bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc Treated Engineered Human Dendritic Cells Capable of Synthesizing High Active Vitamin D and Retinoic Acid Concentrations Enhance Gut-homing Regulatory T Cell Induction
Authors: Ali F N U Shaikh Nisar , Abdalla Maisa I , Cannon Lisa M , Zhang Xiao-Bing , Baylink David J , Tang Xiaolei
Published: August 2023

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the gastrointestinal tract (the Gut). Available IBD therapies can cause severe side effects, and many patients either fail to respond to the treatment or lose therapeutic effects with time. Hence, IBD therapy is still an unmet medical need. We recently proposed to enhance the induction of regulatory T cells (Tregs) expressing gut-homing molecules (gut-homing Tregs) to control chronic gut inflammation in IBD patients without compromising systemic immunity. We have shown that dendritic cells (DCs), engineered to de novo synthesize high concentrations of the active vitamin D (1,25-dihydroxy vitamin D or 1,25[OH]2D) and retinoic acid, increase the induction of gut-homing Tregs phenotypically and functionally in healthy mice and the mice induced for experimental colitis. This current study investigated whether the engineered DCs worked in humans. In addition, we also asked how zinc impacts the function of our engineered DCs because zinc is a biologically essential mineral widely included in daily supplements and has also been shown to tolerize DCs. Our data showed that the engineered DCs, derived from healthy control subjects and IBD patients, stimulated autologous naïve CD4+ T cells to express both regulatory and gut-homing molecules (gut-homing Tregs). In addition, the stimulated CD4+ T cells suppressed the proliferation of autologous effector T cells. Moreover, the addition of zinc enhanced the ability of the engineered human DCs to augment the induction of autologous gut-homing Tregs. Hence, our findings indicate zinc-treated engineered DCs are a promising novel IBD therapy.

This study was partially supported by the AAI Careers in Immunology Fellowship; the US Army Medical Research and Materiel Command grant (W81XWH-15-0240); and the GI Foundation of Loma Linda University Innovation grant (RIG-GI 681207-2967).
Time restricted feeding in diet induced obesity restores diurnal T cell rhythms in the small intestine
Authors: Edell Claudia Jean , Colson Jackson , Maynard Craig L , Pollock Jennifer S
Published: August 2023

Diet induced obesity (DIO) causes circadian disruption and dysregulation of T cells in the gut. Previous studies have also shown that immune cells, including T cells, have rhythms of migration through tissues. We previously found that time-restricted feeding (TRF) in DIO leads to reduced cardiovascular target organ damage. However, it is unknown how TRF in DIO alters intestinal T cells, specifically anti-inflammatory regulatory T cells (FoxP3) and inflammatory Th17 cells (RORgt). We used a 20-week model of high fat (DIO) with TRF intervention with food restricted to the 12-hour active (dark) phase in weeks 18–20. We assessed T cells in the small intestine via flow cytometry in normal diet (ND), DIO, and DIO+TRF groups at Zeitgeber time (ZT) 3 (light period) and ZT13 (dark period). Interestingly, there is a significant diurnal difference of total immune cells (CD45+), total T cells (TCRb+), and CD4+ T cells in the small intestine of ND mice, with increases at ZT13 compared to ZT3. No diurnal rhythm in T cells is seen in DIO mice. DIO+TRF restores a significant time-of-day difference in T cellsin the small intestine. We investigated CD4+ T cell transcription factors, RORgt and Foxp3. RORgt expression was suppressed at ZT13 compared to ZT3 in DIO. DIO+TRF led to increased RORgt+ CD4+ T cells at ZT13 compared to ZT3. The number of Foxp3+ CD4+ T cell was also increased at ZT13 compared to ZT3 in DIO+TRF. CXCR3, a T cell migration marker, was found to be significantly downregulated on CD4+ T cells at ZT13 compared to ZT3 in DIO+TRF mice but not DIO. These findings indicate that intestinal T cell rhythms are lost in DIO mice and TRF restores the T cell rhythm and suggest that the TRF-induced restoration of T cell rhythms in the gut may lead to reduced target organ damage.
Bcl11b regulates multipotency and effector programs in intestinal resident memory CD8+ T cells
Authors: Zelenka Tomas , Helm Eric , Cismasiu Valeriu , Islam Shamima , Silvane Leonardo , Shaw Timothy , Avram Dorina
Published: August 2023

Tissue-resident memory CD8+ T cells (Trm) reside in non-lymphoid tissues and provide potent protection from re-infection. They are multipotent and have diverse functions, including effector roles during re-infection. Using a series of RNA-seq, ATAC-seq, ChIP-seq and CUT&RUN experiments, we investigated Bcl11b-dependent molecular mechanisms governing the differentiation of small intestine Trm cells. Mice with conditionally depleted Bcl11b in activated CD8+ T cells had largely altered memory T cell populations. Particularly the Trm cells and their precursors displayed strong transcriptional deregulation. Regions with decreased ATAC-seq and/or H3K27ac signal in Bcl11b-deficient cells were enriched for DNA binding motifs associated with the multifunctional/multipotent Trm program, whereas regions with increased signal for the motifs associated with the effector program. Correspondingly, genes from the multifunctional/multipotent and effector programs had decreased and increased expression in Bcl11b-deficient cells, respectively. Most of the program genes were bound by Bcl11b and displayed related epigenetic changes in Bcl11b-deficient cells. These results were experimentally validated for Tcf1 and Ahr, which exhibited increased binding at regions with decreased and increased chromatin accessibility, respectively. Moreover, the impact of Tcf1, Blimp1, and Ahr alterations on the deregulated Trm differentiation in the absence of Bcl11b was confirmed by rescue experiments. In summary, we propose that Bcl11b promotes multipotency and restricts effector Trm programs, and is overall essential for small intestine Trm cell differentiation.

