bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identifiation of novel effector regulatory T cells through simulaneous single cell epigenetic and transcriptomic profiling
Authors: Nam Hyo Jeong , Kim Yong-Hee , Lee Woochan , Byun Nari , Yoon Jeong Heon , Kim Yong Chan , Chung Hyunwoo , Jeong Soyoung , Joh Christine , Kang Sung-Jun , Park Kyunghyuk , Cha Bukyoung , Kim Jong-Il , Park Chung-Gyu , Kim Hyun Je 
Published: August 2023

Abstract
Regulatory T cells (Tregs) harness a central role in peripheral immune tolerance. Since its discovery, there have been numerous efforts to dissect the role of Tregs in the context of disease condition. Recently, Treg fields are moving from the academia to industry since many evidences have been accumulated for the potential of treatment of inflammatory disease by administering genetically engineered Tregs. However, in the pre-clinical or clinical trials, this rare cell subset needs to be expanded in ex-vivo condition to achieve a sufficient number for the controlling of the disease. Our group previously established ex-vivo Treg expansion condition with 25 mer oligonucleotide to maintain the stability and function of Tregs. However, non-biased investigations on the perturbation of transcriptomic and epigenetic changes during the culture condition has not been explored. Since cutting edge multiomics tools which can simultaneously profile epigenetic and transcript signatures in a single cell resolution has been commercialized, we adopt 10x multiome technique to investigate our frozen ex-vivo expanded Tregs(eTreg) compared to freshly isolated frozen human Treg(fTreg) from the same donor. Compared to fTreg cells, we have identified a new subset that exists only in the eTreg cells and named it eeTreg. We found that the eeTreg resembles the phenotype of both effector Tregs and tumor-infiltrated Tregs. eeTreg is characterized by a significantly increased expression and chromatin accessibility score of FOXP3, STAT5B, ETS1 and RUNX1, which are known to be the essential transcription factors of regulatory T cell function. In summary, we suggest a novel human treg subset which might be manufactured by manipulating ex-vivo culture condition.
PD1 blockade improves survival during lethal microbial exposure in old mice
Authors: Dahlquist Korbyn J V , Huggins Matthew A , Yousefzadeh Matthew J , Soto-Palma Carolina , Cholensky Stephanie H , Pierson Mark , Hamilton Sara E , Camell Christina D 
Published: August 2023

Abstract
The elderly and those with chronic diseases have a 2 to 10-fold increased risk of hospitalization and mortality following infection as compared with healthy adults. A significant contributor to disease severity is the aging of the immune system, including an expansion of exhausted T cells. These cells simultaneously promote excessive levels of inflammation and display impaired T cell activation and function. How exhausted T cells from aged individuals respond to infection and whether acute restoration of T cell activation further exacerbates the inflammatory environment or generates functional T cells is unclear. Previously, we used a mouse model of normal microbial exposure (NME), where old specific pathogen-free (SPF) mice show 100% mortality following exposure to multiple microbes ordinarily found in pet store mice. In the present study, we show that when exposed to NME, old SPF mice have increased expression of multiple inflammatory pathways and exhibit elevated frequencies of a heterogenous, exhausted CD8+ T cell pool that expresses differing combinations of programmed cell death protein 1 (PD1), TOX, and CXCR5. Pre-treatment or intervention with an anti-PD1 monoclonal blocking antibody during the exposure of old SPF mice to NME significantly improves both antibody production and survival, without altering the acute inflammatory response. Anti-PD1 checkpoint blockade-mediated survival is dependent on CD8+, but not CD4+ T cells. Correspondingly, CD8+ PD1+ T cells from old mice given anti-PD1 monoclonal antibody have increased granzyme B production. These data reveal a new approach for reducing vulnerability to infections in the elderly by targeting CD8+ T cell exhaustion through PD1 checkpoint blockade immunotherapy.
This work was supported by National Institute on Aging grant R00 AG058800 (C.D.C.), R21AG078638, the Fesler-Lampert Chair in Aging Studies, the Glenn Foundation for Medical Research/AFAR Grants for Junior Faculty, Irene Diamond Fund/AFAR Postdoctoral Transition Award (M.J.Y) and the Medical Discovery Team on the Biology of Aging.
PD-1 protects expanding human T cells from premature restimulation-induced cell death
Authors: Lee Katherine , Elster Sara , Lake Camille M , Snow Andrew L 
Published: August 2023

