bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

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

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

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

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

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

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

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.
T cell receptor knockout efficiency utilizing an engineered meganuclease is influenced by stimulation conditions.
Authors: Brown Audrey E , MacLeod Daniel T. , Martin Aaron J. , Wetzel Keith J. , Pham Christina D. , Antony Jeyaraj , McCreedy Bruce , Bartsevich Victor V. , Nicholson Michael G. , Smith Jeff , Jantz Derek
Published: January 2023

Recent reports describing allogeneic chimeric antigen receptor (CAR) T cell therapies have generated tremendous excitement over the possibility of a universal CAR T platform. We are utilizing a highly engineered meganuclease designed to target the T cell receptor (TCR) alpha chain to eliminate expression of the TCR, preventing graft versus host disease following adoptive transfer. Meganuclease cleavage efficiency is highly dependent on stimulation of T cells prior to delivery of the meganuclease. To determine optimal activation conditions for TCR knockout and cell yield, T cells were activated with Dynabeads® Human T cell Activator CD3/CD28, Immunocult™ CD3/CD28 and CD3/CD28/CD2 T Cell Activators, and MACS® GMP TransAct CD3/CD28 reagent prior to nucleofection. T cells were stained prior to and following nucleofection to identify cell activation markers and cytokine receptors (CD69, IL2R, IL7R, IL15R, IL21R). Knockout efficiency was determined by staining for CD3 expression. Our results indicate that Dynabead-stimulated T cells expressed the highest levels of activation markers at 18 hours post-stimulation, and that Dynabeads® and Immunocult™ CD3/CD28/CD2 reagents both promote robust T cell proliferation and support the highest TCR knockout efficiency following our protocol. Furthermore, expression of cytokine receptors varies both during stimulation and post-nucleofection, suggesting an optimal cocktail of cytokines may promote T cell proliferation. Future studies will focus on further optimization of T cell stimulation conditions that may promote TCR knockout and expansion of T cells post-nucleofection.
Development and characterization of Th17-related U-PLEX® assays
Authors: Wallowitz Mikhael L , Ning Qian , Shrestha Arun , Shelburne Chris , Liu Pu , Stewart David , Wolfert Robert , Oberoi Pankaj , Wohlstadter Jacob N
Published: January 2023

Th17-related cytokines mediate a host’s defense mechanisms against various infections and play a crucial role in crosstalk between the immune system and affected tissues. Here we describe the development and characterization of multiplexed Th17-related immunoassays on MSD’s flexible U-PLEX platform.

Monoclonal and polyclonal antibodies were conjugated with biotin and/or SULFO-TAG™ and screened as capture and/or detection reagents. A number of analytical parameters were used to select antibody pairs, and assays were developed by optimizing antibody concentrations, calibrator curves, specificity, matrix tolerance, and assay robustness. Assay performance was verified to be compatible with other U-PLEX assays by running Th17-related multiplexes on the U-PLEX platform with controls and samples.

Calibration curves showed expected signals, sensitivity, precision, and accuracy. Control samples for the assays had CVs < 10% within runs and < 25% between runs. Sensitivities were < 1 pg/mL for the majority of the assays. Dilution linearity and spike recovery studies demonstrated acceptable matrix tolerance and accurate quantification for most of the assays tested across all matrices (typically between 75% – 125%). Cross-reactivity between assays was shown to be typically < 0.5%. Results demonstrated a strong correlation between samples measured on U-PLEX multiplex and streptavidin singleplex panels with r2 values > 0.95 and slopes between 0.8–1.2.

Over 20 Th17-related assays for human and mouse were developed for the U-PLEX platform. These assays enable the measurement of multiple proteins on biological matrices that are relevant to a wide range of life science applications and pre-clinical or clinical studies.
Target specific siRNA-conjugate therapy for experimental autoimmune myasthenia gravis
Authors: Huda Ruksana
Published: January 2023