This work was supported by grants from NIH (R01AI067846, R01AI133623) and Moffitt Cancer Center (P30CA076292).
Pseudotemporal and spatial organization of B cell activation and differentiation in Peyer’s patches
Authors: Wiarda Jayne E , Shircliff Adrienne , Stasko Judith , Ackermann Mark , Sivasankaran Sathesh , Tuggle Christopher K , Loving Crystal
Published: August 2023

Organized lymphoid tissues are important sites of immune induction where B cells activate and differentiate. High-resolution transcriptomic technology has revolutionized current understandings of B cell activation and differentiation in organized lymphoid tissues such as lymph nodes but has not been used to dissect B cell activation and differentiation in Peyer’s patches, important sites of intestinal immune induction. We utilized single-cell RNA sequencing and spatial transcriptomics to define pseudotemporal and spatial organization, respectively, of B cell activation and differentiation in porcine jejunal and ileal Peyer’s patches (JPPs and IPPs). IPPs had greater follicular mass and more defined follicular borders than JPPs, resulting in greater follicular B cell abundance in IPPs and non-follicular B cell abundance in JPPs. Despite follicular morphology and B cell abundances varying between JPPs and IPPs, pseudotemporal trajectories and predicted spatial locations of B cell activation and differentiation were similar between intestinal locations. Transition of resting into follicular B cells was demarcated by increased expression of follicle- and cell cycle-associated genes and movement of cells from interfollicular zones (IFZs) into follicles. Another trajectory indicated transition of resting B cells located in IFZs directly into antibody-secreting cells located in IFZs and crypts, suggesting a route of extrafollicular B cell activation and differentiation. Altogether, results indicate conserved patterns of both follicular and extrafollicular B cell activation and differentiation in Peyer’s patches across intestinal locations, despite differences in cellular abundances and follicular morphology.

Research was funded by (1) USDA-ARS CRIS project #5030-3200-225-00D, (2) the SCINet project of the USDA ARS project number 0500-00093-001-00-D, and (3) 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.
CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance
Authors: Ehrlich Lauren Ilyse Richie , Li Yu , Tipan Pablo , Srinivasan Jayashree , Selden Hilary J
Published: August 2023

Central tolerance ensures autoreactive thymocytes are either eliminated via clonal deletion or are diverted to the regulatory T-cell lineage, thus minimizing T cell autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their TCRs for autoreactivity against the diverse self-antigens displayed by medullary antigen presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection against some self-antigens, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, we use synchronized positive selection assays and 2-photon timelapse microscopy to demonstrate that CCR4 and CCR7 are expressed sequentially after positive selection and promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and promotes medullary entry and negative selection of mature thymocytes. Unlike CCR7 ligands, which are expressed by medullary thymic epithelial cells, CCR4 ligands are expressed by hematopoietic APCs, and we find that CCR4 enforces thymocyte tolerance to self-antigens presented by activated thymic APCs. Our findings demonstrate that CCR7 expression is neither necessary nor sufficient for thymocyte medullary entry and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of central tolerance, respectively, via interactions with distinct APC subsets.

Supported by a grant from the NIH/NIAID (R01AI104870)
Genetic tracing of thymic T cell differentiation
Authors: Jiang Menglin , Keith Julia , Zong Xinying , Hao Xiaolei , Li Yichao , Bai Lu , He Minghong , Guy Cliff , Li Jun , Huang Wenjun , Cheng Yong , Feng Yongqiang
Published: August 2023

Thymic T cell differentiation is controlled by stereotyped processes that remain obscure. Here we develop a novel mouse genetic tool to trace thymic T cells during T cell antigen receptor (TCR) rearrangement. Briefly, we insert inducible CreER into the Rag1 locus that is transiently expressed at early thymic T cell differentiation. In the presence of Rosa reporters, Rag1-CreER mice enable us to irreversibly label T cells between the double negative (DN) and double positive (DP) stages upon acute tamoxifen treatment. In the physiological setting and absence of the perturbations historically used to investigate thymic T cell differentiation, our genetic tracing in combination with flow cytometric measurement of cell fates and functional states maps the kinetics, stoichiometry, and cell fate determination of thymic T cells. We further integrate genetic tracing experiments with unbiased single cell RNA sequencing, TCR sequencing, and the expression of cell surface markers, linking thymic T cell differentiation stages to dynamic gene expression. In particular, we show that regulatory T cells acquire CD73 expression over time as a potential consequence of persistent TCR signaling and expression of lineage determinant Foxp3, marking a maturation process during lineage commitment and functional adaptation. Taken together, our integrated genetic tracing of thymic T cells resolves complex differentiation processes coupled with the alterations of gene expression, selection, and cell fate determination. Our results also serve as a foundation for further exploration of the thymic T cell states, developmental pathways, drivers, and modulators involved in these crucial biological processes.

