bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intrinsic B cell TLR-BCR coengagement induces mature and protective antibody responses in the absence of T cells
Authors: Rivera Carlos Eduardo , Chupp Daniel P , Yan Hui , Fernandez Maria J , Zan Hong , Simon Raphael , Xu Zhenming , Casali Paolo 
Published: February 2023

Abstract
Somatic hypermutation (SHM) and class-switch DNA recombination (CSR) together with plasma cell and memory B cell differentiation are central to the maturation of the antibody response. Prompted by our finding that CSR and plasma cell differentiation can be induced in vitro by a TLR ligand together with BCR crosslinking, we hypothesized that TLR and BCR coengagement can induce mature antibody responses in the absence of T cells. To test this hypothesis, we injected Tcrβ−/−Tcrδ−/− mice, which are devoid of T cells, with NP-LPS or Salmonella Typhimurium flagellin, which coengages B cell TLR4 or TLR5 with BCR. These mice generated NP or flagellin-specific IgG class-switched antibodies and plasma cells. The IgG response to NP evoked formation of germinal center microclusters, a high frequency of point-mutations in IgH V186.2 (region which encodes NP-binding BCRs), including the affinity-enhancing Trp33Leu mutation that underpins the production of high-affinity anti-NP antibodies, and generation of memory B cells. The T cell-independence of the class-switched antibody response was further emphasized by the anti-NP IgG response in immune-deficient NSG mice engrafted with purified mouse B cells. It was mimicked by the specific TLR5-dependent IgG response to S. Typhimurium flagellin in Tcrβ− /−Tcrδ−/− mice. The LPS-immunized and flagellin-immunized Tcrβ−/−Tcrδ−/− mice generated LPS-specific and flagellin-specific IgG antibodies that neutralized E. coli and S. Typhimurium in vitro and protected them from lethal infection in vivo. Thus, TLR-BCR coengagement can induce a class-switched, hypermutated and protective antibody response in the absence of T cells.
Genetic regulation of immune homeostatic lung leukocyte populations influences respiratory virus induced disease in collaborative cross mice
Authors: Hampton Brea K , Jensen Kara L , Whitmore Alan C , Gralinski Lisa E , Leist Sarah R , Linnertz Colton L , Maurizio Paul , Menachery Vineet D , Morrison Clayton R , Noll Kelsey E , Plante Kenneth S , Shäfer Alexandra , Shaw Ginger D , West Ande , de Villena Fernando Pardo-Manuel , Baric Ralph S , Heise Mark T , Ferris Martin T 
Published: February 2023

Abstract
Immune homeostasis is the state that the immune system maintains in the absence of insult. Perturbation of immune homeostasis can impact autoimmunity/allergy and adversely affect infectious responses. To date, much of the analysis of immune homeostasis has focused on systemic immunity, but it is also likely to be important in an organ specific manner. Since the lungs are a major site of environmental exposure and infection, we used the Collaborative Cross (CC) mouse genetic reference population to study the genetic regulation of the breadth of baseline immune cell populations in the lung and identify loci regulating these cells at homeostasis. We found that all 54 immune cell phenotypes measured showed strong genetic variation in cell type abundances. We identified 28 quantitative trait loci associated with variation in 24 immune cell populations or the relationship between cell populations. Further, we identified significant associations between 20 of these loci and responses to either influenza A virus (IAV) or Severe acute respiratory syndrome associated coronavirus (SARS-CoV) disease in the same strains of mice. Notably, a locus mapped for variation in Ly6C+ monocyte abundance was associated with SARS-CoV weight loss, as well as titer at days 2 and 4 post-infection. This locus is also associated with influenza-induced disease, as measured by weight loss post-infection. Our analysis highlights the strong genetic control of immune homeostasis, and the key role that immune homeostasis plays in contributing to downstream infectious responses. In particular, our analysis suggests that the abundance of a variety of lung leukocyte populations prior to infection could serve as predictors of immune responses to respiratory viruses.
Engagement of ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells
Authors: Peng Changwei , Huggins Matthew A , Wanhainen Kelsey M , Lu Hanbin , Georgiev Hristo , Wang Haiguang , Mittelsteadt Kristen L , Hogquist Kristin A , Campbell Daniel J , Silva Henrique Borges da , Jameson Stephen C 
Published: February 2023