Myasthenia gravis (MG) is an autoimmune disease of muscle weakness caused by Acetylcholine receptor- autoantibody and complement mediated damage of neuro-muscular junction (NMJ). We have recently shown a greater advantage of using siRNA over other conventional therapeutic tools in an experimental mouse model of MG (EAMG). However, the major drawbacks of siRNA therapy is reduced efficacy due to the lack of target restricted delivery, where it is necessary to suppress pathogenic response arising predominantly from a particular cell/tissue type. In this study, we used a novel strategy of cell specific new formulation of siRNA or siRNA conjugate delivery to pathogenic cells in EAMG. Our preliminary work shows successful preparation of siRNA conjugate and its functional efficacy over siRNA only treatment in an in vitro rodent cell line. Successful completion of the study may provide a solution for the siRNA or monoclonal antibody therapy hurdle and a new direction to ultimately explore a highly effective and safe therapy for patients with MG.
Anti-MAdCAM-1 therapy does not affect immune surveillance in the central nervous system
Authors: Stuve Olaf , D'Haens Geert R.A.M. , Reinisch Walter , Vermeire Severine , Vogelsang Harald , Allez Matthieu , Desreumaux Pierre , Van Gossum Andre , Baumgart Daniel , Hassan-Zahraee Mina , Cheng John , Yanhua Zhang , Sandborn William , Kaminski Annamarie , Ahmad Alaa , Pradhan Vivek , Cataldi Fabio , Clare Robert , Gorelick Ken
Published: January 2023

Background

Blocking integrins can be associated with an increased risk of progressive multifocal encephalopathy (PML). MAdCAM-1 is an adhesion molecule that is not constitutively expressed in healthy CNS and considered mostly gut-selective. However, MAdCAM-1 is upregulated in choroid plexus epithelium during experimental autoimmune encephalomyelitis (EAE. PF-00547659 is a fully human mAb that is highly selective for MAdCAM-1. The purpose of this study was to investigate its effect on cellular elements of immune surveillance in the CNS and blood of patients with Crohn’s disease (CD).

Methods

All methods were tested in a control cohort of volunteers with CD (Cohort 1). Study patients (Cohort 2) had CD and prior treatment with anti-TNF and immunosuppressant therapies. Patients underwent a lumbar puncture (LP1) followed by 3 monthly injections of 225 mg PF-00547659. After the last dose, a LP2 was performed. CSF was analyzed within 24h by multi-parameter flow cytometry.

Results

In Cohort 1, the lymphocyte subset percentages were the same at both time points. Cohort 2 consisted of 30 patients in whom 2 LPs were performed. Their pre-treatment lymphocyte numbers were lower than those of subjects who had stopped immunosuppressants, but otherwise results were the same. CSF lymphocytes (cells per mL) shown as geometric means (CV%). LP1: 37 patients, total lymphocytes 391 (140%), CD3+/CD4+ cells 246 (152%), CD3+/CD8+ cells 100 (126%), CD4:CD8 ratio 2.46 (57%). LP2: 30 patients, total lymphocytes 513 (137%), CD3+/CD4+ cells 332 (143%), CD3+/CD8+ cells 123 (125%), CD4:CD8 ratio 2.69 (56%).

Conclusion

This is the first evidence that a therapeutic dose of anti-MAdCAM-1 mAb does not affect the number and composition of leukocytes in the CNS.
A predictive disease-specific protein biomarker fingerprint for clinical relapses and glucocorticoids treatment of multiple sclerosis
Authors: Raphael Itay , Chase Carol , Gomez-Rivera Francisco , Forsthuber Thomas G
Published: January 2023

Despite extensive research, multiple sclerosis (MS) remains a disease that lacks a definitive prognostic test to predict imminent disease relapses. Thus, patients may undergo years of unnecessary treatments. Additionally, current treatments for MS vary significantly in efficacy between individual patients, and thus there is a critical need to develop biomarkers for treatment efficacy and resistance. To address these issues, we recently developed a high-throughput quantitative proteomics method to measure changes in proteome expression over the course of the preclinical experimental autoimmune encephalomyelitis (EAE) model. Interestingly, using the EAE model we revealed characteristic CNS-specific protein expression waves prior to the onset of clinical symptoms. Moreover, we have identified key proteins with altered expression that correlated with the therapeutic efficacy of glucocorticoid treatment. Bioinformatics analysis revealed candidate protein biomarkers to predict treatment efficacy and clinical disease course. Importantly, these proteins could be detected in serum and expression trajectories analysis identified a strong correlation of the CNS proteome to their levels in serum. Prospective studies in the EAE model using these candidate protein biomarkers showed their effectiveness in predicting clinical disease and treatment responses. Our studies suggest the utility for establishing homologous protein biomarkers in human MS patients. Finally, our work investigating the CNS proteome over the course of EAE may provide novel insights and molecular targets for disease mechanisms and treatments of MS.
Identifying biomarkers for monitoring progression of multiple sclerosis
Authors: Chase Carol Ann , Raphael Itay , Forsthuber Thomas
Published: January 2023

