bluuebunny/crossref_metadata_2025_split huggingface dataset:
Constitutive LcK activity drives sensitivity differences between CD8+ memory T cell subsets
Authors: Krogsgaard Michelle , Moogk Duane , Zhong Shi , Rittase William , Fang Victoria , Dougherty Janna , Perez-Garcia Arianne , Osman Iman , Zhu Cheng , Varadarajan Navin , Restifo Nicholas P , Frey Alan B 
Published: January 2023

Abstract
CD8+ T cells develop increased sensitivity following antigen experience, and differences in sensitivity exist between T cell memory subsets. How differential T cell receptor (TCR) signaling between memory subsets contributes to sensitivity differences is unclear. We show that constitutive activity of lymphocyte-specific protein tyrosine kinase (Lck) is greater in effector memory T cells (TEM) compared with central memory T cells (TCM), leading to enhanced activation signaling, including Zeta-chain-associated protein kinase 70 (Zap-70) phosphorylation and intracellular calcium influx, and resulting in increased cytotoxic effector function in TEM. We provide evidence that the differences in Lck activity between CD8+ TCM and TEM are due to differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase (Csk). Together, this work demonstrates a role for constitutive Lck activity in controlling antigen sensitivity, and suggests that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM.
TCR Repertoire Analysis of Mouse T Follicular Helper Cells and T Follicular Regulatory Cells Following Immunization
Authors: Yeh Chen-Hao , Kuraoka Masayuki , Lynch Heather , Sempowski Gregory D , Kelsoe Garnett H. 
Published: January 2023

Abstract
Generation of high-affinity and class-switched antibody requires the germinal center (GC) reaction after infection or immunization. Within the B-cell follicles of secondary lymphoid organs, the GC represents a sophisticated collaboration between antigen-specific B cells, follicular dendritic cells, T follicular helper (TFH) cells and T follicular regulatory (TFREG) cells. Despite intensive interest in the development and effector function of TFH and TFREG cells, little is known regarding the selection of T-cell receptor (TCR) repertoire during polyclonal GC reactions. In order to evaluate native, polyclonal TCR responses elicited by a complex antigen, we developed a sorting strategy to isolate TFH/TFREG cell populations that were activated and expanded after s.c. immunization with NP15-OVA. TCRβ VDJ rearrangements were recovered from highly purified TCRβ+CD4+CXCR5hiPD-1+Bcl-6+FoxP3− TFH cells and TCRβ+CD4+ CXCR5hiPD-1+Bcl-6+FoxP3+ TFREG cells, amplified by PCR, and sequenced. Analysis of the antigen-specific TCR repertoire of TFH/TFREG cells provides important insights into the factors influencing T-cell recruitment and clonal expansion following infection or vaccination, especially when linked to contemporary analysis of the GC B-cell repertoire. These findings may inform rational and selective control strategy of the GC reaction. Vaccine development can accordingly focus on modulating TFH/TFREG responses to facilitate optimal adaptive immune responses.
T cell specific dysregulation of Foxo1 results in spontaneous inflammatory disease
Authors: Sullivan Jenna M , Duggan Jeffrey M , Thomas Kerri , Campbell Daniel J. 
Published: January 2023

Abstract
The Foxo (forkhead box O) family of transcription factors is important for maintaining homeostasis of both effector and regulatory T cells (TR). Interestingly Foxo1 is necessary for proper development and function of TR and absence of Foxo1 results in fatal inflammation due to TR dysfunction. In conventional T cells, Foxo1 is phosphorylated downstream of the PI3K/Akt/mTOR signaling pathway resulting in its inactivation and translocation from the nucleus and subsequent altered transcription of downstream targets. While many studies have examined the effects of Foxo1 deficiency, few have addressed the role of proper downregulation of Foxo1. To address this question we have obtained mice that carry a non-phosphorylatable Foxo1 allele (Foxo1CA) knocked into the ROSA26 locus, along with a ‘stop-flox’ cassette that prevents expression. We have crossed Foxo1CA mice to CD4-Cre mice (CD4cre Foxo1CA), resulting in deletion of the ‘stop-flox’ cassette and constitutive activation of Foxo1 in both CD4 and CD8 T cells beginning at the double positive stage of thymic development. Mice which are unable to properly regulate Foxo1 in T cells are characterized grossly by runting, dermatitis and splenomegaly. CD4cre Foxo1CA mice displaying symptoms of inflammation have tissue damage in the lung along with increases in eosinophils, pro-inflammatory cytokines, and increases in class switched antibodies and autoantibodies. In the periphery CD4cre Foxo1CA mice have reduced CD4 and CD8 T cell populations comprised mainly of activated effector T cells and a dramatically reduced TR population. Together this data demonstrates that proper regulation of Foxo1 is essential for T cell homeostasis and prevention of systemic inflammatory condition.
α-Galatosylceramide and retinoic acid regulate splenic follicular B cell differentiation and antibody production in a CD1d-dependent manner
Authors: Ross A. Catharine , Chen Qiuyan , Wei Cheng-hsin 
Published: January 2023

