bluuebunny/crossref_metadata_2025 huggingface dataset:
BD Pharmingen™ Leukocyte Blocking Buffer reduces nonspecific binding of cyanine-like dye conjugates to monocytes and other leukocytes
Authors: Wei Ai-Li , Gao Guo-Jian , Ernst David , Cantor Joseph M , Li Li , Verma Shilpi
Published: August 2023

Nonspecific binding of monocytes to certain cyanine-like dye conjugated antibodies that are commonly used in flow cytometry for cell surface staining is well known. Although lesser in extent than monocytes, other leukocyte subsets can also exhibit nonspecific binding to cyanine-like dye conjugates. This poses a challenge for multicolor flow cytometric analysis utilizing these cyanine-like dye conjugates as dye-mediated nonspecific binding can mask the real expression. Here we demonstrate that the BD Pharmingen™ Leukocyte Blocking Buffer, a blocking buffer solution, eliminates nonspecific binding of cyanine-like dye conjugates to monocytes and other leukocyte populations. In this study we show that human whole blood stained with PE-Cy5, PE-Cy7, APC-Cy7, APC-H7, PE-CF594 and other tandem-dye conjugated antibodies in the presence of this new buffer exhibits the appropriate staining profile of these antibodies to monocytes and other leukocyte subsets. We also observed that nonspecific binding of cyanine-like dye conjugated antibodies to myeloid and lymphoid subsets in mouse lymphoid tissues was diminished. BD Pharmingen™ Leukocyte Blocking Buffer, while reducing nonspecific cyanine-like dye conjugates binding, did not impact the desirable antibody-mediated specific staining. Our data also show that cell viability is not impacted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD and Pharmingen are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. NPM-0263 (v1.0) 1222

CF is a trademark of Biotium, Inc. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
A lymph node slice culture model to characterize T cell activation dynamics and anti-viral responses in human tissue
Authors: George Alex , Davis-Porada Julia , Weng Shannon , Zhao Wenting , Farber Donna L , Sims Peter
Published: August 2023

T cell activation in the lymph node (LN) is critical to initiating adaptive immune responses to vaccination, tumors, and infection. We previously demonstrated that T cells from human tissue sites (blood, LNs, bone marrow, lung) undergo conserved cell state transitions during activation. However, these studies were performed in suspension culture from dissociated tissue, depleting the spatial and signaling contributions of the tissue microenvironment. To address this, we developed techniques for culture and single-cell analysis of human LN slices, which preserve tissue architecture, local signaling, and physiological cell frequencies. Single-cell RNA-seq (scRNA-seq) of matched LN suspension and slice cultures stimulated with anti-CD3/28 antibody tetramers revealed distinct activation patterns. While anti-CD3/28 stimulation is commonly used to study T cell activation, this reagent circumvents antigen presentation and may not fully recapitulate antigen-mediated signaling. Conversely, antigen-dependent stimulation with superantigen mimics require MHCII/TCR binding and may benefit from the intact microenvironment in slice culture. We discovered a distinct metabolic intermediate through scRNA-seq analysis of superantigen-stimulated LN suspension and slice cultures that is not observed with anti-CD3/28 stimulation. Overall, these studies demonstrate the utility of analyzing T cell responses in intact tissue and comparative analysis of distinct mechanisms of activation.

1T32GM145440-01 (MSTP Training Grant) U19AI128949 (NIAID)
A 47-marker immune profiling flow cytometry assay to enable comprehensive antigen-specific immune analysis
Authors: Cohen Michael J , Yao Huihui , Li Stephen , Tracey Lauren , Loh Christina
Published: August 2023

The complex nature of the immune system requires deep interrogation at the single-cell level. Flow cytometry using CyTOF® technology employs antibodies tagged with isotopically enriched metals instead of fluorophores, resulting in discrete signals that facilitate highly multiparametric characterization on a single-cell level. CyTOF technology enables analysis of over 50 parameters simultaneously without the impediments of spectral overlap or autofluorescence, allowing for a reliable evaluation of immune responses.

The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, and validated 30-marker antibody cocktail for single-tube staining of human whole blood or PBMC and acquisition on CyTOF systems. Using the assay and Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations. In this study, we highlight the customizability of the Maxpar® Direct panel by adding an array of 17 surface and intracellular parameters with the Maxpar Direct™ T Cell Activation Expansion Panel and live-cell barcoding. The simultaneous detection of key surface and intracellular markers in this expanded panel enabled comprehensive analysis of immune cell activation and antigen-specific recall responses to PBMC stimulated with a pool of immunodominant microbial peptides.

Taken together, this 47-marker panel and automated analysis unlocked deeper immunophenotyping and functional profiling of activated immune cells and provided insight into the cell-mediated adaptive immune response to foreign targets. Such phenomena are hallmarks for research on infection, vaccine development, and immunotherapy.

