bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel SLC5A6 Mutations Lead to B Lymphocyte Maturation Defects with Metabolic Abnormality Rescuable by Biotin Replenishment
Authors: Hsieh Chu-Han , Chen Peng-Chieh , Shieh Chi-Chang 
Published: January 2023

Abstract
Primary immunodeficiencies (PIDs) comprise inherited genetic mutations with a wide spectrum of clinical immune manifestations. We enrolled a seven-years-old female patient whose clinical laboratory tests showed very low levels of all five isotypes of immunoglobulins but with normal numbers of T cells and B cells. By using whole exome sequencing (WES) on the patient and family members, we identified a novel compound heterozygous gene mutations (termed SLC5A6 mutations) in the patient and her deceased elder sister. We successfully established a CRISPR-Cas9 mouse model bearing the same mutation gene from our patient and provide a further chance to investigate detailed mechanism underlie primary immunodeficiency. We found that biotin deficiency because of SLC5A6 mutations lead to defective B cell maturation and antibody insufficiency. We also found that aberrant cellular metabolism profile due to biotin deficiency restrain the efficiency of plasma cell formation and functions. Furthermore, biotin replenishment can not only rescue cellular TCA cycle but also ameliorate immunodeficient phenotypes including antibody insufficiency in patient and in our mouse model. Our results demonstrated the pivotal role of metabolic reprogramming in B cell terminal differentiation and indicated that SLC5A6 is a causative gene for immunodeficiency.
HIV gp120-specific memory develops from pre-existing memory and naive B cells following vaccination in humans
Authors: Kobie James , Piepenbrink Michael , Fucile Christopher , Bunce Catherine , Man Li-Xing , Liesveld Jane , Rosenberg Alexander F , Keefer Michael , Basu Madhubanti 
Published: January 2023

Abstract
The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naïve B cell receptor, fail to bind Env suggesting that the initial responding B cell population is not exclusively comprised of a naïve population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants from HVTN105, a phase I trial testing AIDSVAX B/E (gp120 protein) combined with a DNA immunogen. Using MiSeq-based deep sequencing VH-lineage tracking we identified several of these mAb lineages in pre-immune peripheral blood. Additionally, we sought to characterize the gp120-specific B cell repertoire prior to immunization, and the features of those pre-immune B cell clonal lineages that ultimately respond to the vaccine. Several of these pre-immune lineages also persisted in the bone marrow, including CD138+ long-lived plasma cell compartment, ~7 months after the final vaccination. Greater gp120 binding activity was observed from the non-naïve pre-immune VH members than their germline VH versions. The majority of the pre-immune gp120-reactive lineages included IgM, however IgG and IgA members were also predominant prior to vaccination. Additionally, the majority of the pre-immune lineage members exhibited somatic hypermutation prior to vaccination, suggesting that the vaccine-induced gp120-specific antibody lineages originated from both naïve B cells and cross-reactive memory B cells.
Supported by grants from the NIH (R01AI117787, R21AI116285).
Sterile liver inflammation and T cell-intrinsic T-bet expression drive the formation of Tissue Resident Memory CD4 T cells that protect against systemic Salmonella infection
Authors: Depew Claire , McSorley Stephen J 
Published: January 2023

Abstract
Infection with Salmonella enterica serovar Typhimurium (ST) can cause invasive Non-Typhoidal Salmonellosis (iNTS), a systemic disease which kills about 150,000 people annually. In a mouse model of systemic salmonellosis, hepatic tissue resident memory (TRM) CD4 T cells provided protection against subsequent infection. However, the signals required to generate these protective CD4 T cells remain poorly defined. To interrogate the process of hepatic CD4 TRM formation we developed a simple Salmonella-specific transfer system. ST-specific (SM1) TCR transgenic CD4 T cells were activated in vitro and adoptively transferred into C57BL/6 mice undergoing acetaminophen overdose. Sterile liver inflammation enhanced the formation of hepatic TRM SM1 T cells which expressed increased levels of T-bet compared to circulating memory cells. Subsequent mixed bone marrow chimera experiments substantiated the requirement for T-bet in hepatic TRM formation as T-bet-deficient CD4 T cells were deficient in resident memory. Bulk RNAseq analysis of hepatic CD4 TRMs uncovered a variety of cytokine and chemokine receptors that correlated with hepatic tissue residency. Using a combination of gene-deficiency and in vivo neutralization approaches, efforts are ongoing to uncover key components of sterile hepatic inflammation that drive CD4 TRM development. Understanding this process is critical to understanding the process of CD4 TRM development and will be foundational for the development of an effective vaccine against iNTS.
Supported by NIAID R01AI139410, NIH T32 Animal Models of Infectious Diseases
Meningeal lymphatic drainage promotes T cell responses against Toxoplasma gondii but is dispensable for parasite control in the brain
Authors: Kovacs Michael , Cowan Maureen N , Still Katherine M , Batista Samantha J , Babcock Isaac , Sibley Lydia , Sethi Ish , Harris Tajie H 
Published: January 2023