Abstract
α-Galactosylceramide (αGalCer) and all-trans-retinoic acid (RA) are each potent immune regulators able to increase immune responses in various conditions. αGalCer is particularly known for its direct activation of invariant NKT cells and B cells. In the present study, we used wildtype (WT) and CD1d knockout (KO) mice to characterize the specificity of αGalCer and its interaction with RA on B cell activation. WT and CD1dKO spleen cells were cultured ± αGalCer (100 ng/ml), or LPS (100 ng/ml) as a direct B-cell stimulus, and RA (20 nM), and B cell proliferation and differentiation were determined by flow cytometry. αGalCer and LPS both markedly increased the CD19+ B cell division. Whereas αGalCer mainly enriched spleen follicular CD23+ B cells, LPS increased the marginal zone CD21+ B cell population. The effects of αGalCer were completely absent in CD1dKO mice. However, although spleen B cells of CD1dKO mice failed to respond to αGalCer, these cells had a higher rate of proliferation compared to cells of WT mice, suggesting that CD1d molecules may have immune tonic effects in addition to reacting to αGalCer. Moreover, RA worked together with αGalCer to increase the proportions of IgG+ and CD138+ cells among CD23+ B cells, suggesting enhanced B-cell differentiation in splenic follicles. An in vivo immunization study using Keyhole limpet hemocyanin (KLH, 50 μg, ip) further confirmed that administration of RA (37 μg, po) and αGalCer (2 μg, sc), given at the time of primary immunization, increased antibody production in both the primary and secondary responses, which was not observed in CD1dKO mice. Thus, immune enhancement by αGalCer requires CD1d, and, when combined with RA promotes follicular B cell differentiation and increased antibody production.
The role of CD69 in generating resident memory CD8+ T cells.
Authors: Walsh Daniel Aaron , Hamilton Sara E , Jameson Stephen C 
Published: January 2023

Abstract
Resident memory CD8+ T cells (TRM) are one of the first lines of defense against previously encountered pathogens. Their advantageous position in parenchymal tissue combined with higher expression of effector molecules makes TRM particularly well suited to combat the early stages of an infection. CD69 is a canonical marker of typical CD8+ TRM. It has the potential to regulate TRM trafficking by competition with S1PR1 (and possibly other mechanisms). However, the functional requirement for CD69 to establish CD8+ TRM is unclear. To test this, we competed LCMV specific CD69 deficient and sufficient CD8+ T cells in vivo during the course of an acute LCMV infection. Unexpectedly, CD69 deficient cells were only weakly disadvantaged at establishing and maintaining residency in most parenchymal organs. However, CD69 deficient cells were heavily disadvantaged when establishing residency in the kidney. These insights lead to a more nuanced model of tissue residency, in which the requirement for CD69 expression may be dictated by factors such as the tissue microenvironment and antigen availability.
Systems-based identification of new adjunct therapies for treatment of malaria-induced cerebral pathology
Authors: Strangward Patrick , Couper Kevin 
Published: January 2023