R21 AI146614
Harnessing spatial transcriptomics to study fate decisions and lineage specification in T cell development
Authors: Patel Shil , Chopp Laura B , Nie Jia , Matta Jennifer , Kumar Parimal , Singh Jatinder , Cowan Jennifer , Shissler Susannah C , Clark Alexander D , Edmondson Elijah , Vacchio Melanie S , Sternberg Lawrence R , Bhandoola Avinash , Kelly Michael C , Bosselut Remy
Published: August 2023

T cells are critical components of the adaptive immune system; they develop from bone-marrow derived precursors that migrate to the thymus. There, precursors undergo a sequence of lineage specification and commitment steps that give rise to multiple T cell lineages serving essential functions in defenses against infection and immune homeostasis. The diversity of thymocytes, and their complex interactions with the thymic stroma, including epithelial cells and other hematopoietic compartments, have hampered our understanding of T cell development. For greater insight into intrathymic developmental trajectories and their transcriptional control, we and others have utilized single cell RNA- and ATAC-sequencing (scRNA-seq and scATAC-seq). However, single-cell approaches do not assess contextual cell-cell interactions. To address this limitation, we used in-situ transcriptomics, specifically the 10× Genomics “Visium” technology, which assays in-situ gene expression in 55-micron spots spanning whole mouse thymus lobes. We will present initial results of these experiments, including mapping of thymocyte differentiation stages, location of developmental bifurcations, and the spatial progression of lineage specific gene expression. Briefly, we use statistical models informed by scRNA-seq data to infer the location, relative to the thymic architecture, of lineage decisions. Additionally, we combine positional information of thymocytes and stromal cells to infer cell-cell interactions that direct lineage decisions. Collectively, this analysis sheds light on the interactions that guide development of the diverse T cell family from a common precursor and characterizes the journey of these cells to reach maturity.
Artificial thymic organoids generated with peripheral blood CD34+ cells represent a quick and powerful tool to determine the nature of severe T cell lymphopenia
Authors: Bosticardo Marita , Pala Francesca , Amini Kayla , Montel-Hagen Amelie , Crooks Gay M , Notarangelo Luigi D
Published: August 2023

The study of early T-cell development in humans is challenging because of the scarce availability of thymic samples and limitations of in vitro T-cell differentiation assays. We have previously shown that artificial thymic organoids (ATOs), generated by aggregating DLL4-expressing stromal cells with CD34+ cells isolated from bone marrow (BM) or mobilized peripheral blood (MPB), are a reliable tool to determine the hematopoietic-intrinsic or extrinsic nature of severe T-cell lymphopenia.

However, BM or MPB samples are not often available and delays in obtaining these samples might dangerously postpone the patient’s clinical management decision. We thus set out to evaluate whether we could obtain reliable results from ATOs generated with CD34+ cells isolated from PB. The main challenge of using CD34+ cells from un-mobilized PB is that the frequency of these cells is much lower than that found in BM or MPB. For the generation of ATOs with BM or MPB CD34+ cells we used a minimum of 5000 cells/ATO, but these numbers would not be feasible for PB samples. We thus generated ATOs with decreased numbers of CD34+ cells, and we determined that 1000 cells/ATO was the minimum necessary to achieve efficient T-cell differentiation. Additionally, we slightly modified the experimental protocol and added SCF for the first 3 weeks of culture, in addition to IL7 and FLT3L, which are present throughout the T-cell differentiation assay. We also established that we could efficiently generate ATOs when starting from fresh blood (up to 3–4 days) and a minimum amount of 5–7 ml, for infants and early pediatric samples. Reduced efficiency in ATO generation was however obtained when isolating CD34+ cells from PB drawn more that 5–6 days earlier or from frozen samples.

This work was supported by the Division of Intramural Research, NIAID, NIH.
A CCL5 chemokine trail laid down by migratory dendritic cells directs CCR5+ antigen-presenting monocytes into draining lymph nodes
Authors: Rawat Kavita , Tewari Anita , Li Xin , Mara Arlind B , King William T , Gibbings Sophie L , Nnam Chinaza F , Kolling Fred B , Lambrecht Bart N , Jakubzick Claudia V
Published: August 2023

Antigen presenting dendritic cells (DCs) and monocytes capture and transport antigens from barrier tissues for presentation to antigen-specific T cells in the draining-lymph nodes (LNs). While DCs enter LNs through afferent lymphatics in a CCR7-dependent manner, how exactly antigen-carrying monocytes reach LNs is less clear since monocytes do not express CCR7 and can also enter LNs via the bloodstream. In steady state, and following injection of several PAMPs, scRNA-seq data on LN mononuclear phagocytes identified LN resident versus migratory type 1 and type 2 conventional (c)DCs despite downregulation of DC subset-defining transcripts, such as Xcr1, Clec9a, H2-Ab1, Sirpa, and Clec10a on migratory cDCs. Migratory cDCs gained expression of transcripts controlling cellular migration such as Ccr7, Ccl17, Ccl22, and Ccl5, while migratory monocytes expressed Ccr5 without Ccr7. Using two tracking methods and a gating strategy that clearly distinguishes migratory CD88hiCD26lo monocytes from CD88−CD26hi cDCs, we found that both captured antigens in the lung and migrated to lung draining LNs. Using global and mixed-chimeric Ccl5-, Ccr2-, Ccr5-, Ccr7-, and Batf3-deficient mice, we found that CCR5+ monocytes follow CCL5-secreting migratory cDCs to reach the draining LN via lymphatic vessels. In a model of asthma, such recruited monocytes regulated the induction of type 2 immunity. Overall, our data suggest that CCL5-secreting migratory cDCs lay down the chemokine trail for CCR5+ antigenpresenting monocytes to reach draining lymph nodes and regulate adaptive immunity.