Abstract
Programmed cell death-1 (PD-1) is a co-inhibitory protein that dampens TCR/CD28 signaling in part by recruiting the phosphatase SHP2 to the immunological synapse. Chronic antigenic restimulation of T cells can induce apoptosis or elicit PD-1-mediated exhaustion, rendering them hyporesponsive and ineffectual. While the checkpoint function of PD-1 has profound implications for cancer immunotherapy, the role of PD-1 in conventional effector T cell responses remains unclear. Restimulation-induced cell death (RICD) is a critical propriocidal apoptosis program triggered in activated T cells upon robust TCR re-engagement, constraining effector T cell expansion and longevity to prevent collateral tissue damage. We hypothesized that PD-1 attenuates RICD sensitivity in human effector T cells by modulating TCR signal strength. Here we show that transient upregulation of PD-1 helps to protect newly activated human CD4+ and CD8+ T cells from premature RICD during clonal expansion, with lesser effects noted in terminally differentiated T cells. Both antibody-mediated blockade of PD-1-PD-L1 and SHP2 inhibition increased RICD sensitivity to a similar extent. Conversely, concomitant addition of recombinant PD-L1 with TCR restimulation significantly reduced RICD in a dose-dependent manner, with more pronounced protection noted uopn CD28 coligation. Our ongoing work aims to define the role of recently discovered downstream PD-1 signaling nodes (e.g. PAG, VRK2) and specific pro/anti-apoptotic proteins in this process. These findings present significant implications for understanding how PD-1 blockade may influence apoptosis sensitivity in both normal and exhausted T cells to alter adaptive immune responses.
Supported by grants from NIH (R35GM139619)
Ets1 represses a T-bet dependent migration program and stabilizes the iNKT1 adhesome
Authors: Morgan Roxroy C , Bartom Elizabeth T , Sigvardsson Mikael , Maienschein-Cline Mark , Kee Barbara L 
Published: August 2023

Abstract
iNKT cells are innate-like T cells that recognize glycolipids presented on MHCI-like CD1d using a semi-invariant TCR. Upon activation, iNKTs rapidly produce cytokines to direct the adaptive response towards a TH1, TH2 or TH17 response. The transcription factor ETS1 is important for iNKT cell development, but its roles in iNKT function and homeostasis are not well characterized. Utilizing a novel mouse model for targeted deletion of Ets1 in iNKT1s, we show that ETS1 limits expression of T-bet, the signature transcription factor of the iNKT1 lineage. Ets1Δ/Δ hepatic iNKT1 cells upregulated a gene program associated with migration and had reduced expression of LFA-1 and ICAM-1, receptors critical for retention in the liver. Remarkably, Ets1Δ/Δ liver iNKT1s remained tissue resident but no longer required LFA-1/ICAM-1 for retention. Heterozygous deletion of T-bet abrogated expression of multiple migration-associated genes, restored LFA-1 and ICAM-1, and re-established reliance on LFA-1/ICAM-1 for retention. Our data demonstrate that ETS1 controls the mechanisms of hepatic iNKT1 cell residency by limiting a T-bet-dependent migration and adhesion program.
Supported by NIH/NIAID grant R01 AI106352
Characterizing the Immune Cell Transcriptomic Response To Low-Dose Ionizing Radiation
Authors: Marr Bryan , Khan Abrar , Blimkie Melinda , Pashangzadeh Salar , Azzam Edward , Laakso Holly , Lee Seung-Hwan 
Published: August 2023

