify distinct markers that can be used to distinguish the different origins and heterogenous TRM phenotypes under steady state and type 2 immune responses.
Cell type-specific differential expression in Alzheimer’s disease and cancer using spatial transcriptomics
Authors: Cable Dylan , Irizarry Rafael , Chen Fei
Published: January 2023

Spatial transcriptomics enables spatially resolved gene expression measurements at near single-cell resolution. The detection of genes that are differentially expressed across tissue context for cell types of interest is an essential challenge for dissecting pathological mechanisms. However, changes in cell type composition across space and measurement contamination from other cell types introduce complex statistical challenges. Here, we introduce a statistical method, Generalized Linear Admixture Models for Differential Expression (GLAMDE), that estimates cell type-specific patterns of differential gene expression while accounting for localization of other cell types. By modeling spatial transcriptomics gene expression as an additive mixture, across cell types, of generalized log-linear functions, we provide a unified framework for identifying gene expression changes for a wide-range of relevant contexts: changes due to pathology, anatomical regions, physical proximity to specific cell types, and cellular microenvironment. Our approach enables statistical inference across multiple samples and replicates. We demonstrate, through simulations and validation experiments on Slide-seq and MERFISH data, that our approach accurately identifies cell type-specific differential gene expression with valid uncertainty quantification. Lastly, we apply GLAMDE to characterize spatially-localized tissue changes in the context of disease. In an Alzheimer’s mouse model Slide-seq dataset, we identify plaque-dependent patterns of cellular immune activity. We also find a putative interaction between tumor cells and myeloid immune cells in a Slide-seq tumor dataset. GLAMDE is available within the spacexr R package.

D.C. was supported by a Fannie and John Hertz Foundation Fellowship and an NSF Graduate Research Fellowship. This work was supported by an NIH Early Independence Award (DP5, 1DP5OD024583 to F.C.), the NHGRI (R01, R01HG010647 to F.C.), as well as the Burroughs Wellcome Fund, the Searle Scholars Award, and the Merkin Institute to F.C.. R.A.I. was supported by NIH grants R35GM131802 and R01HG005220.
Analysis of human BCR-IgH from genomic DNA with UMI incorporation
Authors: Brown Brittany E , Pan Wenjing , Berngruber Chelsea , Byrne-Steele Miranda , Han Jian
Published: January 2023

Immune repertoire analysis can be performed from practically any biological sample as long as viable nucleic acid can be extracted. While using RNA as a template has its benefits, gDNA is often preferred as gDNA is often easier to obtain, especially from difficult samples. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may better reflect the quantity of cells. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement.

We developed a new set of BCR-IgH specific primers to target the gDNA V(D)J region while also incorporating unique molecular indices (UMI) through our proprietary dimer avoided multiplex PCR technology. The inclusion of UMI with gDNA amplification allows each cell to be counted since the relationship of one V(D)J per cell will be maintained and will have a unique barcode added during the processing. The inclusion of UMI also assists with the elimination of PCR and sequencing error, which will lead to more confident analysis of low frequency clones and hypermutations. The primers were first tested on a BCR-IgH synthetic library. The sequencing result demonstrated >85% correlation with the known synthetic library gene expression. These results showed that the new gDNA BCR-IgH panel was sufficient at amplifying all known J gene targets.

Presently, we have also applied this method to gDNA extracted from B cell enriched PBMC samples including the spike-in of a known BCR clone at various concentrations. To our knowledge, this is the first assay of its kind to incorporate UMI into BCR-IgH gDNA to capture both the BCR repertoire and true cell frequency.
Microfluidic cell sorting for gentle isolation of immune cells.
Authors: Jagnandan Nicole , Morachis Jose
Published: January 2023

Isolation of specific immune cells is critical to biomedical research, however, techniques such as droplet-based fluorescence-activated cell sorting can mechanically irritate cells resulting in cell death or unwanted activation of immune cells that affect experimental analysis. Here we show that T cell subsets and neutrophils can be isolated using a low pressure (<2 psi) microfluidic cell sorter (WOLF G2®, NanoCellect®). These experiments show that this 11-parameter cell sorter can isolate complex cell types at high purity and high viability. In addition, due to the low-pressure microfluidic sorting mechanism, laminar flow reduces shear stress relative to traditional droplet sorters and cells remain functional and unaltered for subsequent experiments. Furthermore, we demonstrate high quality single-cell genomics data when combined downstream with single-cell partitioning and plate-based workflows. These findings demonstrate a novel workflow for isolating immune cells using the next generation of cell sorters.
Lumit: a novel bioluminescent, homogenous immunoassay platform for analyte detection
Authors: Bach Maggie L , O’Brien Martha A , Nath Nidhi , Zegzouti Hicham , Lazar Dan , Cali James
Published: January 2023

The detection of a specific analyte in a complex mixture by an immunoassay is a common procedure in many bioanalytical workflows. Immunoassays rely on the interaction between labeled antibodies and the analyte of interest. We have developed Lumit, a novel bioluminescent labeling approach allowing no-wash immunoassays amenable for high-throughput applications.