This work was supported by National Institutes of Health grants R01 HL115334, R01

HL135001, and R35 HL155458 (CVJ)
Investigate the impact of MHC-I cytoplasmic tail on CD8 T cell function and fate
Authors: Sun Yimo , Lizee Greg
Published: August 2023

Mutational studies have revealed that the cytoplasmic tail of MHC-I plays an important role in the generation of CD8+ T cell mediated immunity. Although the MHC-I tail contains highly conserved serine and tyrosine phosphorylation sites that can potentially harbor signals or ‘instructional payloads’, little is known about how these post-translational modifications are induced, or the impact they may have on immune responses. In this study, we established the artificial antigen presenting cells K562 that express HLA-A2 with different cytoplasmic tail mutations on serine and tyrosine sites. We demonstrated that the deletion of exon 7 on HLA-A2 tail led to a higher T cell killing in the 4-hour assay, while mutating serine 335 to glutamic acid (S335E) increased long-term T cell killing. We further found that S335E has the highest expression of HLA-A2 on K562 cell surface compared to wild type and all the other tail mutants. As a result, the robust CD8 T cell killing to S335E K562 cells can be partially explained by the high expression level of HLA-A2 on the cell surface. We then conducted a T cell priming experiment using cytoplasmic tail mutated KG-1 cells. All the tail mutants generated low number of antigen specific CD8 T cells from healthy donor PBMC when compared to the wild type. However, T cells primed by KG-1 that bearing tyrosine 320 mutation (Y320E) expanded at a higher level after sorting. Together, our data showed that the MHC-I cytoplasmic tail might have an impact on CD8 T cell priming and effector function through the conserved amino acids S335 and Y320.
Immune chaperone modulates dendritic cell functions in the tumor microenvironment
Authors: Mazzoccoli Luciano , Iwanowycz Stephen , Ngoi Soo , Hill Megan , Liu Bei
Published: August 2023

Dendritic cells (DCs) are central regulators of the adaptive immune response, thereupon necessary for T cell-mediated cancer immunity. To improve anti-tumor response, strategies to stimulate T cell response create new opportunities in DC cell biology, where DC maturation and antigen presentation are paramount. DC1 subtype is known for antigen-cross presentation to CD8+ T cells and the DC2 subtype to CD4+ T-cells. Previous studies showed that the immune chaperone GP96 plays a pivotal role in the processes of innate receptors. As a subject of interest from our lab, the GP96 client network offers potential targets to be explored for improving DC functions. We reported an increase in DC2 infiltration on the tumor side and a reduction of tumor growth in DC-specific GP96 deficient mice. Notwithstanding, the mechanism of GP96 in regulating DC functions is the subject of our study. By using in vitro approach, our study shows improvement in DC differentiation through gene expression assay, where DC subtypes display differential dependence on ER chaperone proteins. Also, the deletion of GP96 in DCs improves immunostimulatory activation and inhibits regulatory functions. Lastly, GP96 deficient DCs increased OVA antigen uptake and showed less T-cell exhaustion profile from in vivo tumor model. Directly DC activation or bypassing regulatory pathways can unleash T-cell response. Our study provides new insights into the role of GP96 in DCs in directing adaptive immune responses in the tumor microenvironment.

This work was supported by the Pelotonia Institute of Immuno-Oncology (PIIO). The work was also supported in part by NIH/NCI (CA193939) and NIH/NIAID (AI125859) and supported by the Flow Cytometry Shared Resource at The Ohio State University Comprehensive Cancer Center and NIH/NCI P30 (CA138313).
Investigating the role of Quaking in dendritic cells
Authors: Li Yating , Hu Jian , Yee Cassian
Published: August 2023

Dendritic cells (DCs) are essential players in the anti-tumor immunity, which are often found dysfunctional in tumor microenvironment. Impairing DC antigen processing and presentation, which subverts T cell priming, is one of the primary mechanisms by which tumor cells escape the immune surveillance. However, the cellular factors regulating antigen processing and presentation by DCs and how tumor cells render DCs’ T cell priming capacity are not fully understood.

We revealed a potential regulatory role of an RNA-binding protein, Quaking (QKI), in antigen processing and presentation by DCs. Consistent with our previous studies in neural stem cells and microglia, we found QKI could regulate the phagocytosis by promoting the formation of phagosome and endosomes in DCs. QKI-deficient DCs showed reduced CD8+ T cell priming capacity in vitro. Furthermore, knocking out QKI in DCs accelerated the tumor growth in vivo. Together, these suggest the pivotal role of QKI in DC function and further enhance our understanding of how antigen processing and presentation is regulated in DCs.

