bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.