For Research Use Only. Not for use in diagnostic procedures.
Unraveling T cell mitochondrial dynamics using imaging flow cytometry
Authors: D’Cruz Louise M , Shi Xiaoshan , Widmann Stephanie J , Tyznik Aaron J
Published: August 2023

Mitochondria are highly mobile, dynamic subcellular organelles that mediate energy generation in mammalian cells. In response to changes in energy and stress, mitochondria can either join or divide, a process known as fusion or fission, respectively. Mitochondrial morphology plays a critical role in the metabolic state of T cells and can affect T cell function in health and disease. Despite increased interest in mitochondria dynamics, a robust and efficient method to simultaneously image mitochondria morphology, analyze T cell phenotypes and sort cells has not been feasible. Here, we used the state-of-the-art BD CellView™ Image Technology on the BD FACSDiscover™ S8 Cell Sorter to detect changes in mitochondria morphology with high resolution and profile T cell phenotypes associated with specific mitochondrial shapes. With the imaging capabilities of the BD FACSDiscover™ S8 System, we visualized live mitochondria and nuclei using three-color staining in a high-throughput manner. We differentiated effector T cells in vitro and imaged increased fission-like mitochondria in these cells. With the cell sorting feature of the BD FACSDiscover™ S8 System, we sorted effector T cells based on their cell surface phenotype and distinct mitochondrial characteristics and evaluated their mitochondrial gene signature. Thus, we provide an advanced and comprehensive workflow to unravel mitochondrial dynamics and isolate cells with a particular phenotype for downstream in-depth characterization enabled by the BD FACSDiscover™ S8 System. As many cellular processes are linked to mitochondrial dynamics, this technique provides a powerful tool to understand how this critical organelle is regulated in a variety of human diseases.
Development of a 40+ color full spectrum flow cytometry panel for in-depth immunophenotyping of chief cell subsets in non-human primate peripheral blood mononuclear cells
Authors: Fears Alyssa , Geisbert Thomas , Woolsey Courtney
Published: August 2023

Leveraging full-spectrum technology, we designed and optimized a 40+ color flow cytometry panel for the analysis of non-human primate peripheral blood mononuclear cells (PBMCs). Non-human primate models of disease allow for the closest approximation of human outcomes to vaccination, disease, and treatment due to similarities in physiological host responses, which increase predictive accuracy. This first panel of its kind achieves comprehensive profiling of immune subpopulations by the inclusion of surface, intracellular, and cytokine markers representative of differentiation, activation, functionality, and proliferation to deeply interrogate the immune response to infectious disease and vaccination. The optimized panel is additionally appropriate for the discovery of new immune phenotypes important for protection against pathogens. After initial optimization for use with cynomolgus macaque (Macaca fascicularis) and rhesus macaque (Macaca mulatta) PBMCs, the panel was further adapted for use with African green monkey (Chlorocebus aethiops) PBMCs to allow for cross-comparison between three frequently implemented non-human primate models of infectious disease.

This study was supported by the US Army Medical Research Acquisition Activity contract number W81XWH1910027 to TWG and Department of Health and Human Services, National Institutes of Health grant number UC7AI094660 for BSL-4 operations support of the Galveston National Laboratory.
Spatially resolved immune receptor repertoires from Visium transcriptomic libraries
Authors: Montagne Janelle Marie , Sidiropoulos Dimitrios N , Mo Guanglan , Zhu Qingfeng , Anders Robert A , Zheng Lei , Jaffee Elizabeth M , Larman H Benjamin , Fertig Elana J , Kagohara Luciane T
Published: August 2023

Recent advances have enabled the integration of T cell receptor (TCR) sequencing into spatial transcriptomics. Current approaches yield critical information about in situ T cell responses directly from relevant tissues, yet are largely limited to alpha/beta TCRs, leaving the clonal dynamics of B cells and gamma/delta T cells underexplored. We therefore modified our previously reported ultra-efficient immune receptor repertoire sequencing (IRR-seq) method, Framework Region 3 AmplifiKation Sequencing (FR3AK-seq), for compatibility with the 10X Genomics Visium spatial transcriptomics platform. Here we report simultaneous acquisition of alpha/beta TCR, gamma/delta TCR, and immunoglobulin (B cell receptor, BCR) heavy/light chain sequences from a single multiplexed FR3AK-seq reaction using Visium transcriptomics-generated cDNA libraries. More specifically, we used the Visium spatial transcriptomics protocol to generate cDNA from serial sections of a lymph node with a metastatic lesion obtained from a patient with pancreatic ductal adenocarcinoma previously treated with immunotherapy. Notably, we detected sequences from all IRR chains as expected from the abundant immune infiltrates within these sections. Furthermore, we found that multiplexed FR3AK-seq yields technically reproducible repertoires, with the TCR beta chain repertoires also comparable to those obtained using a previously published method. We anticipate that this simple extension of the Visium technology will accelerate the simultaneous and comprehensive examination of in situ IRR dynamics beyond alpha/beta T cells.
Decoupling peptide binding from T cell receptor recognition with engineered chimeric MHC-I molecules
Authors: Papadaki Georgia , Ani Omar , Florio Tyler , Young Michael , Danon Julia , Sun Yi , Dersh Devin , Sgourakis Nikolaos
Published: August 2023

Human leucocyte antigens (HLAs) display a repertoire of epitopic peptides on the cell surface for T cell recognition. Amino acid polymorphisms in the peptide-binding groove of different HLA allotypes result in distinct antigenic repertoires, providing a basis for adaptive immunity to emerging pathogens at the population level. T cell receptors (TCRs) recognize pHLA antigens through interactions with both peptide and heavy chain residues. In this work, we use structural data from peptide:MHC-I and pMHC:TCR structures to identify residues important for peptide and/or TCR binding, and we outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Moreover, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules displaying tumor-associated antigens with modified TCR interaction surfaces, which could further serve as platforms to elicit alloreactive T cell responses against self-antigens that are overexpressed in cancer. Finally, we gain a deeper understanding of the evolution and functional divergence of the different HLA allotypes by demonstrating an example of convergent evolution.