There are currently no reliable methods for assessing the progression of multiple sclerosis (MS) from the relapsing-remitting to the secondary progressive form. This gap in knowledge hinders the ability for therapeutic intervention and ultimately results in continued relapses and physiological deterioration. To begin to address the urgent need for biomarkers of progressive MS we investigated proteome changes over the disease course of progressive experimental autoimmune encephalomyelitis (EAE) in NOD mice as a preclinical model of the disease. Our lab has pioneered a novel high-throughput quantitative proteomic technique which we used to quantify expression levels of central nervous system (CNS) proteins over the course of monophasic EAE in C57.BL/6 mice. We utilized bioinformatics tools to prioritize key proteins whose expression level correlated specifically with the progressive phase of disease in the NOD EAE model. Importantly, we were able to detect corollary changes in these CNS-specific proteins in the serum, pointing to a minimally invasive means of monitoring disease progress and measuring drug efficacy. Our studies will provide a proof-of-concept for identifying homologous human biomarkers to guide treatment in individual patients. Furthermore, our results may provide insights into mechanisms that contribute to disease pathology and offer additional therapeutic targets for slowing the progression of MS.
Metabolic Reprograming of Alloantigen-Activated T Cells after Hematopoietic Cell Transplantation
Authors: Yu Xue-Zhong , Mehrotra Shikhar , Chi Hongbo , Nguyen Hung D.
Published: January 2023

T cells are the driving force in the induction of graft-versus-host disease (GVHD); yet little is known about T-cell metabolism in response to alloantigens after hematopoietic cell transplantation (HCT). The current study demonstrates that donor T cells undergo reprograming after allogeneic HCT by switching from fatty acid β-oxidation (FAO) and pyruvate oxidation via the TCA cycle to aerobic glycolysis, thereby increasing dependence upon glutaminolysis and the pentose phosphate pathway. Glycolysis is required for optimal function of alloantigen activated T-cells and induction of GVHD, as inhibition of glycolysis by targeting mTORC1 or pfkfb3 ameliorates GVHD mortality and morbidity. Thus, donor T cells use glycolysis as the predominant metabolic process after allogeneic HCT, which can be a valid therapeutic target for the control of GVHD.
Transient depletion of donor CD4+ T cells early after HCT allows recipient tissue PD-L1 to tolerize infiltrating CD8+ T cells and to prevent acute GVHD while preserving GVL effect
Authors: song qingxiao , Ni Xiong , Cassady Kaniel , Deng Ruishu , Jin Hua , Zhang Mingfeng , Forman Stephen , Martin Paul , Wang Jianmin , Zeng Defu
Published: January 2023

Prevention of graft-versus-host-disease (GVHD) while preserving graft-versus-leukemia/lymphoma (GVL) effect remains the Holy Grail for allogeneic hematopoietic cell transplantation (HCT). Purified donor CD4+ T cells induce strong but CD8+ T cells induce little acute GVHD (aGVHD); however, the mechanisms remain unknown. Recipient tissue expression of PD-L1 (B7H1) down-regulates aGVHD via simultaneous PD-L1 interactions with CD80 and PD-1 on donor T cells, but whether this mechanism impacts on GVL effect remains unclear. In the current studies, with MHC-mismatched HCT model of C57BL/6 donor to BALB/c recipient, we observed that 1) although sorted wild-type (WT) CD8+T cells induced no signs of aGVHD, purified PD-1−/− CD8+ T cells induced lethal aGVHD; 2) while WTCD8+ T or very low-dose (0.1×106) CD4+ T cells alone induced little signs of aGVHD, co-transplantation of them induced lethal aGVHD; 3) depletion of CD4+ T cells by anti-CD4 mAb early after HCT prevented aGVHD in wild-type but not in PD-L1−/− recipients, and the GVHD prevention is associated with increase of serum IFN-γ, upregulation of host tissue PD-L1, upregulation of CD80 and PD-1 by CD8+ T cells, as well as increase of apoptosis and anergy of tissue infiltrating CD8+ T cells; 4) interestingly, the depletion of donor CD4+ T cells augmented donor CD8+T cell proliferation without increasing anergy or apoptosis in the lymphoid tissue and preserved strong GVL effect. These results indicate that depletion of donor CD4+ T cells early after HCT leads to increasing susceptibility of donor CD8+ T cells to host tissue PD-L1-mediated apoptosis and anergy in GVHD target tissues but not in the lymphoid tissues, such that GVHD is prevented and GVL effect is preserved.
Inhibition of Endogenous Memory CD8 T Cell Infiltration into Cardiac Allografts Subjected to Prolonged Cold Ischemic Storage is Required to Promote Long-Term Graft Survival Naoki Kohei, Hidetoshi
Authors: Fairchild Robert L , Kohei Naoki , Tsuda Hidetoshi , Tanaka Toshiaki , Iida Shoichi , Abe Toyofumi , Kish Danielle , Ayasoufi Kathy , Valujskikh Anna , Fairchild Robert L
Published: January 2023

Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class I MHC within 24 hrs after graft reperfusion in mice. We have reported that prolonged cold ischemic graft storage (CIS) provokes intense inflammation within hours after allograft reperfusion and promotes CTLA-4Ig-resistant endogenous memory CD8 T cell rejection of the allograft by day 28 post-transplant (whereas CTLA-4Ig prolongs survival of allografts subjected to minimal CIS > day 60). We tested strategies inhibiting endogenous memory CD8 T cell infiltration into cardiac allografts subjected to prolonged CIS to promote long-term allograft survival. Peri-transplant anti-LFA-1 mAb and anti-CD154 mAb treatment of recipients of allografts subjected to minimal CIS prolonged survival of 60% of allograft > 100 days. In contrast, this treatment prolonged only 20% of allografts subjected to prolonged CIS beyond day 80 post-transplant, with rejection accompanied by the appearance of high titers of donor-specific antibody (DSA >10,000 vs. <100 in long-term survivors). Use of a new regimen: peri-transplant recipient treatment with anti-LFA-1 mAb, anti-TNFa mAb, and anti-CD154 mAb plus additional doses of anti-CD154 mAb on days 14 and 16 post-transplant promoted long-term survival of 60% allografts subjected to prolonged CIS past day 100 post-transplant. Recipients with surviving allografts accepted donor skin allografts, but rejected third-party skin allografts. These studies indicate long-term survival of allografts subjected to prolonged CIS by using strategies that include reagents inhibiting endogenous memory CD8 T cell infiltration into the allografts and reagents that inhibit the production of DSA.
Understanding the effects of an inflammatory milieu on the development of mesenchymal stem cells: implication for treating inflammatory diseases
Authors: Sherman Lauren S. , Ponzio Nicholas M , Rameshwar Pranela
Published: January 2023

Mesenchymal stem cells (MSCs), multipotent cells found in various adult tissues, are an attractive source of cells for cellular therapy and regenerative medicine. Reasons include their ease to expand, plasticity to generate cells of all germ layers, reduced ethical concerns, and ability to be available as ‘off the shelf’ cells for immediate use in transplantation. MSCs can respond differently to varying microenvironments to perform distinct immune functions. The microenvironment can also affect the developmental state of MSCs. Better understanding of how the microenvironment influences MSC multipotency is crucial for effective translational use of these cells in the clinic. This study tested the hypothesis that the changes in an inflammatory microenvironment will influence MSC function. To study these effects, an in vitro model of GvHD was developed by culturing MSCs in a three-dimensional model – rather than in a traditional monolayer – under physiological conditions. The GvHD model was generated based on a modified two-way mixed lymphocyte reaction. The cells were then assessed for phenotype, proliferation, and viability. Following growth in suspension, the MSCs continued to express an MSC phenotype. Proliferation and viability studies suggested that, although the cells were proliferating, they underwent cell death at a quicker rate than their two-dimensional culture counterparts. When cultured in the GvHD model, MSCs exhibited increased viability as compared to culture in the three-dimensional system alone. This in vitro GvHD model will elucidate factors within the inflammatory milieu that alter MSC multipotency. Identifying these factors will allow for more controlled and effective clinical use of MSCs.
Evaluation of T-Track® CMV to assess the functionality of Cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients
Authors: Boeckl Katharina , Banas Bernhard , Böger Carsten , Lückhoff Gerhard , Krüger Bernd , Barabas Sascha , Starke Astrid , Bendfeldt Hanna , Schemmerer Mathias , Köstler Josef , Wagner Ralf , Deml Ludwig , Leicht Joachim , Krämer Bernhard K.
Published: January 2023