Abstract
Toxoplasma gondii is an intracellular protozoan parasite that causes chronic brain infection in a wide range of mammalian hosts. Animal studies have demonstrated that continuous T cell recruitment to the brain is necessary for parasite control. However, it has remained unclear how T cells outside the central nervous system sense and respond to brain-derived microbial antigen. Here, we test the hypothesis that the newly described meningeal lymphatic system promotes T cell immunity during T. gondii brain infection. We find that chronic brain infection is associated with significant expansion of parasite-specific T cells in the cerebrospinal fluid (CSF)-draining deep cervical lymph nodes. T cell activation at this site is correlated with parasite burden in the brain and peaks during the late stages of chronic infection. Flow cytometric analysis of CSF reveals a population of activated dendritic cells that is not present in the CSF of naïve mice. Mature dendritic cells are also mobilized in the meninges, specifically in areas where CSF protein can be sampled. Disrupting meningeal lymphatic drainage by ligating the collecting vessels leads to impaired T cell responses in the deep cervical lymph nodes. Surprisingly, in spite of reduced T cell activation and IFN-γ production at this site, T cell responses in the brain remain intact, likely due to ongoing antigenic stimulation in lymph nodes that drain non-central nervous system tissue. Overall, we provide evidence that meningeal lymphatic drainage supports robust parasite-specific T cell responses in the deep cervical lymph nodes. Nonetheless, we find that drainage of central nervous system material to this site is dispensable for host-protective T cell responses in the brain.
Supported by grants from NIH (5T32AI007496, 5T32GM007267, R01NS091067, R01NS112516, R56NS106028, F30AI154740)
Selective Reprogramming of Peritoneal Macrophages by IFN-γ During Acute Toxoplasma gondii Infection
Authors: Martin Andrew Thomas , Yarovinsky Felix 
Published: January 2023

Abstract
Host resistance to intracellular pathogens is dependent on the pleiotropic cytokine interferon gamma (IFN-γ) which activates myeloid cells, such as macrophages, and results in pathogen clearance. Macrophages safeguard most regions of the body participating in tissue homeostasis, disease surveillance, pathogen elimination, and resolution of inflammation. The peritoneal cavity is patrolled by two unique subsets of resident macrophages: monocyte-derived small peritoneal macrophages (SPMs) and embryonically-seeded large peritoneal macrophages (LPMs). To determine the roles of SPMs and LPMs during intracellular infections, we infected mice intraperitoneally with the intracellular parasite Toxoplasma gondii, a robust driver of IFN-γ-mediated type 1 immune responses. We observed that T. gondii triggers a distinctive response in LPMs and SPMs responding to parasitic infection. Through the use of neutralizing antibodies, recombinant IFN-γ, and mice conditionally lacking expression of the IFN-γ receptor in macrophages, we have shown that the direct effects of IFN-γ are both required and sufficient for selective reprogramming of peritoneal macrophage subsets in response to T. gondii infection. Molecular mechanisms underlying the macrophage subset-specific effects of IFN-γ were further dissected in vivo and in vitro through imaging and omics approaches. Our results revealed the peritoneal macrophage subset-specific responses to IFN-γ that are essential for host resistance to T. gondii.
Supported by the URMC T32 Immunology Training Grant AI00728, NIH R01 GR501266, and the Burroughs Wellcome Fund.
Predicted Cellularity using RNASeq-Based Cellular Deconvolution Differentiates Periprosthetic Joint Infection from Non-Infectious Arthroplasty Failure
Authors: Fisher Cody , Krull Jordan , Bhagwate Aditya , Greenwood-Quaintance Kerryl , Abdel Matthew P , Patel Robin 
Published: January 2023