Supported by grants from NIAID (5R01AI143997), NIGMS (5R35GM125034), and NIDDK (5U01DK112217)
CD8+ T-cell memory induced by SARS-CoV-2 mRNA vaccination is maintained by clonal replenishment
Authors: Aoki Hiroyasu , Kitabatake Masahiro , Abe Haruka , Shichino Shigeyuki , Hara Atsushi , Ouji-Sageshima Noriko , Ito Toshihiro , Matsushima Kouji , Ueha Satoshi
Published: August 2023

mRNA vaccines against the Spike glycoprotein of severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2) elicit strong CD8+ T-cell responses. However, in repetitive vaccination, it is unknown whether T-cell clones induced by the first dose respond repeatedly or new clones are mobilized and respond after each dose. Here, we temporally tracked the CD8+ T-cell repertoire in individuals who received three doses of the BNT162b2 mRNA vaccine. CD8+ T-cell clones that expanded after the first, second, or third dose were identified, and their kinetic profiles throughout the vaccination were analyzed. The extent of clonal expansion after the first vaccination was significantly less than that after the second or third vaccination. After the second vaccination, clones that had expanded after the first dose (“first responders”) were less responsive, while a new set of clones (“second responders”) greatly expanded and surpassed the first responders. After the third vaccination, the second responders expanded again, while the first responders did not. Moreover, we observed expansion of another set of clones that had not responded to the previous doses (“third responders”). Single-cell mRNA/protein/TCR analysis after the second vaccination revealed that the first-responder clones exhibited a terminally differentiated phenotype, whereas the second-responder clones exhibited an actively proliferating phenotype. These results suggest that CD8+ T-cell memory induced by repetitive mRNA vaccination is maintained by the mobilization of newly generated clones with proliferative potential.

This work was supported by Japan Society for the Promotion of Science (JSPS) under Grant Number 17929397 and 20281832, and Japan Agency for Medical Research and Development (AMED) under Grant Number JP22fk0310509, JP22ama221306, and P21gm6210025.
The Comparison of the Duration of Immunity Induced by Pertussis Vaccines and Infection in a Baboon Model
Authors: Wang Yihui , Merkel Tod
Published: August 2023

Whooping cough is a highly contagious, acute respiratory illness caused by the Gram-negative bacteria, Bordetella pertussis. Disease is often mild in older children and adults but can be severe or fatal in young children and infants. It is endemic worldwide and a major cause of vaccine-preventable deaths despite high vaccine coverage rate. A steady increase in the number of reported pertussis cases has been evident over the last 20 years. The apparent correlation of this resurgence and the replacement of whole-cell (wP) vaccines with acellular (aP) vaccines suggests the current aP vaccines may not control pertussis as well as the wP vaccines they replaced. Some evidence indicates the aP vaccines may induce protection with shorter duration of immunity relative to wP vaccines.

There is increased interest in developing next-generation vaccines that induce longer lasting protection. However, immunological correlates predicting durable and protective immunity remain unknown. Studying duration of immunity in a human population is problematic due to potential asymptomatic exposures and the difficulty in sample accessibility. In an ongoing, long-term study, using a baboon model we are comparing the duration of immunity and immunological correlates of protection in a controlled population comparing aP, wP vaccines and natural infection. Extensive sampling was undertaken around the vaccinations to characterize the vaccine-mediated immune responses. Animals enrolled in the study will be followed for years with sampling twice yearly. With strict control of Bordetella exposure, the data generated from the study will provide a clearer understanding and a basis of comparison for the duration of aP, wP, and infection-induced immunity.
Adenoviral vector vaccines are effective mucosal booster modalities to elevate systemic and establish mucosal immunity against SARS-CoV-2
Authors: Lapuente Dennis , Viherlehto Vera , Fuchs Jana , Willar Jonas , Antao Ana V , Eberlein Valentina , Uhlig Nadja , Issmail Leila , Schmidt Anna , Oltmanns Friederike , Peter Antonia Sophia , Mueller-Schmucker Sandra , Irrgang Pascal , Fraedrich Kirsten , Cara Andrea , Hoffmann Markus , Pöhlmann Stefan , Ensser Armin , Pertl Cordula , Willert Torsten , Thirion Christian , Grunwald Thomas , Überla Klaus , Tenbusch Matthias
Published: August 2023

Licensed SARS-CoV-2 vaccines induce robust systemic immune responses, but mucosal immunity in the respiratory tract in form of secretory immunoglobulin A (IgA) or tissue-resident memory T cells (TRM) is not established. Inducing such front-line immunity by mucosal boosting might reduce the individual risk of breakthrough infections and thereby likely mitigate infection dynamics on population level as well. The following project evaluates preclinical and licensed adenoviral vector (Ad) platforms as mucosal SARS-CoV-2 boosters.