Abstract
Low-dose exposure to ionizing radiation is increasingly common in medical, industrial, and public settings worldwide. Previous studies examining the impacts of low-dose radiation (LDR) exposure on genetic material, cellular responses, and health have been inconclusive and conflicting. The National Academy of Science, Engineering and Medicine has outlined that future LDR research should utilize single-cell omics technologies and computational workflows to define the molecular signatures of LDR exposure. In partnership with the Canadian Nuclear Laboratories (CNL), we have used single-cell RNA sequencing (scRNA-seq) to characterize the in-vivo immune cell response to chronic LDR. Mature adult (18 weeks-old) C57BL/6 female mice were whole-body-exposed to 60Co gamma radiation for 7 days, at dose rates of 0.06 mGy/h or 0.6 mGy/h, to achieve cumulative absorbed doses of 10 mGy (n=6) or 100 mGy (n=6), respectively. scRNA-seq of cells isolated immediately post-irradiation identified subtle candidate transcriptomic changes caused by LDR exposure in splenic and bone marrow cell populations. As the first scRNA-seq study of in-vivo LDR exposure, we reveal a more detailed description of the cellular response to LDR exposure than available in the current literature. Therefore, dissemination of these results will advance radiation biology and will be valuable for evaluating Nuclear Safety guidelines.
The research is funded by the CANDU Owner’s Group and the Mitacs Accelerate program.
Hypoxia promotes cytolytic activity of CD8 T cells and pathogenesis in cutaneous leishmaniasis
Authors: Fowler Erin , Amorim Camila Farias , Hales Emily D S , Varkey Aditi , Salem Mariam , Xin Gang , Collins Patrick L , Novais Fernanda 
Published: August 2023

Abstract
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, which is critical to develop therapies to ameliorate pathology. 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 the skin 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 but not dLN have a hypoxic signature. In vivo, we observed that CL 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, and significantly decreased CD8 T cell production of IFN-γ. In vivo, blocking hypoxia-inducible factor (HIF), a master regulator of hypoxia, with acriflavine decreased lesion development in mice. Mice with CD8 T cell deficient in HIF-1α produced significantly less GzmB in the skin lesions compared to wildtype mice. Together, our results suggest that the hypoxic signaling through the transcription factor HIF in CL lesions is necessary to convert CD8 T cells into pathogenic CD8 T cells in the skin lesion.
Supported by grants from NIH (1R01AI162711)
Protective Role of IL-17 in Pulmonary Tuberculosis Relapse
Authors: Clark Lucinda Puebla , Martinez-Martinez Yazmin Berenice , Huante Matthew B , Chauhan Sadhana , Files Megan A , Endsley Janice J 
Published: August 2023

Abstract
Tuberculosis (TB) recurrence following drug treatment and apparent clinical cure affects an estimated 700,000 people annually and is increasingly associated with relapse of initial Mycobacterium tuberculosis (Mtb) infection. Cellular immunity by T helper (Th) cells, including Th17 populations is important during active TB. The contribution of Th17 and other Th populations to immune pressure and Mtb clearance following TB chemotherapy is poorly understood. We used Mtb-HN878 for a paucibacillary model of TB infection in WT, IL17RA-KO, and second study using WT and conditional Th17-KO (CD4-Cre-RORγtflox/flox) mice to identify the cellular sources and protective role of IL-17 in TB relapse following drug therapy. Assessment of disrupted lung by multi-parameter flow cytometry demonstrated that Th17 populations expand during active TB, retract after drug therapy, and subsequently display a moderate re-expansion during relapse. Flow cytometric analysis also revealed expansion and contraction of other IL-17+ cells among both the non-lymphocyte and CD3− lymphocyte populations in the lung during active, post-drug, and relapse phase of TB. Compared to WT, the lung of IL-17RA-KO and conditional Th17-KO mice had greater mycobacterial burden during TB relapse. These results demonstrate a critical role for Th17 cells and IL-17 signaling in protection against Mtb regrowth following TB chemotherapy. Further, it suggests that both lymphoid and non-lymphoid cells of the innate immune system also contribute to IL-17-mediated protection during active and relapse TB.
NHI grant R01AI147948
TcpC affects the grazing resistance of Escherichia coli to the social amoeba Dictyostelium discoideum
Authors: Hamilton Sarah , Akingbehin Victoria , Palm Cheyenne , Snyder Michelle 
Published: August 2023