Lumit immunoassay technology is based on an adaptation of NanoBiT® luciferase, a structural complementation system that consists of the Large BiT (LgBiT; 18 kDa) subunit and a small, complementary peptide, Small BiT (SmBiT; 11 amino acids), that has low affinity for LgBiT. Antibodies labeled with LgBiT or SmBiT subunits are brought into proximity due to binding to a target analyte. This facilitated complementation reconstitutes an active luciferase and generates light proportional to the amount of analyte present. This detection chemistry has several advantages over traditional ELISA workflows, including a no-transfer and no-wash protocol suitable for automation, fast detection time (30 – 90 minutes), and a broad dynamic range mitigating the need for sample dilutions.

Here, we present a variety of applications of Lumit technology to detect analytes in immunology research. Examples include detection of cytokines from activated cultured immune cells using labeled primary antibodies, the detection of antibody-Fc receptor binding, the detection of protein phosphorylation using labeled secondary antibodies, and detection of the PD1: PDL1 interaction using labeled anti-tag antibodies.
Buoyancy Activated Cell Separation (BACS™) Microbubbles for Lysis and Ficoll Free Processing of Leukopaks
Authors: Grimley Edward C , Roussey Jon , Ku Chia , Snow Tiffany , Wegner Casey , McNaughton Brandon H
Published: January 2023

Leukopaks are highly-enriched apheresis product containing large numbers of mononuclear cells. In applications where large numbers of primary immune cells are required, leukopaks offer an ideal starting material. For this reason, leukopaks are a vital source material for immunophenotyping studies, cell therapy manufacturing, and pharmaceutical development.

The enrichment of leukocytes from leukopaks have traditionally required an upfront density gradient separation to remove red blood cells (RBCs) which has made leukopak processing time consuming, inefficient, and inconsistent. To overcome these obstacles, Akadeum has developed a gentle and effective approach for density gradient and lysis-free processing of leukopaks that is based on our proprietary Buoyancy Activated Cell Sorting (BACS™) Microbubbles.

Here we report on the isolation of untouched T cell and CD4+ T cell populations directly from leukopak material. Untouched T cells and CD4+ T cells were isolated in roughly 30 minutes using our quick and easy protocol. T cells were isolated with a final purity of 91.8% while CD4+ T cells were isolated with a final purity of 93.9%. In addition to high purity, we achieved greater than 90% recovery of both cell populations. These data clearly demonstrate the value of Akadeum’s easy, gentle, and effective sample preparation platform.
The immune dysregulations in inflammatory arthritis immune related adverse events
Authors: Zeng Hu , Zhu Xingxing , Li Yanfeng , Jaquith Jane , McCarthy-Fruin Kathleen , Thanarajasingam Uma
Published: January 2023

The immune checkpoint inhibition therapy induces various immune related adverse events (irAEs). The immunological basis of irAEs remains unclear. We recruited a cohort of patients with inflammatory arthritis irAE (IA-irAE), the most common form of rheumatic irAEs, together with matched patients with conventional rheumatoid arthritis (RA) and cancer patients without irAEs. Comprehensive clinical and immunological investigations reveal an imbalance of T cell exhaustion and senescence in IA-irAE patients, and immunological similarities between IA-irAE patients and seronegative RA patients. These data suggest that IA-irAE and seronegative RA may have shared immunological mechanisms. Thus, our study identifies potential immunological underpinnings of IA-irAE and the relationship between IA-irAE and conventional RA. The results may have implications for managing irAE and seronegative RA patients.

Supported byR01AR0077518 Arthritis National Research Foundation
ST8Sia6 expression in beta cells mitigates onset of autoimmune diabetes in the murine NOD model
Authors: Choe Justin , Belmonte Paul , Rajcula Matthew , Theodore Keith , Lee Hyun Se Kim , Shapiro Michael J , Shapiro Virginia
Published: January 2023