In addition, QKI is often found to be downregulated in tumor-infiltrating DCs, as compared to the DCs in normal tissues. How tumor cells impair DC function by suppressing QKI expression is currently under investigation. This will reveal a new mechanism by which tumor cells modulate anti-tumor immunity and can provide new strategies to target DCs for improving cancer immunotherapy.
Characterization of the B Cell Receptor Repertoire and Discovery of New Immunoglobulin Genes in the Domestic Ferret
Authors: Hebert Luke S , Lavinder Jason J , Soto Gabriel , Ross Ted , Moody Tony A , Ippolito Gregory C , Seeger Allison E
Published: August 2023

Mustela putorius furo uniquely reflects physiological and immunological changes in human aging such as immune imprinting from influenza infection, yet key ferret immunobiology knowledge gaps limit the scope of molecular analyses. First, ferret germline B cell receptor genes require identification. Additionally, the V, D, and J gene repertoire expressed by B cell receptors in antigen-experienced cells needs characterization.

Illumina NGS was used to generate B-cell receptor mRNA data sets. A candidate list of gene segments was assembled including ferret genomic accessions homologous to known immunoglobulin genes and previously-known ferret immunoglobulin genes. NGS reads were queried against the candidate list using IgBLAST. IgBLAST hits were focused into consensus sequences using principal components analysis and K-means clustering. To identify IgG subclasses, putative ferret genome sequences sharing human IgG C genes were used to query Oxford Nanopore data that covered the FR3 through C gene area of transcripts.

We report two new functional IGHV, two IGKV, and one IGLV gene segments in the ferret, with more sequences likely to surface in downstream analyses. We also corroborate most already-proposed functional V, D, and J gene segment sequences. Ferret orthologues to human IGHV subgroups commonly involved in stem-directed, broadly neutralizing antibody responses were identified. For the first time, productive IgG3 was described in the ferret model and distinguished from productive IgG1.

Our report of novel ferret immunoglobulin genes will disinhibit the ferret as a model organism for the study of infectious disease. Specifically, it will inform the immunogenicity of candidate HA antigens in preclinical vaccine studies.

Research reported in this abstract was supported by NIH NIAID contract 75N93019C00050.
Investigating the functional roles of TNFSF13 family members in Cartilaginous Fishes
Authors: Hill Thomas , Redmond Anthony , Dooley Helen
Published: August 2023

The TNFSF13 cytokines BAFF and APRIL along with their cognate receptors BAFFR, TACI, and BCMA play critical parts in the development, function, and maintenance of B cells in mammals. Despite the well-studied roles that BAFF and APRIL play in the immune systems of higher vertebrates, their evolutionary history and functions in non-mammalian organisms are not well understood. Recently, our lab has demonstrated that collectively the TNFSF is evolutionarily ancient, with most of its members having evolved over 450 million years ago in the jawed vertebrate ancestor. Interestingly, we found that despite the evolutionary ancient position of cartilaginous fishes, their repertoire of TNFSF is just as complex as higher vertebrates with evidence of lineage specific TNFSF members that are absent in more evolutionary recent taxa. Specifically, cartilaginous fishes possess two additional TNFSF13 family members, namely BALM (TNFSF13C) and a retrotransposed duplicate of BAFF (iBAFF). Thus, cartilaginous fishes possess four TNFSF13 members whose functional roles remain unexplored. Here we demonstrate the tissue specific expression of BAFF, iBAFF, BALM, and APRIL, as well as their anticipated receptors BAFFR, TACI, and BCMA in the nurse shark. Additionally, we explore receptor-ligand interactions using a yeast surface display assay aiming to understand the role(s) each plays in the development and maintenance of shark B cells. Such information is key to understanding the evolutionary history of this key cytokine family.
The biology and evolution of the unconventional γμ T cell in non-eutherian mammals
Authors: Morrissey Kimberly A , Stammnitz Maximilian , Miller Robert D
Published: August 2023

There are three lineages of T cells in mammals, αβ, γδ and γμ T cells, classified by the composition of their T cell receptors (TCR). γμ T cells are present only in marsupials and monotremes. The γμTCR has a number of unique features, most distinctly is the three extracellular-domain TCRμ chain. In this study we investigate the evolution of the TCRμ genes and characteristics of γμ T cells in different species. γμ T cells appear to have tissue specific residency. In adult opossums, Monodelphis domestica, they are restricted to the spleen, liver, throughout the gut, and mesenteric lymph nodes. By comparison, in the Tasmanian devil, Sarcophilus harrisii, there was also tissue specific residency in liver, spleen, bone marrow, and skin. In opossums and Tasmanian devils, the TRM locus is organized as discrete, tandem clusters, and there is evidence of lineage specific cluster duplication. In a monotreme, the platypus, TRM is less organized, although there is evidence of ancestral cluster-like patterns. Our results support conserved patterns of tissue specific residency of the unconventional γμ T cell in marsupials and help to reconstruct the evolution of this unique T cell type.