Abstract
Resident memory T cells (TRM) provide localized protective immunity in non-lymphoid tissues, but the signals and interactions within non-lymphoid tissues involved in generating TRM are incompletely defined. Inducible T-cell costimulator (ICOS) is a costimulatory molecule with well elaborated roles in regulating differentiation of CD4+ T cell populations, its function in CD8+ T cells is largely unexplored. With acute infection models, we found that ICOS-deficient CD8+ T cells showed minimal defects in the initial effector expansion and the generation of recirculating memory subsets. However, consistent with its high expression in TRM, ICOS deficiency significantly compromised the establishment of CD8+ TRM cells in diverse non-lymphoid tissues. We further confirmed that rather than pre-programming the TRM fate during initial T cell priming, the interaction between ICOS ligand (ICOSL) and ICOS in the non-lymphoid tissues during CD8+ T cell recruitment is critical for efficient tissue retention and acquisition of tissue residency signatures. ICOS signaling through PI3K pathway is key for TRM generation and ICOS stimulation promotes downregulation of KLF2, a transcription factor that regulates T cell trafficking, in CD8+ T cells after entering non-lymphoid tissues. Altogether, our data illustrate that engagement of ICOS is one of the critical local costimulatory cues that promote the establishment of tissue-resident populations, with potential implication for therapeutic manipulation.
The actin-disassembly protein glia maturation factor γ enhances actin remodeling and B cell antigen receptor signaling at the immune synapse
Authors: Deretic Nikola , Bolger-Munro Madison , Choi Kate , Gold Michael R 
Published: February 2023

Abstract
Signaling by the B cell antigen receptor (BCR) initiates actin remodeling. The assembly of branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, allowing B cells to form membrane protrusions that can scan the surface of antigen-presenting cells (APCs). The resulting Arp2/3 complex-dependent actin retrograde flow promotes the centripetal movement and progressive coalescence of BCR microclusters, which amplifies BCR signaling. Glia maturation factor γ (GMFγ) is an actin disassembly-protein that releases Arp2/3 complex-nucleated actin filaments from actin networks. By doing so, GMFγ could either oppose the actions of the Arp2/3 complex or support Arp2/3 complex-nucleated actin polymerization by contributing to the recycling of actin monomers and Arp2/3 complexes. We now show that depleting GMFγ in human B cell lines impairs the ability of B cells to spread on antigen-coated surfaces, decreases the coalescence of BCR microclusters into a central cluster at the B cell-APC contact site, and reduces APC-induced BCR signaling. These effects of depleting GMFγ are similar to what occurs when the Arp2/3 complex is inhibited. This suggests that GMFγ cooperates with the Arp2/3 complex to support BCR-induced actin remodeling and amplify BCR signaling at the immune synapse.
Single cell analysis of iNKT cell phenotype, functionality and metabolism
Authors: Kane Harry , LaMarche Nelson , Brenner Michael , Lynch Lydia 
Published: February 2023

Abstract
iNKT cells have been identified as potent regulators of diverse physiological responses ranging from tumor immunity to homeostatic regulation of adipose tissue. Recent studies have also established that iNKT cells are heterogeneous, and include distinct NKT1, NKT2, NKT17 and NKT10 subsets. To date, these subsets have been broadly characterized using various transcriptomic-based approaches, providing a basis for further analysis and exploration. However, many studies characterizing the iNKT cell transcriptome have been limited to resting iNKT cells, or have focused on iNKT cell development, and thus have not captured the dramatic changes in iNKT phenotype and function that have been observed following antigen exposure. Here, we took advantage of single cell RNA-Sequencing to characterize adipose and splenic iNKT cells before and after activation, and performed integrated analysis of novel and published data to construct a comprehensive overview of the iNKT cell response to antigen. We find that all iNKT cells undergo rapid and profound transcriptional remodeling in response to αGalCer, and identify conserved features of iNKT cell activation, including dynamic reprogramming of cellular metabolism. We also find differences between activated iNKT cell subsets, and we demonstrate that oxidative metabolism functionally segregates NKT2 and NKT17 cells from NKT1 cells. Furthermore, we find that key features of iNKT cell activation are blunted in regulatory iNKT cell populations, which are enriched in adipose tissue, and we identify a conserved network of gene signatures associated with NKT10 cells. Overall, this study provides novel insights into the biology of iNKT cells, and the interplay between their biology and metabolism.
LINGO2 expression on T cells promotes anti-helminth immunity
Authors: Belle Nicole Maloney , Pastore Christopher , Jean Erin , Herbert DeBroski 
Published: February 2023