In this study, we harnessed immunomodulatory Siglec-sialic acid interactions to mitigate autoimmune rejection of beta cells in Type 1 Diabetes (T1D). Mice engineered to express the sialyltransferase ST8Sia6 in beta cells (βST mice) were backcrossed to the non-obese diabetic mouse model of T1D and followed for disease; 60% of female littermates spontaneously developed diabetes by 300 days, compared to minimal occurrence (5%) in female βST mice. Histologic analysis of pancreas sections from non-diabetic βST and littermate mice suggested a similar degree of immune infiltration at 4 and 8 weeks of age, but the severity of insulitis was drastically reduced in βST mice beyond 20 weeks of age as compared to littermates. IHC for various immune populations suggested no difference in composition of insulitis at 8 weeks between βST and littermate mice. ELISA for serum anti-insulin antibodies demonstrated no difference in concentration between female βST and littermate mice. After female euglycemic βST and littermate mice at 16 weeks of age were challenged with anti-PD-L1 monoclonal antibodies, both groups resulted in similarly rapid induction of disease, suggesting that ST8Sia6 dependent protection occurs peripherally. Shut-off of ST8Sia6 expression in euglycemic βST mice at 20 weeks of age did not abrogate protection from disease, supporting persistent immune tolerance beyond this time. Flow analysis of islet infiltrating immune cells at 300 days confirmed reduced number of infiltrating immune cells in βST mice. Thus, induced expression of ST8Sia6 has been shown to mitigate autoimmune attack in the NOD mouse and presents a novel path forward for improving the success of curative islet transplant into T1D patients.

Supported by funding from the Mayo Clinic (Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic MSTP) and the NIH (5R21AI135858-02, 1F30DK127564).

Supported by funding from the Mayo Clinic (Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic MSTP) and the NIH (5R21AI135858-02, 1F30DK127564).
Induction of tolerance to a CD4 T cell hybrid insulin peptide epitope prolongs islet graft survival and suppresses autoreactive CD8 T cells
Authors: DiLisio James E , Jamison Braxton L , Neef Tobias , Miller Stephen D , Baker Rocky , Haskins Kathryn
Published: January 2023

Autoreactive T cells are thought to drive autoimmune diabetes by recognizing beta cell-derived peptide antigens. We previously discovered that hybrid insulin peptides (HIPs) are potent neoantigen peptide ligands for a subset of autoreactive CD4 T cells in both the NOD mouse model and human type 1 diabetes patients. Inducing tolerance to prominent T cell autoantigens through antigen-specific immunotherapy could prevent disease onset or recurrence after islet transplantation without the need for broad immunosuppression. Here we show that tolerogenic nanoparticle (NP) delivery of the 2.5HIP, a dominant CD4 T cell HIP epitope in the NOD mouse, can prolong islet graft survival in transplanted diabetic NOD mice. Tolerance induction to the 2.5HIP not only suppressed 2.5HIP tetramer+ CD4 T cells but also autoreactive CD4 and CD8 T cells specific for different islet antigens. 2.5HIP NP treatment induced a dysfunctional state in graft-infiltrating T cells characterized by increased expression of anergic markers on CD4 T cells and reduced inflammatory cytokine production in 2.5HIP tetramer+ CD4 T cells as well as IGRP (islet specific glucose-6-phosphatase catalytic subunit-related protein) tetramer+ CD8 T cells. In conclusion, we demonstrate that antigen-specific immunotherapy aimed at inducing tolerance to a single CD4 T cell HIP epitope can prolong islet graft survival and suppress autoreactive CD8 T cells in the NOD mouse model of autoimmune diabetes.

Supported by grants from NIH (R01 DK122566, R01 DK081166, T32 DK120520) and JDRF (2-SRA-2018-566-S-B)
Tolerogenic Immune Modifying Nanoparticles (TIMP) Encapsulating Multiple Diabetogenic Epitopes Prevent Onset of Type 1 Diabetes in NOD Mice
Authors: Genardi Samantha , Podojil Joseph R , Kakade Sandeep , Boyne Michael , Elhofy Adam , Miller Stephen D
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease characterized by inflammatory lesions within islets (insulitis) and β cell loss which is associated T cell and B cell responses specific for pancreatic islet β cell proteins. Data show that treatment with poly(lactide-co-glycolide) (PLGA) nanoparticles (i.e. TIMP/COUR’s CNP) containing a single diabetogenic peptide blocks T1D induced by transfer of CD4+ BDC2.5 or CD8+ NY8.3 TCR transgenic T cells to NOD-scid mice. In contrast, Ag-specific tolerance approaches targeting single β-islet cell protein epitopes, e.g. GAD65 or Insulin, have failed to achieve efficacy in both wildtype NOD mice and clinical trials. The possibility exists that T1D initiation and progression is due to activation of T cell populations specific for multiple diabetogenic epitopes. To test this hypothesis, recombinant Chormogranin A, GAD65, and Insulin proteins were encapsulated within CNPs to assess Treg/Tr1 cell induction, inhibition of Ag-specific T cell responses, and blockade of T1D in NOD mice. While treatment of NOD mice with CNPs containing a single protein inhibited the corresponding Ag-specific T cell response, the inhibition of T1D development only occurred if all three diabetogenic proteins were included within the CNPs (CNP-T1D). CNP-T1D-induced blockade of T1D was characterized by Treg/Tr1 cell induction and a significant decrease in both peri-insulitis and immune cell infiltration into pancreatic islets. We have recently published that CNP treatment is both safe and induces Ag-specific tolerance in Phase I/IIa celiac disease clinical trials. Therefore, utilization of this technology including multiple diabetogenic proteins is a promising Ag-specific tolerance approach to T1D treatment.
An IL-2 mutein promotes Foxp3+ Treg-mediated suppression of dendritic cell activation in response to inflammatory stimuli
Authors: Jamison Braxton L , Campbell Daniel J
Published: January 2023