Abstract
Cerebral Malaria (CM) is a severe neurological complication of Plasmodium falciparum infection characterised by abnormal posturing, seizures and coma. Current treatment involves the administration of anti-parasitic drugs, and fails to prevent mortality in 10–20% of cases. Furthermore, up to a quarter of effectively treated individuals exhibit long-term neurological dysfunction after an episode of CM. Crucially, we lack understanding of the factors that govern a successful or unsuccessful outcome to current treatment. Therefore, the aim of this study was to utilise the murine experimental model of CM (ECM) to further our understanding of the mechanism(s) that determine successful drug therapy of CM, in order to aid development of specific adjunct therapies.
C57BL/6 mice were infected with Plasmodium berghei ANKA and then treated with anti-parasitic drugs at the onset of mild neurological dysfunction.
Survival was ~80% in drug treated mice, with histological examination indicating attenuation of oedema as a key feature in mice that respond successfully to drug therapy. Additionally, whole brain transcriptomics demonstrated unique cerebral RNA signatures in mice at the point of drug treatment, post successful drug treatment and in the absence of drug treatment. Moreover, these data implicated several pathways relating to endothelial permeability as significant in mice that succumb to ECM.
The results of this study provide important new information on pathways amenable to therapeutic modulation to aid treatment of CM.
NK T cells promote IL-21 secretion and enhance Ab production and affinity maturation during Plasmodium yoelii infection
Authors: Wikenheiser Daniel , Stumhofer Jason 
Published: January 2023

Abstract
NK T cells are a class of lipid antigen-restricted CD4+ T cell possessing qualities of both innate and adaptive immune cells. In addition to rapid production of various effector cytokines, NK T cells have also been shown to play a role in modulating the germinal center (GC) reaction directly by differentiation into NK-Tfh cells, or indirectly by promoting dendritic cell (DC) maturation. Due to their ability to enhance DC expression of MHCII and B7-family co-stimulatory molecules, synthetic glycolipids that target NK T cells are being considered as adjuvants in a number of vaccine approaches designed to facilitate conventional CD4+ T cell activation. During Plasmodium infection, a pathogen for which a vaccine is greatly needed, NK T cells have been shown to be responsive to parasite-derived glycolypids. To assess the contribution of NK T cells to the GC reaction following Plasmodium infection, NK T cells were depleted during murine Plasmodium yoelii infection. Here, we demonstrate that NK T cell depletion did not result in the production of fewer conventional Tfh cells or GC B cells. However, depletion of NK T cells resulted in decreased frequency and total number of IL-21–producing CD4+ T cells. This resulted in NK T cell depleted mice producing a lower quantity and quality of MSP-119-specific IgG Abs when compared to control mice. Ultimately, the decrease in Ab titer was attributed to significantly fewer MSP-119-specific IgG antibody-secreting cells in the bone marrow of NK T cell depleted mice. Collectively, this data suggests NK T cells play an important role in shaping the GC reaction during P. yoelii infection. Successful modulation of NK T cell activity could prove a useful strategy in future Plasmodium vaccine design.
Inflammatory monocytes mediate competition between genetically diverse malaria parasites during mixed-strain Plasmodium infections.
Authors: Jacobs Nathan Thomas , Barra Jessie , Antia Rustom , Lamb Tracey J 
Published: January 2023

Abstract
In areas of intense malaria transmission, many individuals receive multiple infectious bites per night, leading to overlapping infections with multiple strains of Plasmodium. As the transmission likelihood of a given strain is influenced by blood-stage parasite densities, parasites that can multiply faster and avoid immune clearance mechanisms are more competitively fit and are likely to be favored by evolution. Competitive ability has previously been linked to the virulence of infection, but mechanistic details are unclear. Here, we demonstrate a role for innate phagocytic cells, specifically CD11b+ Ly6Chi inflammatory monocytes, in mediating competition between two clones of the rodent malaria parasite Plasmodium chabaudi during a mixed infection. PcAS and PcCB grow to similar densities and cause similar degrees of anemia in separate single infections, but differ in the inflammatory and immunoregulatory responses they elicit. In mixed infection, PcAS densities were more adversely impacted than those of PcCB when compared to single infections, but competitive suppression was abrogated by depletion of macrophages and monocytes prior to infection using clodronate-loaded liposomes. Flow cytometry analysis of splenic populations revealed that the CD11b+ Ly6Chi inflammatory monocyte population during mixed infection more closely resembles that of an infection with PcCB than with PcAS. Taken together, our results indicate that competition between genetically diverse malaria parasites can be mediated by innate immunity independent of parasite virulence, and that parasites gain a competitive advantage by polarizing the immune system towards a response to which they are adapted.
Germinal-center derived, somatically hypermutated ­Plasmodium-specific IgM+ memory B cells are optimally responsive to a secondary blood stage malaria infection
Authors: Krishnamurty Akshay T , Thouvenel Chris , Hondowicz Brian , Keitany Gladys , Crompton Peter , Rawlings David J , Pepper Marion 
Published: January 2023