Abstract
T helper type 2 (TH2) cells are critical for clearance of parasitic helminth infections. Their coordinated efforts harness cellular and molecular pathways to expel worms, resolve intestinal inflammation and drive tissue repair. Yet the mechanisms that control TH2 cell activation, maintenance, and downregulation remain unknown. We recently demonstrated that Leucine rich repeat and Ig domain containing 2 (LINGO2) can function as a receptor for the mucosal reparative cytokine, Trefoil factor 3, to limit epithelial damage, colitic disease and immunopathology. Because LINGO2 is broadly expressed in both hematopoietic and non-hematopoietic cell lineages, this study specifically addressed whether LINGO2 expression in the T cell compartment served a biologically important function in host protection against the parasitic helminth Trichuris muris. Data show that mice selectively deficient for LINGO2 in T lymphocytes (CD4Cre+ LINGO2flox/flox) had significantly higher numbers of adult worms in the cecum, parasite eggs in the stool and colon immunopathology compared to CD4Cre+ or LINGO2flox/flox controls. Intriguingly, after Trichuris infection, CD4Cre+ LINGO2flox/flox mice had significantly fewer GATA3+ TH2 cells and expressed significantly increased interferon gamma levels in the mesenteric lymph node. Taken together, this work highlights a previously unrecognized cell-intrinsic role for LINGO2 in controlling T cell responses that critically shapes the outcome of helminth infection.
A novel SLP-76 signaling pathway T-cells that controls cancer immunotherapy
Authors: Tu Thai Hien , Hulea Laura , Rudd Christopher E. 
Published: February 2023

Abstract
The receptors and signaling pathways that regulate T-cell activation are central in the control of immune responses against tumors. In addition to proximal kinases such as p56lck and downstream immune cell adaptors, we have identified a new signaling pathway in T-cells that involves the direct regulation of the nuclear pore complex (NPC) by the binding of immune adaptor SLP-76 to SUMO-RanGAP1 of the NPC (Mol Cell 2015 59(5):840–9). This interaction represents a second checkpoint for NFATc1 nuclear entry and accounts for some 40–50% of transcription factor entry into the nucleus. RanGAP1 binds to a motif in SLP-76 involving the lysine at residue 56 where its mutation ablates complex formation. In this new study, we report the generation of K56E SLP-76 knock-in (KI) mice which are markedly impaired in the control of tumor growth. This loss of control was accompanied by a marked reduction in the presence CD8+ effector TILs and in the production of interferon-g1 and CD8 cytolytic effector molecules, perforin and granzyme B (GZMB). Further, K56E T-cells showed an impairment in metabolism. Seahorse analysis showed a &gt;70% decrease in glycolysis in response to TCR ligation due to this single mutation in a single adaptor protein. Moreover, K56E T-cells showed a decrease in the expression of Glut1 and in c-Myc translocation into the nucleus of T-cells. By contrast, the expression of other mediators such as PPARα and CPT-1A in fatty acid oxidation were unaffected. Our findings identify a novel SLP-76-RanGAP1 signaling pathway in T-cells that controls transcription factor entry into the nucleus and which modulates immunity against tumors.
The role of tyrosine phosphatase SHP-1 in regulatory T cell function and immune tolerance
Authors: Gu QinLei , Parlak Mahmut , Lorenz Ulrike 
Published: February 2023