We show that nasal instillation of Ad5- and Ad19a-Spike following a systemic DNA or mRNA priming results in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with a licensed mRNA vaccine, the mucosal boost with Ad vectors induced CD103+ CD69+ tissue-resident memory CD8+ T cells and high levels of Spike-specific IgA in the respiratory tract as well as an enhanced mucosal neutralization of variants of concern. Moreover, the protective efficacy of the mRNA prime/intranasal Ad5 boost strategy was not inferior to two intramuscular mRNA immunizations.

In a more translational approach, we employed Janssen’s Ad26.COV2.S as a mucosal booster delivered by a medical mucosal atomization device after a systemic mRNA prime of mice. Concerning systemic responses, the mucosal boost was similarly immunogenic compared to an intramuscular boost with the same vaccine. However, only a mucosal delivery provoked TRM and IgA in the respiratory tract as well as a robust mucosal neutralization of Omicron variants.

In conclusion, a mucosal booster immunization is a promising approach to establish mucosal immunity in addition to systemic responses in an mRNA vaccine-primed population.
mRNA vaccination induces persistent pharyngeal mucosal B cell responses to SARS-CoV-2 in children
Authors: Xu Qin , Mudd Pamela , Behzadpour Hengameh , Newman Daniel , Bellusci Lorenza , Grubbs Gabrielle , Tang Juanjie , Liu Can , Milanez-Almeida Pedro , Martins Andrew , Kardava Lela , Moir Susan , Khurana Surender , Schwartzberg Pamela , Manthiram Kalpana
Published: August 2023

We recently reported that SARS-CoV-2 infection triggers robust, persistent adaptive immune responses in the tonsils and adenoids of children. However, whether mRNA vaccination induces similar immune responses and how this compares to natural infection in children are unknown. To evaluate these questions, we collected peripheral blood (PBMCs), tonsils, and adenoids from 30 children undergoing tonsillectomy/adenoidectomy primarily in 2022. From questionnaires and serology, we identified 12 vaccinated subjects who had not been infected and compared them to 24 COVID-19 convalescent children from our previous study collected in 2020–2021. We found SARS-CoV-2-specific germinal center and CD27+ switched memory B (BSM) cells, including cells recognizing the spike protein receptor binding domain (RBD) of the original WA-1 strain and RBD from Omicron, in pharyngeal tissues of vaccinated children. PBMCs trended towards having higher percentages of S1+ RBD+ B cells post-vaccination than post-infection, while adenoids had more S1+ RBD+ B cells post-infection. SARS-CoV-2-specific BSM cells were dominated by IgG isotypes in all tissues, but a higher proportion of IgA+ cells were seen post-infection. Furthermore, S1+ RBD+ BSM cells showed greater CXCR3 expression post-infection in all tissues. In contrast, S1+ RBD+ BSM cells showed higher expression of activation and atypical markers post-vaccination, particularly in PBMCs, implying different characteristics of B cell memory depending on the type of exposure. Our results demonstrate tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after vaccination and provide further evidence that mRNA vaccination induces broad protective B cell memory.

This work was supported in part by intramural funds of NIAID, NIH.
Particulate characteristics of engineered nanoparticle vaccines modulate vaccine immunogenicity in an intranasal route
Authors: Dong Chunhong , Wang Bao-Zhong
Published: August 2023

The intranasal route is an ideal vaccination route for preventing infectious respiratory diseases due to its capability of generating both mucosal immunity—the first line of defense against respiratory virus invasion—and systemic immunity. Protein antigens are poorly immunogenic when administered intranasally. Previously we found that polycationic polyethyleneimine (PEI)-hemagglutinin (HA) nanoparticles significantly enhanced influenza HA immunogenicity in an intranasal route. Herein, we prepared hybrid PEI-HA-lipid nanoparticles and investigated how particulate features influence their immunogenicity.

We found that a phosphatidylserine-based lipid layer coating around PEI-HA nanoparticles made the particle negatively charged and eliminated the induced antibody responses, reflecting the importance of surface charges in the antibody induction. However, further incorporation of PAMP adjuvants (MPLA and cGAMP) substantially boosted humoral and cellular responses both mucosally and systemically. MPLA and cGAMP-co-loaded hybrid nanoparticles generated the most lung-resident CD8+ T memory cells among all groups. These nanoparticles showed distinct antigen delivery behavior and early immune responses in mouse lungs and NALTs. Only adjuvanted particles induced significant BMDC maturation and inflammatory cytokines secretion in vitro cultures.

Studies on these particle vaccines’ protective efficacy are ongoing in mice. The role of particulate features in programming the immune responses would be further analyzed. Our study will provide perspectives for the development of intranasal particulate vaccines against infectious respiratory diseases.