Abstract
Uropathogenic Escherichia coli (UPEC) is among the most common causative agents of urinary tract infections (UTIs). UPECs survive in hosts using various mechanisms, including by secreting a protein known as TcpC, which is a Toll/interleukin-1 receptor (TIR) domain containing protein. In mammals, TIR domains are important components of Toll like receptor (TLR) signaling pathways that activate innate immune responses. TcpC interacts with components of TLR signaling, inhibiting activation of the immune system and preventing the detection and destruction of the bacteria in mammalian hosts. We hypothesize that TcpC also affects the survival of UPECs in model systems, including the social amoeba Dictyostelium discoideum and planaria flatworms. To test this hypothesis, we are using mutant strains of the UPEC CFTO73 that have altered expression of TcpC to analyze the effects of TcpC on bacterial growth and virulence in model organisms. Our initial tests with D. discoideum indicate that the presence of TcpC in UPEC strains may increase their resistance to grazing and phagocytosis by the social amoeba D. discoideum. If TcpC affects survival of UPECs in model systems such as D. discoideum, this may open new avenues for characterization of the molecular mechanisms used by bacterial TIR domains to evade innate immune cells.
VA was supported by NSF LSAMP Award #1619676 and CP was supported by the NIH Bridges to Doctorate Scholars Program, NIH- R25 GM119970.
Antibiograms of select anaerobic bacteria over 10 years
Authors: Sterner Rosalie , Shannon Samantha , Estes Lynn , Norgan Andrew , Schuetz Audrey 
Published: August 2023

Abstract
Antibiotics are critical complements to the immune response in combatting infection. Anaerobic bacteria are increasingly common causes of infection, and antibiotic resistance of anaerobes has been rising. Most laboratories do not perform antimicrobial susceptibility testing of anaerobes, and even fewer have compiled antibiogram data. Here we investigated antimicrobial susceptibility results of anaerobes from January 2012 to December 2021 obtained from inpatients at Mayo Clinic using reference agar dilution testing. Actinomyces (60–70 isolates annually), Bacteroides fragilis (30–80), Finegoldia magna (40–60), and Cutibacterium acnes (70–110) were included. Penicillin, clindamycin, metronidazole, ertapenem, piperacillin-tazobactam, and moxifloxacin were tested. Clinical and Laboratory Standards (CLSI) breakpoints were followed. B. fragilis showed ≥90% and Actinomyces 100% susceptibility to piperacillin-tazobactam and ertapenem. All F. magna and &gt;95% B. fragilis were susceptible to metronidazole. The most striking fluctuations in percent susceptibility from year to year were observed for clindamycin, with Actinomyces ranging from 66–79%, B. fragilis 34–67%, and F. magna 47–85%. In the absence of local anaerobe antibiograms, providers may refer to such antibiogram data to guide empiric anti-anaerobic therapy at their institutions. In conclusion, anaerobe antibiotic susceptibility patterns must be monitored to ensure appropriate antibiotic empiric therapy and stewardship.
Mathematical modeling suggests heterogeneous replication of Mycobacterium tuberculosis in rabbits
Authors: Ganusov Vitaly V , Kolloli Afsal , Subbian Selva 
Published: August 2023