Abstract
Our immune system relies on multiple levels of controls to balance its response to danger while maintaining self-tolerance. The functional interactions between conventional T cells (Tcon) and regulatory T cells (Treg) are crucial in this process. Previous studies from our lab revealed that the tyrosine phosphatase SHP-1 plays a critical role in immune tolerance, and while its regulatory role in Tcon cells is widely studied, the role of SHP-1 in Treg is much less understood.
Here, we investigated the role of SHP-1 in Treg cells by using a genetic mouse model carrying a Treg-specific deletion of SHP-1 to address two questions: (I) how SHP-1 affects the activity of Treg cells; and (II) how SHP-1-deficient Treg cells shape the overall immune response.
(I) A characterization of SHP-1-deficient Treg cells demonstrated an increase in the expression of a subset of Treg signature markers compared to SHP-1-sufficient Treg cells. Upon stimulation, SHP-1 deficiency in Tregs increases IL-17 cytokine expression. Moreover, we observed that SHP-1 decreases AKT phosphorylation in Treg cells, consistent with previous studies in Tcon cells from us and others. Finally, we found that SHP-limits Treg suppressive activity in vitro.
(II) Next, we asked whether SHP-1 deficiency in Treg cells affects the overall immune system and found a significant increase in CD44hi CD4+ and CD8+ T cells, indicating a secondary in vivo effect of SHP-1-deficient Treg cells on SHP-1-sufficient Tcon cells. Using murine models of autoimmune diseases, we are currently addressing the underlying mechanism of this phenotype.
In summary, our studies suggest an important regulatory role of SHP-1 in Treg activity and function thereby additionally contributing to the overall immune balance.
A superior dead cell removal platform using Akadeum’s BACS microbubbles
Authors: Grimley Edward C , Roussey Jon , Petlakh-Co Nadia , Wegner Casey , McNaughton Brandon 
Published: February 2023

Abstract
Single cell sequencing (SCS) is one of the most commonly used analytical techniques for cellular characterization and has become a mainstay in immune profiling. As SCS has gained in popularity and improved in sensitivity, it has become apparent that ambient nucleic acids can have a major impact on the quality and reproducibility of obtained SCS results. Since the bulk of ambient nucleic acids in a sample come from dead or dying cells that have lost membrane integrity, one way to dramatically reduce ambient nucleic acids and decrease background noise in SCS is to remove dead cells from the sample prior to sequencing.
To address the challenge presented by contaminating dead cells, Akadeum has developed a Buoyancy Activated Cell Sorting (BACS™) microbubble approach for depleting dead cells from biological samples. Leveraging the power of gravity to drive self-isolation, Akadeum’s BACS microbubbles represent some of the gentlest isolations available; therefore, they are ideally suited for applications where cell viability is integral to success of the experiment. Additionally, BACS microbubbles from Akadeum are quick and easy to use since they require only minimal manipulation and do not rely on any extra equipment or consumables.
Depletion of dead cells is achieved through the selective capture of cells with exposed phosphatidylserine with Annexin V conjugated BACS microbubbles. Once mixed with the sample, the Annexin V BACS microbubbles capture dead cells and float them to the sample surface for removal leaving behind healthy and untouched cells. Samples exposed to the Annexin V BACS microbubbles demonstrated significantly reduced dead cell populations and ultimately yielded more reliable results.
Detection and characterisation of lung-resident memory T cells in human and non-human primates
Authors: Humphries Duncan Charles , O‘Connor Richard , Chabaud-Riou Martine , Boudet Florence , Finlayson Keith , Dhaliwal Kevin , Pavot Vincent 
Published: February 2023

Abstract
Rationale: Lung-resident memory T cells (TRM) play a critical role in protecting against respiratory infection. Maintained in a quiescent state, TRM are poised for rapid effector function upon reactivation with cognate antigen. Kronoscan is a clinic-ready, fibre-based, optical imaging system developed by the University of Edinburgh, capable of measuring both fluorescence intensity and lifetime. The aim of this study is to develop a staining protocol to enable the detection of human and non-human primate (NHP) pulmonary TRM populations in situ.

Methods:
Human lungs (deemed non-suitable for transplantation) following ex vivo lung perfusion (EVLP) and NHP lungs (cynomolgus macaque obtained from Charles River Laboratories) were enzymatically digested to achieve a single cell suspension. Cells were stained with cross-reactive antibodies prior to flow cytometric analysis. Fluorescence intensity and lifetime imaging was also performed using Kronoscan on MACs-enriched, antibody-stained, pulmonary T cells.