NSF IOS-2103367 DGE-1939267
Changes in MMΦ Phenotype Influence Early Life Development of the Enteric Nervous System
Authors: Joyce Elisabeth L , Schill Ellen M , Gaddipati Shreya , Floyd Alexandria N , Rusconi Brigida , McDonald Keely G , Newberry Rodney D
Published: August 2023

Vancomycin is a common medication given to premature infants. While protective, this treatment is associated with increased risk of developing disease later in life, including late-onset sepsis and necrotizing enterocolitis (NEC). Previous studies demonstrated that neonatal mice exposed to vancomycin in the early life period exhibit disruptions in the structure and function of the enteric nervous system (ENS). It remains unclear whether these disruptions are a direct consequence of dysbiosis or an indirect result achieved via crosstalk with immune cells. Muscularis macrophages (MMΦs) are a subset of intestinal macrophages identified to interact closely with enteric neurons. We hypothesized that vancomycin impacts ENS structure and function in early life by altering the phenotype of MMΦs. Neonatal mice were fed vancomycin (83mg/kg/day) or water daily from day of life (DOL) 1–10 and sacrificed for analysis on DOL 10. Single cell RNA sequencing performed on isolated MMΦs (CD45+ CXCR1+CD11c−/lo) demonstrated that vancomycin-treated mice had macrophages with a more inflammatory phenotype. Consistent with our RNA seq, MMΦs in mice treated with vancomycin had changes in their morphology consistent with activated macrophages/microglia. Flow cytometry revealed that vancomycin treatment significantly altered the proportion of MMΦs that were yolk sac derived compared to the bone marrow. To investigate how early life bone marrow derived MMΦs contribute to postnatal ENS development, we utilized CCR2 minus;/minus; mice, which cannot recruit macrophages to the colon. These mice exhibited significantly fewer neural cell bodies, suggesting that antibiotic-driven changes in MMΦ origin may influence early postnatal development of the ENS.
Rapid adipose tissue expansion induces proliferation of memory adipose tissue T cells
Authors: Jacks Ramiah , Lumeng Carey N
Published: August 2023

Obesity is recognized as a pro-inflammatory state featuring immune dysregulation and metabolic disease. Obesity-induced inflammation is observed in the adipose tissue of humans and mice. Adipose tissue T cells (ATTs) are dynamically shifted from Th2 cells in the lean state to an influx of CD8+ and Th1 T cells that promote adipose tissue inflammation in the obese state. Knowing this, many gaps remain in our understanding of ATT generation, maintenance, expansion, and regulation in adipose tissue. Specifically, it is unknown if naïve T cells are recruited to and stimulated within adipose tissue or if resident memory ATTs, which are abundant in the lean state, are stimulated during the initiation of the obese state. We hypothesized that memory ATTs respond to the early fat expansion elicited upon obesity induction. To test this, we used a short-term DIO male mouse model and assessed ATT proliferation during high-fat diet (HFD) feeding. Mice on HFD gained significantly more total body, epidydimal white adipose tissue (eWAT), and inguinal WAT (iWAT) weight compared to normal diet mice. We observed no increase in frequency or proliferation of naïve ATTs with HFD. Conversely, we observed significant proliferation of memory ATTs with 1 week of HFD in the eWAT, iWAT and omental WAT. This proliferation was restored to normal diet levels by 2 weeks of HFD. These data suggest that memory ATTs uniquely and intensely respond to signals present during HFD-induced rapid adipose tissue expansion. Ongoing studies seek to determine if there are sex differences by examining ATT dynamics in female mice. Further, we seek to determine which signal(s) drive memory ATT proliferation to identify targets to suppress ATT initiation of obesity-associated inflammation.

Supported by grants from NIH (K12 GM111725, R01 DK090262).
Immunotherapy of melanoma using TRP2 peptide-loaded biodegradable nanoparticles combined with poly-IC
Authors: Park Ji-Hyun , Lee Hee-Sung , Kim Sang-Hyun , Park Chan-Su , Lee Chong-Kil
Published: August 2023

Vaccination with tumor epitopes associated with MHC-I is an attractive approach directed at inducing tumor-specific CTLs. However, challenges remain in improving the therapeutic efficacy of peptide epitope vaccines, including the low immunogenicity of peptide epitopes and insufficient stimulation of innate immune components in vivo. Herein, we report an innovative strategy to overcome the limitations. The essential feature of this strategy is vaccination using tumor epitope-loaded biodegradable nanoparticles (NPs) in combination with poly-IC. Carboxylated NPs were prepared using poly(lactic-co-glycolic acid), covalently conjugated with anti-H-2Kb mAb, and then attached with H-2Kb molecules. Native peptides associated with the H-2Kb molecules of H-2Kb-attached NPs were exchanged with melanoma-specific peptide TRP2(180–188). TRP2 peptide-loaded NPs in combination with poly-IC efficiently induced TRP2-specific CTLs when immunized into melanoma-bearing mice as well as normal mice. Accordingly, TRP2 peptide-loaded NPs in combination with poly-IC exerted significant anti-tumor activity in mice implanted with B16-F10 melanoma cells. In addition, TRP2 peptide-loaded NPs in combination with poly-IC exerted potent anti-metastatic activity in mice i.v. injected with B16-F10 melanoma cells. These findings show that TRP2 peptide-loaded NPs combined with poly-IC could be an effective means in treating melanoma via the induction of TRP2-specific CTLs.
IAV/bacteria coinfection causes changes in pathology in an in vitro model of the lung epithelium
Authors: Zambrano Raphael Angelo I , Fitzgerald Ethan , Jamieson Amanda
Published: August 2023