Supported by grants from NIH/NIAID (R01AI101047, R01AI116835, R01AI143844)
Extracellular immune checkpoint molecules released from HTLV-1-infected cells mount immune suppression in the context of neuroinflammation
Authors: Joseph Julie , Premeaux Thomas , Pinto Daniel O , Rao Ahbishek , Guha Shrobona , Panfil Amanda , Carey Alison , Ndhlovu Lishomwa , Bergmann-Leitner Elke , Jain Pooja
Published: August 2023

HTLV associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neuroinflammatory demyelinating condition of the spinal cord. We have previously shown that aberrant expression and activity of immune checkpoint (ICP) molecules – PD-1/PD-L1, negatively associates with the cytolytic potential of T cells in individuals with HAM/TSP. Interestingly, ICPs can exist in soluble cell-free form and can be carried on extracellular vesicles (EV) and exosomes (small EVs, <200nm) while maintaining their immunomodulatory activity. Therefore, we investigated the role of soluble/EV membrane-bound ICPs in HTLV-1 associated neuroinflammation. We first profiled a range of ICP mediators in sera of HAM/TSP patients, and in purified exosomes from HAM/TSP-derived HTLV-1-producing (OSP2) cells. ICPs, such as BTLA, LAG-3, and PD-L2, were elevated in the plasma samples of patients at the levels comparable to their presence in EVs from OSP2 cells, primarily in exosomal fractions. These exosomal ICPs were found to be functional and adversely affected healthy CD8 T-cell functions. Viral proteins and cytokines (IFNγ) were also found to be present in purified exosomes. IFNγ exposure boosted release of ICP molecules while antiretroviral drugs significantly inhibited this process. HTLV-1 b-Zip protein (HBZ) has been linked to factors that enhance EV release and its knockdown here led to reduced expression of several endosomal sorting complex required for transport (ESCRT) associated genes as well as abrogated release of ICP molecules, suggesting a transcriptional control. Collectively, these findings highlight potential new immunotherapeutic avenues to be explored that may slow or reverse the risk or progression of HAM/TSP.

NIH/NINDS: R01 NS097147 to P.J.
Reactive persulfide controls intestinal inflammation by suppressing CD4+ T cell proliferation
Authors: Tayama Shunichi , Kitamura Yuya , Hiraide Kyoga , Li Jing , Yang Ziying , Kawajiri Akihisa , Sato Kosuke , Okuyama Yuko , Kawabe Takeshi , Akaike Takaaki , Ishii Naoto
Published: August 2023

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation where CD4+ T lymphocytes play an essential role. Accumulating evidence suggests that immune responses driven by CD4+ T cells are critically regulated by various metabolic pathways including oxidative phosphorylation and glycolysis. In addition, we have recently identified a novel, sulfur metabolic pathway that is controlled by mitochondrial cysteinyl-tRNA synthetase (CARS2/CPERS) through the production of a highly reactive sulfur metabolite cysteine persulfide (CysSSH). However, the functional significance of this metabolic signaling in CD4+ T lymphocytes remains unclear. Here we show using Cars2+/− animals that CARS2/CPERS-dependent persulfide metabolism restrains CD4+ T cell proliferation in a cell-intrinsic manner. Under steady state, Cars2+/− mice exhibited a spontaneous accumulation of effector/memory CD4+ T cells in the colon with age. In lymphopenic conditions, Cars2+/− CD4+ T cells showed enhanced cell cycle entry and an exacerbated form of colitis, the latter response being rescued by treatment with an endogenous persulfide donor. Furthermore, re-analysis of publicly available gene datasets of human CD4+ T lymphocytes revealed that downregulation of CARS2 was associated with pathogenesis of IBD, and indeed addition of persulfide donor inhibited CD4+ T cell proliferation in vitro. Together these observations reveal that CARS2/CPERS-dependent persulfide metabolism is essential for homeostasis of intestinal effector/memory CD4+ T cells, and further suggest that dysregulation of the same metabolic pathway can lead to development of gut inflammation in both mice and humans.

Supported by grants from Japanese Society for the Promotion of Science KAKENHI grant numbers JP20J20344
Inhibition of LRRK2 Kinase Activity with a Novel Inhibitor (CS 82) Inhibits NLRC4 Inflammasome Activation and Reduces Intestinal Inflammation Severity
Authors: Deloer Sharmina , Fuss Ivan , Suebsuwong Chalada , Kumar Kunal , Peter Inga , Devita Robert J , Strober Warren
Published: August 2023

In the last twenty years Leucine Rich Repeat Kinase 2 (LRRK2) has received immense attention due to its association with several diseases, for example, neurodegenerative disease PD (Parkinson’s Disease), inflammatory bowel disease (IBD), Crohn’s disease (CD), infectious disease Leprosy etc. Not only do genetic mutations make this protein a hotspot for various diseases but also it shows some pleotropic characteristics which might influence several diseases. LRRK2 directly controls several proteins for example Rab GTPase proteins, NLRC4 inflammasomes etc. NLRC4 also showed correlation with auto-inflammatory diseases such as CD, Macrophage Activation Syndrome (MAS) etc. Here we report that NLRC4 dependent cytokine storm retarded through LRRK2 kinase inhibition as well as reduces severity of intestinal inflammation. For LRRK2 kinase inhibition a novel kinase inhibitor (Termed CS 82) has been used. In vitro studies disclosed that this inhibitor suppress LRRK2 phosphorylation as well as its ability to phosphorylate the LRRK2 targets, Rab 10 and 12; in addition, they suppress human dendritic cell production of TNFa stimulated by several known LRRK2 activators including the Dectin-1-ligand, zymosan- depleted S. cerevisiae extract. Finally, the inhibitors exhibited powerful suppression of the ability of LRRK2 to mediate NLRC4 inflammasome production of IL-1beta. LRRK2 deficiency showed inhibitor has no effect on NLRC4 dependent IL-1b cytokine productions. These findings suggest that inhibition of LRRK2 kinase activity retarded NLRC4 inflammasome activation which reduces IL-1b cytokine productions and intestinal inflammation severity.
Monoclonal anti-toxin therapy supports the protective innate immune response following Clostridioides difficile infection
Authors: Denny Joshua E , Alam Zahidul , Mdluli Nontokozo , Maslanka Jeffrey , Lieberman Linda , Abt Michael
Published: August 2023