Abstract
Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBCs). Secondary responses by MBCs traditionally are thought to involve the production of class-switched, high affinity antibodies that can neutralize an invading pathogen if pre-existing antibody concentrations are not sufficient to control infection. In infections with high pathogen loads, like blood stage malaria, MBC induction may therefore be critical and has been correlated with protection. What types of MBCs are important for mediating this protection remains unknown. Recent studies have expanded upon conventional MBC paradigms and phenotypically and functionally heterogeneous MBCs have been described in mice and humans. To gain insight into the types of MBCs that form and function during primary and secondary Plasmodium infection, we developed a novel B cell tetramer against the Plasmodium blood stage protein, Merozoite Surface Protein 1 (MSP1). In a murine model of Plasmodium infection three distinct MSP1-specific MBCs form: an IgG+, IgD+ and IgM+population, which are all maintained for long periods of time. These three subsets are phenotypically and genetically distinct, have different developmental histories and respond to a secondary iRBC challenge in unique ways. Remarkably, somatically hypermutated MSP1-specific IgM+ MBCs outcompete IgG+ MBCs early during a secondary challenge resulting in the expansion of newly formed plasmablasts and increased levels of serum MSP1-specific IgM antibodies. As a result, this study highlights a previously unrecognized role for IgM+ MBCs during an infection and should be considered a target population when developing an anti-malarial vaccine.
Regulation of host immune responses to Schistosoma mansoni by the inhibition of the parasitic NAD salvage pathway.
Authors: Schultz Michael David , Dadali Tulin , Botta Davide , Manouvakhova Anna , Sosa Melinda , McKellip Sara N. , Woods LaKeisha , Tower Nichole A. , Ross Larry J. , Rasmussen Lynn , White Lucile , Padmalayam Indira , Augelli-Szafran Corinne , Everts Maaike , Sorci Leonardo , Bostwick James R. , Suto Mark J. , Lund Frances E 
Published: January 2023

Abstract
T regulatory cells (Tregs) are a subset of FoxP3+ lymphocytes that maintain immune homeostasis through immunosuppressive signaling. In many helminth infections, one mechanism of immune evasion involves the influx and maintenance of Tregs. Multiple mechanisms can control Treg homeostasis including a Nicotinamide adenine dinucleotide (NAD) dependent apoptotic pathway. In most organisms, the two pathways of NAD synthesis include de novo synthesis from amino acid precursors, and the salvage pathway in which nicotinamide-containing precursors are recycled into NAD. A comparative genome analysis of S. mansoni, the parasite that causes Schistosomiasis, enabled the assembly of a putative NAD biosynthetic pathway. Only orthologues of NAD salvage-specific genes were identified and expression was confirmed by PCR. This suggests that S. mansoni decreases host NAD levels in a salvage-specific manner and rescues Tregs from NAD-dependent cell death. As such, we hypothesized that inhibition of NAD biosynthesis through the salvage pathway would result in impaired NAD uptake by S. mansoni and restoration of NAD-mediated cell death of Tregs. Indeed, co-culture with S. mansoni prevented NAD-induced toxicity and death of Tregs. Blockade of the NAD salvage pathway via the inhibition of Schistosoma mansoni NAD catabolizing enzyme (SmNACE) restored NAD-mediated cell death of Tregs. Furthermore, SmNACE inhibition decreased intracellular NAD levels and reduced metabolism and viability of the parasite. Collectively, our data suggest that inhibition of NAD biosynthesis blocks immune evasion and metabolism of S. mansoni, and that targeting the NAD salvage pathway is a promising therapeutic approach for the treatment of Schistosomiasis.
Distinctive signalling in NK cells during cytotoxicity to the yeast Cryptococcus neoformans
Authors: Xiang Richard F , Stack Danuta , Li Shu Shun , Ogbomo Henry , Mody Christopher H 
Published: January 2023

Abstract

Introduction
Cryptococcus is a fungal pathogen that subverts the adaptive immune response. Fortunately, NK cells are capable of directly recognizing, triggering cytotoxicity and killing Cryptococcus. Although, NK cytotoxicity against tumor and virally infected targets are well studied, less is known about anti-fungal signaling. In this study, we investigated a novel NK cytotoxicity pathway involving Rac, Src family kinases (SFK), and integrins.