Infection by the Influenza A Virus (IAV) remains a persistent issue contributing to increased risk of morbidity and mortality, especially for susceptible individuals. IAV infection can lead to increased susceptibility to bacterial infections, which further exacerbate disease. Therapeutics targeting pathogens are often ineffective as pathogens can mutate to avoid potential vaccines and antimicrobials. Improving our understanding of IAV/bacteria-host interactions can allow us to improve host-specific approaches to tackle potential morbidity and mortality brought on by coinfections that are primarily found in epithelial cells. Lung epithelial cells not only serve as an integral part of the respiratory system, but are also crucial in host defense against respiratory infections. Therefore, we adopted an Air-Liquid Interface (ALI) strategy for the culture of human bronchial epithelial cells (HBEC) to mimic the structure and function of respiratory epithelial tissue. ALI cultures were then subjected to single and coinfections by IAV and Streptococcus pneumoniae to assess the resulting pathology. We discovered that the specific type of infection influenced the type of programmed cell death that occurred, ranging from apoptotic to necroptotic. Additionally, coinfection with IAV and bacteria caused a unique gene expression profile. These findings demonstrate that the presence of specific pathogens causes a significant change in the cell death characteristics as well as transcriptional profile of lung epithelial cells. We hope that the progress of our investigation further elucidates consequences of host-pathogen interactions that can contribute to developing novel therapeutic strategies for complex lung infections.

Supported by grants from the NIH, NHLBI R01HL126887 (A.J.), P20GM121344 Pilot Project (A.J.), and under award number RL5GM118975 (R.Z.) of NIGMS.
Mosquito saliva alters draining LN architecture and function
Authors: Brisse Morgan E , Valenzuela Jesus , Olivera Fabiano , Hickman Heather D
Published: August 2023

Current murine models of most arbovirus infections result in the development of viremia and antiviral adaptive immune responses. However, delivery of virus by injection via syringe does not fully encompass all aspects of the response that develops after viral delivery via mosquito bite. One reason for this is likely to be the lack of additional immunomodulatory responses specifically induced by mosquito saliva as previous studies have demonstrated profound proviral and immunosuppressive properties of saliva. While the adaptive antiviral response develops in the lymph node draining the site of infection, the impact of mosquito saliva on this critical lymphoid organ has not been extensively explored. Further, the lymphatic system acts both as centralized hubs for immune cell activation and as dissemination pathways to distal tissues, meaning that immunomodulation of the lymphatic system likely affects infection and immunity systemically. Therefore, we explored the effect of mosquito saliva on the structure and function of the draining lymph node in the presence of absence of the mosquito borne arbovirus Zika virus (ZIKV). Preliminary confocal microscopic analyses of lymph nodes after saliva injection into the skin revealed changes in nodal architecture and specific cellular populations. Future studies will explore the impact of saliva specifically on lymphatic endothelial cells within the lymph node. These studies should provide additional insight into the mechanisms of bite-induced immunomodulation in secondary lymphoid organs and the impact on the developing antiviral response.

Supported by the Intramural Research Program of NIAID, NIH (H.D.H.)
Design and Characterization of Bivalent Molecules Against SARS-CoV2
Authors: Ahmad Javeed dhobi , Jiang Jiansheng , Perks Richard W , Lafont Bernard , Burdette Tracey , Reed Johnson , Muslinkina Lisa , Boyd Lisa , Natarajan Kannan , Margulies David
Published: August 2023

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compelled extraordinary attention to the development of therapeutic reagents. The favorable physical properties of nanobodies make them attractive candidates for investigations of their ability to block viral entry. By X-ray crystallography we determined the structural basis of the interaction of a panel of synthetic nanobodies (Sb14, Sb16, Sb45 and Sb68) with the SARS-CoV-2 receptor binding domain (RBD): binary complexes of Sb16–RBD and Sb45–RBD; a ternary complex of Sb14–RBD–Sb68, and Sb45–RBD–Sb68. Sb16 and Sb45 bind the RBD with identical footprints with ACE2, but their complementarity determining regions (CDR)2 and CDR3 are oriented diametrically oppositely. The ternary structure of the Sb45-RBD-Sb68 complex indicates that Sb45 binds squarely at the ACE2 interface, whereas Sb68 attaches toward the ACE2 interface’s perimeter. The ternary configuration suggests that several sybodies may capture a sizable portion of the RBD’s surface. These sybody structures provided structural insights that were used to create novel, highly potent biparatopic nanobodies and bivalent molecules with ACE2 and ACE2 binding scaffold mimicking miniproteins that bind the RBD with high affinity and neutralize SARS-CoV-2 and variants.

Supported by the Intramural research program of the NIAID/NIH
Altered epithelial function in the stria vascularis and hearing loss in a mouse model of congenital cytomegalovirus infection associated hearing loss
Authors: Smith Matthew , Britt William
Published: August 2023