Clostridioides difficile causes over 200,000 hospitalizations and 13,000 deaths annually in the United States. C. difficile infects the large intestine following perturbation of the microbiome to cause pathology that ranges in severity from diarrhea to pseudomembranous colitis. Virulence factors Toxin A and Toxin B are the primarily drivers of disease. These toxins damage the epithelial barrier leading to pathologic inflammation and bacterial translocation. Monoclonal antibodies (mAbs) that neutralize Toxin A and Toxin B, actoxumab and bezlotoxumab, respectively, significantly reduce disease severity in a murine model of C. difficile infection, however the impact of toxin neutralization on the induction of the innate immune response following infection is unknown. The goal of this study was to define the quality of the host innate immune response in the context of anti-toxin mAb therapy. At day 2 post-infection, C. difficile infected, mAb-treated mice had significantly less disease despite no differences in C. difficile burden compared to isotype-treated mice. Further, no difference in neutrophil infiltration or production of IFNg or IL-17 from innate lymphoid cells (ILCs) was observed within the intestinal lamina propria following infection. However, C. difficile-infected, mAb-treated mice had increased IL-22-producing ILCs and increased eosinophil infiltration in the intestine compared to iso-treated mice. Both IL-22+ ILCs and eosinophils promote protective host response following C. difficile infection. These findings indicate that activation of natural host protective mechanisms remain intact in the context of treatment that neutralizes toxin but does not alter C. difficile burden in the intestine.

Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA
Use of dirty mice to study skin resident memory T cells in cancer treatments and graft-versus-host-disease
Authors: Fu Xiaopeng , Shah Pranali Nitin , Hsieh Pei-Chen , Romar George , Divito Sherrie
Published: August 2023

Graft versus host disease (GVHD) is a significant cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). While donor T cells have been shown to be instrumental in disease pathogenesis, prior studies of host T cells are limited, as host T cells are assumed to be depleted by conditioning (chemotherapy ± irradiation) used in HSCT. Recent data suggest that host skin resident memory T cells survive HSCT conditioning in humans and contribute to GVHD, but deeper mechanistic interrogation is necessary. Herein, we developed a novel mouse model using dirty mice to study host skin resident memory T cells in chemotherapy and GVHD. Laboratory mice cohoused with pet-shop mice developed a robust memory T cell population including CD44high CD62Lhigh central memory T cells, CD44high CD62Llow effector memory T cells, and CD44high CD62Llow CD69+ CD103+ and CD103− skin resident memory T cells of both CD4+ and CD8+ T cell subsets. Similarly to human data, both CD4+ and CD8+ skin resident memory T cells in dirty mice survived induction chemotherapy and HSCT conditioning while other T cell populations in blood, lymph node and spleen were depleted. In a reduced intensity conditioning chemotherapy-based fully mismatched model of GVHD, dirty mice developed higher GVHD score than clean control mice, with some dirty mice developing early vitiliginous skin change that was not observed in clean controls. Taken together, dirty mice appear to be an ideal model to test skin resident memory T cells in cancer treatments and GVHD, and more broadly in inflammatory skin diseases.

Supported by grants from Brigham Research Institute (126409)
Dissecting the role of TGF-b in the skin
Authors: Ottaviani Valentina , Chen Wanjun , Joller Nicole
Published: August 2023

The skin is outermost barrier in the body. Its only breaches are represented by hair follicles, which represent the only point of entrance for all the skin-sitting pathogens and commensals. Therefore, they can be defined as the most immunologically active sites of the skin. As such, tight regulation is needed at these sites and it is achieved not only through immune cells, but also non-hematopoietic cells like keratinocytes and fibroblasts. Example of immune cells are regulatory T cells, which are not only activated by, but also produce large amounts of TGF-b, a multifunctional cytokine, present in the body in 3 isoforms (TGF-b1,2 and 3). These isoforms have been reported to be fundamental for skin development during organogenesis and additional findings report about their role in driving cell localization in the skin as well as involvement in tissue repair. Though, litte is known about their immunological role in the skin. Upon generating a working protocol to separate skin layers and isolate cells from them, we were able to start to define the cell composition of each layer both at steady state and during inflammation. Moreover, we could detect the three TGF-b isoforms in each layer at both gene and protein level further discriminating between hematopoietic and non-hematopoietic sources. Finally, the importance of TGF-b in the skin was confirmed by experiments on TGF-b receptor I depleted mice. For the future, we are planning on defining exactly which cells secrete and respond to TGF-band confirm these findings by generating conditional KO mice. Additionally, the role of TGF-b will be assessed in world-wide skin diseases.