Methods
The involvement of Rac, SFK, integrin-linked kinase (ILK), and integrins were explored using small molecule inhibitors, siRNA knockdown, activated Rac pull down assays, and immunoblots.

Results
Our studies found that Cryptococcus activated both SFK and Rac signaling pathways. Small molecule inhibitors of SFK and Rac blocked Erk and PI3K activity. siRNA knockdown of Rac also inhibited PI3K activity, suggesting that Rac was an upstream non-canonical activator of PI3K. Moreover, individually Rac and SFK were not sufficient for activation of PI3K. Inhibition of Rac also resulted in a 90% loss of anti-cryptococcal cytotoxicity. Additionally, inhibitors of ILK prevented Rac activation and caused an 80% reduction in cryptococcal killing. Lastly, siRNA knockdown of beta-1 integrins caused an inhibition of ILK activation and 50% reduction in cryptococcal killing.

Conclusion
We found beta-1 integrins to be a fungal receptor and Rac an activator of PI3K. These findings build a signaling model of anti-fungal killing that is different from the canonical tumor killing model that involves beta-2 integrins and Rac downstream of PI3K. Our model provides various potential therapeutic targets that could enhance fungal clearance mediated by the innate immune system.
γδ T cells support gut Ag-reactive colitogenic effector T-cell generation by enhancing Ag presentation by CD11b+ DCs in the mesenteric LN
Authors: Do Jeong-Su , Visperas Anabelle , Freeman Michael L , Jang Eunjung , Kim Sohee , Malissen Bernard , Min Booki 
Published: January 2023

Abstract
T cells expressing the γδ TCR are dominant T-cell subsets in the intestinal immune system. We previously demonstrated that γδ T cells play important roles in augmenting Th17-type colitogenic immune responses in a T cell-induced colitic inflammation model. However, its underlying mechanism remains poorly understood. In this study, an in vitro coculture system using effector T cells enriched in gut Ag-reactive cells was employed as a readout tool to search for gut Ag presenting APCs. We found that the presence of γδ T cells dramatically enhances gut Ag presentation within the mLN in mice. Mesenteric MHCII+ CD11c+ DCs from TCRβ−/− induce massive proliferation of colitogenic T cells but not DCs from γδ T cells deficient mice. Gut Ag presentation by CD11b+ DC subsets was particularly controlled by γδ T cells. Interestingly, γδ T cell entry to the lymph nodes was essential to improve the Ag presentation. Therefore, our results highlight that γδ T cells play a previously unrecognized role to support colitogenic immunity by regulating gut Ag presentation in the draining LN.
Methods to Simplify the Design of Multi-color Panels.
Authors: Tyznik Aaron J , Rohrer Jurg , Widmann Stephanie , Rabenstein Jacob 
Published: January 2023

Abstract
Flow cytometry has long been a critical tool for immunologists. As the number of parameters used in flow cytometry continues to increase, one of the more difficult aspects of this practice is panel design. Fully resolving dim populations of interest can be challenging. Low antigen density, fluorescence spillover, available fluorochromes and limited catalog offerings are just a few of the factors that make panel design difficult and lead to sub-optimal resolution. Here, we present a basic set of principles that can help simplify panel design. Following these principles helps reduce the number of iterations needed to maximize the resolution for populations of interest. Additionally, we present a basic methodology that can be used to characterize your flow cytometer and generate a matrix showing the relative impact of fluorochrome combinations on population resolution. Using this Resolution Impact Matrix [RIM], low complexity, no/low compensation panels can be more easily created. Similarly, when performing higher order multicolor experiments, this characterization is critical to help design the optimum panel for that instrument. When used together, these two methods can help simplify the panel design process and lead to higher quality data.