Abstract
Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains the major health problem. We performed experiments with rabbits infected with a novel, virulent clinical Mtb isolate of the Beijing lineage HN878 carrying an unstable plasmid pBP10, allowing us to quantify how quickly HN878 replicates and dies in lungs of rabbits. In our in vitro experiments we found that pBP10 is more stable in HN878 strain than in a more commonly used laboratory-adapted H37Rv strain (the segregation coefficient being s=0.10 in HN878 vs. s=0.18 in H37Rv). Interestingly, the kinetics of the frequency of plasmid-bearing bacteria in lungs of Mtb-infected rabbits did not follow an expected monotonic decline; the percent of Mtb-bearing cells increased between 4 and 8 weeks post-infection and remained stable between 12 and 18 weeks post-infection despite a large increase in bacterial numbers in the lung at these time points. Mathematical modeling suggested that such a non-monotonic change in the percent of plasmid-bearing cells can be explained if the lung Mtb population consists of several (at least 2) sub-populations with different replication kinetics: one major population expanding early and being controlled, while another, a smaller population expanding at later times causing a counterintuitive increase in the percent of plasmid-bearing cells. Given that HN878 forms well circumscribed granulomas in rabbits, our results suggest independent bacterial dynamics in subsets of such granulomas. Our model predictions can be tested in experiments in which HN878-pBP10 dynamics in individual granulomas is followed over time.
1R01AI158963-01A1
Elucidating the role of the host protein, FBXO22, in the infection of macrophages with the zoonotic bacterial pathogen, Brucella
Authors: Mazumdar Varadendra Balaji , Joshi Kirnami , Nandi Binita Roy , Radhakrishnan Girish K 
Published: August 2023

Abstract
Brucella spp. are facultative intracellular bacterial pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes. Successful invasion and chronic intracellular persistence indicate that Brucella manipulates various host cellular processes to create a replication permissive niche. The essential mechanisms and host factors that support the invasion and intracellular replication of Brucella remain poorly understood. RNA interference (RNAi) is a potent molecular tool for understanding gene function by downregulating the target gene expression through mRNA degradation. We used siRNA-based screening to identify the host factors that are involved in Brucella-macrophage interaction. Macrophages were treated with gene specific siRNAs, followed by infection with B. neotomae/B. melitensis and quantification of intracellular Brucella. The screening identified several host factors that are involved in the Brucella infection of macrophages. Subsequently, we performed detailed characterization of one of the identified host proteins, FBXO22. Downregulation of FBXO22 resulted in diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages that resulted in induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins, for degradation through the SCF complex.
Funding from Department of Biotechnology (DBT) (BT/PR12301/ADV/90/176/2014)
Blimp-1 is a negative regulator of IL-2 signaling in T cells
Authors: Roy Suyasha , Ren Min , Christensen Steve , Li Peng , Spolski Rosanne , Phelan James , Staudt Louis , Leonard Warren J 
Published: August 2023

Abstract
Interleukin-2 (IL-2) is a T cell growth factor with immunomodulatory effects and pleiotropic actions on T cells. IL-2 signaling occurs via the intermediate IL-2Rb and IL-2Rg and high-affinity IL-2Ra receptors, fine-tuning tolerance and effector functions. To investigate key regulators of the IL-2 signaling pathway, we performed genome-wide CRISPR-Cas9 screening in IL-2-dependent ED40515(+) cells, which were derived from a patient with HTLV-I associated Adult T Cell Leukemia. We found a significant dropout of sgRNAs for genes known to be important in IL-2 signaling, whereas sgRNAs targeting Prdm1 were highly enriched, with its being the top enriched hit in the screen. Prdm1 encodes Blimp-1, which is known to inhibit IL-2 production in T cells; however, its precise role in IL-2 signaling pathway remains elusive. Overexpression of Prdm1 resulted in lower expression of IL-2R subunits (CD25, CD122, CD132) and decreased IL-2-induced phosphorylation of STAT5, AKT, ERK in mouse CD4+ T cells. Correspondingly, conditional knockout (CKO) of Prdm1 in mouse CD4+ T cells and Tregs led to enhanced expression of IL-2R subunits, pSTAT5, pAKT and pERK. In an influenza infection mouse model, CD122 expression and pSTAT5 levels were higher in T follicular helper (TFH) cells from Prdm1 CKO mice as compared to WT TFH cells. Further, adoptive transfer of Prdm1 CKO Tregs into Rag1−/− mice resulted in augmented IL-2 signaling and ameliorated inflammation in colitis. Concordantly, CRISPR/Cas9-mediated deletion of Prdm1 in human CD4+ T cells and Tregs resulted in increased expression of IL-2R subunits and IL-2-induced phosphorylation of STAT5, AKT and ERK. These results suggest that Blimp-1 is a negative regulator of IL-2 signaling in mouse and human T cells.
Supported by intramural funding from NIH