Abstract
Differentiation of aseptic and septic causes of arthroplasty failure is essential due to their different treatment schemes. We hypothesized that predicted cellularity profiling of sonicate fluid using the RNASeq-based the cellular deconvolution tools CIBERSORTx and ABIS-seq can differentiate between PJI and non-infected arthroplasty failure (NIAF). Profiles were created for 93 sonicate fluid samples (40 from NIAF and 53 from PJI patients) that had been subjected to RNASeq analysis. CIBERSORTx provided 22 total predicted cell types; 12 predicted cellular abundances were differentially expressed in PJI versus NIAF, including increased neutrophils, activated mast cells, and eosinophils (p≤0.0004) and decreased M0 macrophages, M2 macrophages, and Treg cells (p≤0.0001). ABIS-seq provided 17 total predicted cell types; seven predicted cellular abundances were differentially expressed in PJI versus NIAF, including increased neutrophils, Vd2 γδ T cells, and basophils (p-values of &lt;0.0001, 0.0003, and 0.0263, respectively) and decreased nonclassical/intermediate (NC+I) monocytes, mucosal-associated invariant T (MAIT) cells, and myeloid dendritic cells (mDC) (p-values ≤0.0001). Receiver operative characteristic area under the curve (AUC) analysis identified cells types most predictive of PJI (CIBERSORTx: neutrophils [AUC = 94], activated mast cells [AUC = 93]; ABIS-seq: neutrophils [AUC = 88]) and NIAF (ABIS-seq: NC+I monocytes [AUC = 85], MAIT cells [AUC = 80], mDCs [AUC = 81]). PJI and NIAF samples were differentially clustered by principal component analysis using both tools. Overall, cellularity profiling using RNASeq-based cellular deconvolution can differentiate between PJI and NIAF sonicate fluid samples.
Work supported by NIAMS (NIH R01 AR056647). CF was supported by the Mayo Clinic Graduate School of Biomedical Sciences and the Ph.D. Training Grant in Basic Immunology (NIH R25 GM055252 24).
Type I Interferon remodels intrahepatocytic signaling to promote T cell dysfunction during liver stage Plasmodium infection
Authors: Minkah Nana K , Reynolds Laura , Okolo Vera , Kappe Stefan 
Published: January 2023

Abstract
Malaria is caused by Plasmodium parasites and accounted for 627,000 deaths in 2021. Upon deposition into the dermis, Plasmodium parasites traffic to, infect, and develop within hepatocytes before egressing to cause symptomatic erythrocytic infection. We recently reported that liver stage Plasmodium replication induces type I Interferon (IFN-I) signaling that compromises anti-malaria T cells. We now show that this IFN-I-mediated, inferior T cell response is mostly limited to hepatic CD8 T cells and is characterized by diminished cytokine production, impaired homeostatic CD8 T cell survival, and the expression of receptors and transcription factors associated with T cell exhaustion. We set out to identify the cells and molecular events involved in the induction of T cell exhaustion during the transient and non-persistent liver stage Plasmodium infection and observed that infected mice lacking IFNAR expression solely on hepatocytes do not generate significant frequencies of exhausted T cells. Furthermore, transcriptomic analyses of hepatocytes isolated during infection indicate that IFN-I signaling coincides with a remodeling of the hepatocyte transcriptome such that antigen processing and presentation, chemokine transcripts and immunoregulatory receptors and soluble factors are upregulated. We hypothesize that IFN-I signaling reshapes the hepatocyte transcriptome to promote an immunosuppressive liver microenvironment that impairs anti-malaria T cells. Together, our studies represent the first description of a negative role for liver stage IFN-I signaling on anti-malaria adaptive immunity and may necessitate a paradigm shift in the rational design of efficacious anti-malaria whole parasite vaccines.
Supported by 1U01AI42001
Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry
Authors: Chanda Monica Kasturi , Shudde Claire E , Piper Taylor L , Zheng Yating , Courtney Adam H 
Published: January 2023