Results:
Human and NHP TRM were primarily identified using CD69 and CD103 and co-expressed a number of chemokine receptors (CXCR3, CCR10), adhesion molecules (CD49a, CD49d) and the immune checkpoint inhibitor PD-1. Differences in phenotype were observed between CD4+ and CD8+ TRM. TRM could be identified via fluorescence intensity and lifetime imaging using Kronoscan.

Conclusions:
A cross-reactive antibody panel capable of detecting human and NHP pulmonary TRM was successfully developed. Fibre-based fluorescence lifetime imaging may provide a valuable tool for assessing pulmonary TRM populations in situ, reflecting the level of immunity following infection and vaccination.
An increasingly important role for salivary antibodies in public health surveillance
Authors: Augustine Swinburne A. J. , Eason Tarsha , Wade Tim , Griffin Shannon , Dufour Al 
Published: February 2023

Abstract
Rapid, sensitive, and specific salivary IgG antibody tests can reveal past infections that could assist in the determination of the true prevalence of microbial infections in population studies. Multiplex immunoassays combining IgM, IgA, and IgG antibody responses against multiple pathogens simultaneously, can provide a wealth of information to delineate acute from chronic infections. Previously, we developed and applied salivary antibody multiplex immunoassays against a range of environmental pathogens including noroviruses (GI.1 and GII.4), hepatitis A virus, Helicobacter pylori, Campylobacter jejuni and Toxoplasma gondii to examine infections associated with recreating in contaminated waters. Here, we employ the methods to study saliva samples collected from individuals recreating at an Iowa, USA, riverine beach. Results showed that nearly 80% of beachgoers had salivary antibodies to at least one of the targeted pathogens at the beginning of the study. Most of these exposures were to noroviruses (GI.1: 59.4%, GII.4: 58.8%) and T. gondii (22.8%). Of the individuals who returned the requested samples for each collection period, 6.1% immunoconverted to one or more pathogen, mostly to noroviruses (GI.1: 3.82%, GII.4: 2.3%) and T. gondii (1.5%). Outcomes of this effort illustrate that this rapid immunological assay serves as a viable, high-throughput, noninvasive and cost-effective tool for providing valuable information on the occurrence of known and emerging pathogens (e.g., SARS-CoV-2) in population surveillance studies.
Improved inter-instrument consistency and high precision for CHO cell bioprocessing using the Cellaca&[trade] MX high-throughput cell counter
Authors: Bell Jordan , Huang Yongyang , Hoover Willis , Kuksin Dmitry , Qiu Jean , Chan Leo 
Published: February 2023

Abstract
CHO cell bioprocessing is a common application for producing biologics, antibodies, and proteins for therapeutic products. An important aspect of the CHO bioprocess is the characterization of a cell culture’s concentration and viability to ensure that the cells are in optimal condition for production. Traditionally, CHO cells have been measured using a manual hemocytometer or automated cell counter with trypan blue staining. However, these methods have limitations in throughput and instrument-to-instrument consistency.
Here, we evaluate the performance of the Cellaca™ MX high-throughput cell counter for implementation into the CHO cell bioprocess. We investigate the precision, instrument-to-instrument consistency, linearity, and proportionality following ISO cell counting standard 20391-2:2019. We demonstrate close agreement between multiple Cellaca™ MX instruments using both CHO cells with Trypan Blue (5 instruments) and beads (32 instruments). We also report system-wide precision, which includes variation between multiple counts, consumables, instruments, and days (in the case of beads). Furthermore, we include the results of several comparison experiments in which samples were counted using Cellaca™ MX, hemocytometer, “Cell Counter V” (a competing instrument), as well as the Celigo® Imaging Cytometer. Finally, we demonstrate the use of the ISO cell counting standards to evaluate the linearity, precision, and proportionality index of the Cellaca™ MX. These results show that Cellaca™ MX can count trypan blue-stained CHO cells in brightfield in less than 1 min for 24 samples, and that the consistency, comparability, and precision of the Cellaca™ MX are significantly improved over the traditional methods.