Intramural Research Program of NIDCR
Murine CD4+ T cells exhibit sexually dimorphic responses to estrogen signaling
Authors: McNeer Sarah K , Kocinski Adrian D , Goodman Wendy A
Published: August 2023

Many autoimmune diseases exhibit sexual dimorphism. 17β-estradiol (estrogen, “E2”), a steroid sex hormone primarily known for its reproductive roles, has also been shown to modulate CD4+ T cells. E2 signals through two nuclear receptors, ERα and ERβ, which regulate gene transcription through direct DNA binding and other mechanisms. We previously showed that in the SAMP/YitFC model of Crohn’s-like ileitis, loss of ERβ enhanced inflammation selectively in female mice. We also found that loss of ERβ resulted in decreased expression of Foxp3 in CD4+ T cells, suggesting that ERβ is required for differentiation of Foxp3+ Tregs. These prior findings suggest a pro-inflammatory role for ERα and anti-inflammatory role for ERβ, potentially in a sex-specific manner. In the current study, we investigated mechanisms of E2 signaling in CD4+ T cells isolated from males vs. females. Using standard in vitro assays, we found that the proliferation of TGFβ-induced regulatory T cells (Tregs) was enhanced in female cells co-treated with E2 compared to male cells, suggesting that E2 enhances Treg differentiation more strongly in females. Interestingly, using the CD45RB T cell transfer model of colitis, we found that recipients of female donor cells exhibited more severe disease compared to recipients of male cells, suggesting that female-derived CD45RBhigh T cells are more colitogenic than male cells. Future studies will determine the effect of E2 signaling on both effector and regulatory CD4+ T cell populations, identifying the contributions of ERα vs. ERβ-specific signaling in these T cell subsets and the functional impact of these signaling pathways in males versus females.

Supported by grants from NIH (R01DK128143, R03DK123579, T32AI089474)
Reprogramming adult hematopoietic progenitors towards fetal-restricted lymphopoiesis via Cbfb2 gene dosage
Authors: Frascoli Michela , Berthelette Alyssa , Taniuchi Ichiro , Kang Joonsoo
Published: August 2023

Hematopoietic Stem Cells (HSC) hold the potential to self-renew and differentiate into most immune cell subsets throughout life. However, some specialized immunocytes like microglia, B-1 B cells and γδ T cells emerge only during fetal life, and they cannot be generated by adult HSC. We discovered that CBFβ isoform 2 (CBFβ2), an evolutionarily conserved component of the RUNX transcription factor complex, reprograms adult HSC into acquiring fetal hematopoietic potential. While Cbfb2 heterozygous mice generated normal numbers of mature IL-17 producing γδ T (Tγδ17) cells, we found that they lacked DN1d cell containing Tγδ17 precursors. Thus, Cbfb2 gene dosage is critical for imprinting Tγδ17 differentiation potential in distinct precursor cells. In line with this reprogramming, Cbfb2 haploinsufficient adult bone marrow (BM) progenitors, which are normally inefficient at Tγδ17 cell generation, became permissive for innate T cell development. Analysis of transcriptional profiles of HSCs and lymphoid progenitors (LMPPs and CLPs) from adult BM revealed that, among hemopoietic progenitors, LMPPs from Cbfb2 heterozygous mice were the most affected by Cbfb2 gene dosage and upregulated genes implicated in embryonic hematopoiesis. Using In Utero Transplantation of adult hematopoietic progenitors into the fetal liver of E14.5 pregnant dams’ fetuses, we were able to successfully reconstitute Tγδ17 cells and confirm an unprecedent bias in Tγδ17 development underpinned by reduced CBFβ2-dependent transcriptional activity. Our findings identify gene network architectures that impose temporal restriction on hematopoietic pluripotency and that could be exploited for the generation of fetal-derived lymphocytes in adult life.
The human preBCR uses CDR-H3 amino acid composition to control early B cell selection and prevent oncogenesis
Authors: Khass Mohamed , Burrows Peter D , Capriotti Emidio , Schroeder Harry J
Published: August 2023

Human B cell repertoire selection depends on the composition of the complementary determining heavy chain (CDR-H3), which lies at the center of the antigen binding site. During B cell development, preB cells express a preB cell receptor (preBCR), which is composed of μH chain bound to surrogate light chain (SLC). In mouse, the VpreB R101D57R51 motif of the SLC selects against μH chains with hydrophobic CDR-H3s. Abnormal selection at the preBCR stage predisposes to autoreactive B cell survival and leukemia. We sought to study preBCR selection in healthy human individuals versus acute lymphoblastic leukemia (ALL) patients to dissect the mechanism of formation of the human protective CDR-H3 repertoire. We hypothesized that expression of a preBCR with an abnormal amino acid content prevents differentiation of the ALL blasts into a late preB and then a mature B cell, promoting oncogenic survival. We sorted early B cells from bone marrow swabs of healthy controls and ALL patients. We extracted RNA, deep sequenced preB cells and leukemic blasts and then analyzed their CDR-H3 based on amino acid content using bioinformatics and structure modeling. Our results suggest that in healthy individuals, similar to mouse, the human preBCR selects for CDR-H3 based on amino acid content with a preference for tyrosine (Y) and negative selection of hydrophobic antigen binding sites. The CDR-H3s of ALL leukemic blasts have amino acid characteristics similar to the negatively selected antigen binding sites of healthy individuals. In conclusion, this type of analysis might be used to monitor optimal immunoglobulin repertoire diversification.