Abstract
Immunotherapies have transformed the treatment of cancer through the development of drugs that allow T cells to recognize and kill malignant cells. With this has come a need to evaluate how these drugs affect T cell function. We developed an imaging cytometry-based method that can simultaneously assess T cell activation and the functional ability of these activated T cells to kill cancer cells. We used the Nur77-GFP reporter, which expresses GFP upon T cell receptor signaling, as a readout of T cell activation. We combined this reporter system with an MC38 colon adenocarcinoma cell line expressing a TCR-specific antigen that is used as a model tumor antigen (TA). We show that in T cell-cancer co-culture systems, we are able to quantify the number of activated cytotoxic CD8+ T cells and their ability to recognize and kill cancer cells by imaging cytometry. We tested the sensitivity of this platform by quantifying the proportion of antigen-specific activated T cells in co-cultures with varying ratios of unresponsive T cells, mimicking heterogenous lymphocyte populations. To test our system in a pharmacological context, we evaluated the effects of a SRC family kinase inhibitor on T cell activation and cancer cell death. In each case, we were able to quantify the proportion of activated T cells and the extent of TA-specific cancer cell death. Our findings show that the Nur77-GFP reporter system combined with imaging cytometry is a fast and reproducible system to evaluate how different treatment conditions influence TA recognition by T cells using T cell-cancer co-cultures. The simultaneous analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics.
Supported by The Elsa U. Pardee Foundation (AHC), the V Foundation Scholar Award (AHC), the Cancer Biology Training Program T32CA009676 (MKC), the Pharmacological Sciences Training Program T32GM140223 (CES), and a Charles W. Edmunds Pharmacology Fellowship (YZ). This study was also supported by resources provided by the U-M Flow Cytometry Core and the U-M Rogel Cancer Center (P30CA046592).
Use of novel fluorescent dyes enables in depth analysis of checkpoint inhibitors CTLA-4 (CD152) and PD-1 through spectral flow cytometry
Authors: Ellsworth Jessica , DeFelippi Kendall , Strenger-Smith Jazmine , Zahner Sonja 
Published: January 2023

Abstract
PD-1 and CTLA-4 (CD152) are prominent checkpoint inhibitors in T cell responses and their inhibition has emerged as a successful route to boost anti-tumor immune responses in cancer patients. Clinical trials involving CAR-T cell therapy with anti-CTLA-4 and PD-1 antibodies seem to be improving the tumor directed T cell response.
Regulation of surface vs cytoplasmic CTLA-4 pools is crucial to balancing stimulatory and inhibitory signals in T-cell responses. Surface CTLA-4 contributes to tumor escape by inhibiting T cell responses, and upregulation of CTLA-4 expression from the cytoplasm to the surface is highly regulated. Therefore, it might be of interest to monitor these CTLA-4 pools as this might indicate the efficacy of CAR-T cell therapeutic approaches. Here we show how novel fluorescent dyes enable the composition of a spectral flow cytometry panel to interrogate the presence of surface versus cytoplasmic CTLA-4 pools and the dynamics of PD-1 expression in a single panel. Combining these parameters into a single panel allows for less workflow steps and permit conservative sample use.
Advancements in single cell multiomic profiling of antigen-specific T cells with dCODE Dextramer&[trade] (RiO) and BD® AbSeq Reagents on the BD Rhapsody&[trade] Single-Cell Analysis System
Authors: Sakofsky Cynthia , Jacobsen Kivin , Lopez-Salmeron Vadir , Nakamoto Margaret , Brix Liselotte , Ayer Aruna 
Published: January 2023