Foxp3+ regulatory T (Treg) cells are essential for maintaining immune tolerance and the development of strategies that boost Tregs is a promising approach for the treatment of autoimmune disorders. We previously developed a murine Fc-fused IL-2 mutein (Fc.Mut24) with decreased affinity for IL-2Rβ resulting in increased CD25 dependency and potent Treg selectivity. High-dose Fc.Mut24 administration prevents autoimmunity in the non-obese diabetic (NOD) mouse; however, the mechanisms by which Fc.Mut24 affects Treg suppressive function remain unclear. In this study, we have performed a phenotypic analysis of Treg and classical dendritic cells (DC) from IL-2 mutein-treated mice. We have found that Fc.Mut24 treatment increases CTLA-4 recycling/expression on Treg with a concurrent decrease in PD-1 expression specifically on effector regulatory T (eTreg) cells. Both CD8+ cDC1 and DCIR2+ cDC2 subsets from Fc.Mut24-treated mice have reduced expression of the co-stimulatory molecules CD80/CD86 following lipopolysaccharide (LPS) injection. Furthermore, RNA-sequencing reveals that Tregs regulate the transcriptional response of DCs to LPS and may preferentially suppress cDC1 activation in vivo. These results suggest that a major mechanism of action by IL-2 mutein may be promoting CTLA-4-mediated transendocytosis of CD80/CD86 while simultaneously reducing PD-1: PD-L1 co-inhibitory signaling between eTreg and DCs. Our findings address unanswered questions about the role of IL-2 in modulating Treg suppressive function and have important implications for the use of Treg-selective IL-2 therapeutics in the clinic.

B.L.J. is supported by a postdoctoral fellowship from the Washington Research Foundation.
Chromatin profiling of renal allograft-infiltrating cells reveals epigenetic regulation of CD8+ T cell exhaustion in chronically rejected human kidney allografts
Authors: Yi Stephanie Grace , Zou Dawei , Dai Yulin , Zhao Zhongming , Gaber Osama , Chen Wenhao
Published: January 2023

Purpose

Our single cell RNA sequencing (scRNA-seq) analysis found that T cells in chronically rejected kidney allografts and peripheral blood mononuclear cells (PBMCs) of the same kidney transplant recipients (KTR) exhibited a spectrum of dynamic cell states; herein, we investigate how T cell states are regulated at the epigenetic level during chronic rejection.

Methods

H3K27me3 and H3K27ac CUT&Tag was performed in live T cells isolated from PBMCs and allografts of 3 KTR with failed transplants. All transplants had chronic cellular and antibody mediated rejection on pathology.

Results

With CD8+ T cell infiltration into allografts, H3K27me3 was deposited at the gene loci associated with T cell quiescence and stemness (TCF7, BACH2, FOXP1, IL7R, etc.), whereas H3K27ac was mostly deposited at the gene loci involved in T cell effector (GZMB, ID2, ZEB2, etc) and exhaustion (TOX, TOX2, TIGIT, PDCD1, HAVCR2, etc) programs.

Conclusion

Our prior scRNA-seq analysis revealed that most CD8+ T cells in PBMCs maintain TCF7+ stem-like cell phenotypes. However, most CD8+ T cells infiltrated in chronically rejected allografts lose stemness, becoming either TCF7− GZMBhi effector or TCF7− PDCD1hi HAVCR2hi exhausted T cells. Herein our CUT&Tag analysis reveals that chromatin remodeling mediates such dynamic changes of T cell states. Of note, although T-cell exhaustion occurs in chronically rejected allografts, it only represents dynamic changes of T cell states. We propose that TCF7+ allogenic T cells in the periphery maintain stem-like property. Without proper epigenetic silencing of their function, TCF7+ allogenic T cells produce not only exhausted progeny but also effector cell progeny to mediate allograft rejection.

Supported by internal