R21AI163555
β-catenin engages IKZF factors to control lymphopoiesis
Authors: Cosgun Kadriye Nehir , Jumaa Huda , Robinson Mark E , Xu Liang , Xiao Gang , Arce David Fonseca , Khanduja Dhruv , Chan Lai N , Lee Jaewoong , Schjerven Hilde , Jellusova Julia , Müschen Markus
Published: August 2023

β-catenin-signaling promotes proliferation and survival in epithelial, neuronal, and mesenchymal lineages, but is dispensable for B- and T-cell development. β-catenin activation is a common oncogenic driver throughout all types of cancer with the exception of B- and T-lymphoid malignancies. Unlike epithelial tissues, we found that B- and T-cells consistently lack β-catenin expression. Instead, both B- and T-cells were highly sensitive to β-catenin-activation and critically depend on its negative regulation by GSK3β-dependent phosphorylation and degradation. Inhibition of GSK3β induced dramatic nuclear accumulation of β-catenin, anergy and cell death. In contrast, genetic deletion of β-catenin enabled clonal expansion of premalignant B- and T-cells.

In epithelial cells, β-catenin and TCF-family factors form complexes for transcriptional activation of MYC. Instead of TCF-factors, our interactome studies in B- and T-cells revealed that, β-catenin formed complexes with lymphoid-specific Ikaros zinc finger (IKZF) transcription factors for transcriptional repression of MYC at a recently discovered MYC ‘blood enhancer cluster’ (BENC). Hence, activation of β-catenin engages repressive complexes with Ikaros factors to regulate lymphopoiesis through transcriptional control of MYC.

We propose that β-catenin-accumulation functions as sensing mechanism of pathological B- and T-cell signaling and that this mechanism can be leveraged for targeted eradication of pathological (autoreactive or malignant) clones. To engage this mechanism, targeted accumulation of β-catenin can be achieved by GSK3β-inhibition and represents a selective vulnerability of pathological B- and T-cell clones.
Fluctuation of cell numbers and kinetics reveals dynamical hematopoiesis
Authors: Jia Dongya , Mukherjee Ratnadeep , Altan-Bonnet Grégoire
Published: August 2023

Fluctuations of multiple blood cell types (B cells, neutrophils, reticulocytes, platelets etc.) have been reported in many hematological disorder and leukemia patients. It remains largely elusive about the mechanisms underlying cell fluctuation. To quantify the dynamics of blood cells, we performed a longitudinal studies of blood profiling using identical healthy mice. We combine a pulse protocol of 5-iodo-20-deoxyuridine (IdU) injections with subsequent analysis by mass cytometry (CyTOF and CyTEK) and mathematical modeling of the cell dynamics. We demonstrate that cell abundance exhibits large fluctuations, and a 10-fold change has been observed for the blood neutrophil numbers in healthy mice. Although individual cell type is fluctuating, different cell types exhibit strong correlation/anti-correlations, such as the anti-correlated cell numbers of B cells and neutrophils. The large fluctuation of cell numbers motivated us to characterize cell kinetics, such as the proliferation rate. Through the IdU pulse experiments, we show that cell proliferation rates also exhibit large variation across identical mice. Strikingly, we observe a broad involvement of cell types in exhibiting strong positive correlations of their proliferation rates. Through mathematical modeling, we show that the burst of B cell proliferation often reduces the plasticity of the hematopoietic system and result in regular oscillatory behaviors of B cells, neutrophil etc., which is consistent with the previous reports. A better understanding of the fluctuation of cell numbers and kinetics can afford a better administration of therapies for blood disorder patients.
The obesity-induced expression of T-bet in B cells promotes metabolic dysfunction and alters antibody responses
Authors: Vanz Carlo , Dudley Elizabeth , Enslow Benjamin , Leadbetter Elizabeth
Published: August 2023

Obesity is a leading risk factor for premature mortality and poor medical outcomes due to its increasing global prevalence and its profound impact on the human body. Obesity also has a significant immune component as the establishment of a chronic inflammation in obese individuals is a significant driver of metabolic dysfunction. Additionally, obesity alters anti-tumor immunity, and it impairs protective antibody responses during infection or vaccination. The outcome can be extremely catastrophic, as highlighted in the COVID-19 pandemic with obese patients being particular susceptible to the disease.

This relationship warrants more investigation into the obesity-associated changes occurring in B cells, central players in both the obesity-associated chronic inflammation and in the production of antibodies.

Our lab has identified a subset of B cells which is expanded in both humans and mice during obesity and is characterized by the expression of the transcription factor T-bet.

In vivo studies using obese mice fed a high fat diet revealed that these Tbet+ B cells promote inflammation and metabolic dysfunction through the production of proinflammatory antibodies. Additionally, blocking the expression of T-bet in the B cells of obese mice resulted in significant changes in antibody responses, with a greater expansion of germinal center B cells and an increased ability to generate antigen-specific B cells following immunization.

Based on our findings, T-bet+ B cells represent an interesting link between obesity, inflammation, and antibody responses. Identifying the mechanisms regulating their function will provide new potential ways to reduce inflammation and enhance protection against infectious diseases in obese patients.