Abstract
The advancement of immunotherapies and drug development relies on the characterization of antigen-specific T cells. However, due to their low frequency among T cells and their inherent weak affinity to bind known MHC-peptide complexes, detection of these cells has been difficult. Furthermore, pairing this information with the corresponding variable (V), diversity (D), and joining (J) [V(D)J] sequences of the antigen-specific T cell receptors has also been challenging due to hurdles in sequencing these large regions, particularly at the single-cell level. Here, we have expanded our previous work of combining two powerful technologies, Immudex dCODE Dextramer™ (RiO) Reagents and the BD Rhapsody™ Single-Cell Analysis System, to detect and characterize low-frequency antigen-specific T cells, including the full sequences of the V(D)J gene segments of the T cell receptors, as well as profile transcriptome and protein expression. Specifically, thousands of sorted PBMCs were multiplexed to provide high-throughput detection of individual antigen-specific CD8+ T cells in combination with the corresponding full V(D)J sequences of the T cell receptors. In addition, we simultaneously obtained gene expression data for over 400 immune-related mRNAs as well as cell phenotypes using a panel of 30 cell surface BD® AbSeq Protein Markers. Together these data can be used to define T cell phenotypes associated with T cell activation states, alongside antigen specificity of enriched CD8+ dextramer+ cells from PBMC populations. This study showcases the importance of multiomic analysis for high-resolution T cell profiling that has broader implications and utility in immuno-oncology, infectious diseases and autoimmunity.
A Rapid, Calcium-independent Mojosort &[trade] Dead Cell Removal Kit for Improved Live Cell Enrichment and Downstream Application
Authors: Zhang Hong , Ruiz Jen , Yamamoto Kenta , Taylor Kevin , Wiethe Carsten , Yang Xifeng 
Published: January 2023

Abstract
High sample cell viability is crucial for obtaining high-quality data for various downstream applications. Commonly used methods include density centrifugation, flow cytometry sorting, and Annexin V-based magnetic cell separation. However, above entioned methods have several limitations that ultimately leads to fewer live cell recovery. Here we present MojoSort™ dead cell removal kit, which relies on Ca2+-independent, Phoshatidylserine (PS)-binding for dead and apoptotic cell removal. Unlike Annexin V that requires specialized, high Ca2+-containing buffers that can potentially induce cytotoxicity, MojoSort™ dead cell removal kit can be performed in the buffer with or without Ca2+ that also contain chelators such as EDTA. This enables 20% improved live cell recovery compared to other commercially available, Annexin V-based magnetic cell separation kits, while demonstrating 90–95% live peripheral blood mononuclear cell (PBMC) purity post-enrichment from 50% starting viability. Single-cell cellular Indexing of transcriptomes and epitopes by sequencing (CITE-seq) demonstrated effective removal of a distinct dead cell cluster comprised of cells with high mitochondrial gene read counts, while preserving all common PBMC lineages and surface marker expression levels. In summary, MojoSort™ dead cell removal provides a rapid, effective live cell enrichment option that overcomes shortcomings associated with other routinely used methods for downstream applications that require high sample viability for success.
“Therapeutic targeting of tumor necrosis factor like weak inducer of apoptosis (TWEAK) in psoriasis”
Authors: Gupta Rinkesh Kumar , Gracias Donald , Figueroa Daniela Salgado , Miki Haruka , Miller Jacqueline , Fung Kai , Ay Ferhat , Burkly Linda C , Croft Michael 
Published: January 2023

Abstract
Excessive activity of TNF and IL-17 in keratinocytes can drive psoriatic skin inflammation and their targeting is highly advantageous in psoriasis patients, but whether these cytokines act with other inflammatory molecules is unknown. Previously, we demonstrated with knockout mice that the TNF-related cytokine TWEAK (TNFSF12) was a major driver of skin inflammation in the imiquimod model of psoriasis. Here, we found that mice carrying a deletion of TWEAK’s receptor, Fn14 (TNFRSF12A), specifically in keratinocytes, exhibited reduced psoriasis features, including less epidermal hyperplasia and poor expression of genes associated with human psoriasis. Further, RNA-seq analysis in human keratinocytes demonstrated that TWEAK strongly synergizes with either TNF or IL-17A in upregulating the transcripts for psoriasis-associated chemokines and cytokines, implying that TWEAK co-operates with TNF and IL-17 to enhance feedback inflammatory activity. Correspondingly, administration of anti-TWEAK antibody demonstrated therapeutic efficacy in the psoriasis mouse model and was equally as effective as antibodies to IL-17A or TNF in reducing clinical and immunological features of psoriasis-like skin inflammation, while co-neutralization of TWEAK with either cytokine had no additional therapeutic effect, reinforcing the conclusion that all three cytokines act together. We conclude that TWEAK blockade could be another potential therapeutic avenue in patients with psoriasis that might be equally as useful as blocking TNF and IL-17. Future studies targeting TWEAK in clinical trials for psoriasis may then be warranted.
Supported by NIH grant AR072640