Congenital Human Cytomegalovirus (HCMV) infection is a leading cause of sensorineural hearing loss (SNHL) in children. The mechanism of HCMV damage to the developing auditory system that leads to SNHL is not understood and effective treatments are lacking. We developed a murine model of HCMV infection that results in neurodevelopmental abnormalities and SNHL, thus providing a useful model to study the mechanisms of SNHL associated with cCMV infection. We have shown that MCMV induces expression of proinflammatory molecules and immune cell activation in the cochlea associated with histopathologic changes and SNHL. The stria vascularis (SV) plays a critical role in hearing by; (1) maintaining an epithelial barrier needed for generation of the high electrical potential of endocochlear fluid surrounding the hair cells; (2) expression of a system of ion channels that create a high K+ concentration in the endocochlear fluid; and (3) generation of a blood labyrinth barrier (BLB) that surrounds the vasculature in the SV and functions like the blood brain barrier. Utilizing several assays, our studies showed that MCMV infection altered each of these functions. The transcription and protein expression in the SV required for tight junctions in the epithelial barrier and ion channels were decreased early after infection and in SV from infected mice with SNHL compared to those without SNHL. In addition, the resident macrophages that contribute to the BLB had altered phenotypes. Functionally, penetration of a cell-impermeable tracer into the SV could be demonstrated in MCMV infected mice. In all, our data show that MCMV dysregulates expression of key proteins in the SV that in turn impact the capacity of the SV to maintain the cochlea’s unique environment.
Rab27-dependent extracellular vesicle releasing participates in dengue virus non-structural protein 1 secretion
Authors: Wan Shu-Wen , Cheng Miao-Huei , Lee Yan-Hua
Published: August 2023

Dengue disease is one of important arthropod-borne disease in the world and causes around 20,000 deaths annually. Dengue virus (DENV) non-structural protein 1 (NS1) is the major secreted non-structural protein from infected cells and highly associated with the pathogenesis of severe dengue. Several studies have shown that the secreted NS1 contribute to increase of vascular permeability, coagulopathy, activation of immune cells as well as inflammation. These findings strongly indicate the pathological roles of secreted NS1; however, the secretory pathway is not fully clear. Accumulating evidence have shown that virus-associated extracellular vesicles (EVs) not only contain viral components including viral proteins, genetic material or virion particle but also host components. Rab27 is one of several GTPases essential to regulate EV releasing by affecting docking of multivesicular body (MVB) to the target plasma membrane. Here, we show that NS1 can be exported from DENV-infected cells by EVs. NS1 as well as EV biogenesis-associated proteins ALG-2-interacting protein X (ALIX) and neutral sphingomyelinase2 (nSMase2) co-accumulate in extracellular vesicles. We next determine the topology of NS1 and demonstrate that NS1 is associated at the surface of EVs. In addition, the secretion of DENV NS1 is regulated by Rab27. The EV biogenesis and secretion inhibitor, Tipifarnib treatment decreases the levels of NS1 and nSMase 2 but not the levels of ALIX in the EVs. Taken together, this study not only provide the new insights of mechanisms in DENV NS1 secretion through Rab27-mediated vesicle trafficking but also valuable therapeutic targets to dengue disease.

Supported by grants from Ministry of Science and Technology of Taiwan (MOST 111-2320-B-006 -051 -MY3)
Long-term metabolomic alterations during post-acute sequelae of COVID-19
Authors: Wong Andrea C , Devason Ashwarya , Umana Iboro C , Cox Timothy , Dohnalová Lenka , Meyer Nuala J , Abramoff Benjamin , Cherry Sara , Thaiss Christoph , Levy Maayan
Published: August 2023

A large fraction of COVID-19 patients does not fully recover from the acute infection with SARS-CoV-2 but develops long-term symptoms that persist for several months. The pathobiology of such post-acute sequelae of COVID-19 (PASC, “long COVID”) remains largely enigmatic. This project is based on a discovery made by a plasma metabolomics exploration of individuals with acute SARS-CoV-2 infection, post-COVID patients that have fully recovered from the disease, and PASC patients that have persistent manifestations across a wide range of organ systems. We have found key metabolites that are altered by acute SARS-CoV-2 infection and do not recover in individuals with PASC, while returning to normal levels in post-COVID individuals who do not develop long-term symptoms. These metabolites offer a pathway-level hypothesis for a new molecular etiology underlying PASC. We have shown that viral RNA is sufficient to alter these metabolites and suggest that this can explain some of the symptoms reported by patients with long covid.

A.C.W. is supported by training grant T32-AI-055400-19. C.T. is a Pew biomedical scholar and a Kathryn W. Davis Aging Brain scholar and is supported by the NIH Director’s New Innovator Award (DP2-AG-067492), the Agilent Early Career Professor Award, and grants from the Edward Mallinckrodt Jr Foundation, the IDSA Foundation, the Thyssen Foundation, PennCHOP Microbiome Program, Penn Institute for Immunology, Penn Center for Molecular Studies in Digestive and Liver Diseases (P30-DK-050306), Penn Skin Biology and Diseases Resource-based Center (P30-AR-069589), Penn Diabetes Research Center (P30-DK-019525), Penn Institute on Aging and the Borrelli Family Pilot Grant in Lynch Syndrome. M.L. is supported by the NIH Director’s New Innovator Award (DP2-AG-067511), an American Cancer Society Scholar Award, The Pew Scholar Award, the Searle Scholar program, the Edward Mallinckrodt Jr Foundation and grants from the Abramson Cancer Center, The Penn-CHOP microbiome program, Penn Coronavirus Center Pilot, Penn Institute for Immunology, Penn Center for Molecular Studies in Digestive and Liver Diseases, Penn Center for Precision Medicine, Penn Institute on Aging, Penn Center of Excellence in Environmental Toxicology (P30-ES-013508) and the Borrelli Family Pilot Grant in Lynch Syndrome.