Background

Impairment of Cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) by immunosuppressive therapy is a major cause for uncontrolled CMV replication and related clinical complications.

Objective

The aim of the study was to assess the suitability of the immune monitoring tool T-Track® CMV to determine CMV-reactive CMI in hemodialysis patients.

Study Design

Positive and negative agreement of T-Track® CMV with CMV-serology have been examined in a cohort of 124 hemodialysis patients of whom 67 (54%) revealed a positive CMV serostatus and compared with that of QuantiFERON®-CMV and a mixture of 6 CMV iTAG tetramers.

Results

Positive T-Track® CMV, QuantiFERON®-CMV and CMV tetramer results were obtained in 60/67, 45/62 and 40/52 CMV-seropositive hemodialysis patients resulting in 89.6%, 72.6% and 76.9% positive agreement with CMV-serology, respectively. Positive T-Track® CMV responses were dominated by pp65-reactive cells (58/67 (86.6%)), whereas IE-1 responsive cells were observed in 33/67 (49.3%) CMV-seropositive patients. T-Track® CMV, QuantiFERON®-CMV and CMV tetramers showed 78.9% (45/57), 87.3% (48/55) and 93.3% (42/45) negative agreement with serology respectively. Notably, T-Track® CMV-positive tests in seronegative patients were mostly caused by IE1-reactive cells at low spot counts.

Conclusion

T-Track® CMV represents a sensitive assay format, enabling functional assessment of the network of clinically relevant subpopulations of CMV-responsive cells. Thus, T-Track® CMV may represent a valuable tool to identify patients at increased risk for CMV-related clinical complications and may help to guide personalized antiviral and immunosuppressive therapy.
Defining the optimal plasmacytoid dendritic cell graft source for hematopoietic stem cell transplantation
Authors: Hassan Mojibade , Lin Kaifeng , Li Jingxia , Harris Wayne , Waller Edmund K
Published: January 2023

Purpose

Allogeneic hematopoietic stem cell transplantation (HSCT) can cure patients with blood disorders and malignancies. Stem cell source can be bone marrow (BM) or peripheral blood mobilized by granulocyte-colony stimulating factor (G-CSF). Plasmacytoid dendritic cells (pDC) reduce the incidence and severity of GvHD. The BMT CTN 0201 study indicates overall survival, relapse, and the incidence of acute GvHD are similar in patients that receive BM versus G-CSF mobilized peripheral blood (G-PB) grafts, but increased incidence of chronic GvHD in patients who receive G-PB grafts. To explore the effect of graft source on the functional properties of pDC, we compared the effect of G-CSF mobilized pDC to BM pDC in the regulation of GvHD in a murine allogeneic HSCT model.

Methods

1) Expression of surface markers on BM and G-CSF treated pDC were assessed by flow cytometry. 2) We performed an HSCT from C57BL/6 mice into B10.BR recipients. Groups include HSC only, HSC + T cells, and HSC + T cells + BM pDC or G-CSF mobilized pDC. Hematopoietic chimerism, GvHD, and overall survival were assessed.

Results

1) BM pDC express CCR9 (marker for tolerogenic pDC) and CD62L at a higher frequency, suggesting enhanced homing to the gut, thymus, and lymph nodes. G-CSF mobilized pDC express maturational markers MHC II and CD86 at a higher frequency, suggesting effectiveness in activating T cells. 2) Mice that received transplants consisting of only HSC rejected the transplant. Mice that received transplants with T cells had greater than 90% donor hematopoietic cells. GvHD more than 40 days post transplant was highest in mice that received G-CSF mobilized pDC. Survival without pDC was under 60%. Groups that received pDC had an overall survival of greater than 80%.
IDH Mutant Gliomas Escape Natural Killer Cell Immune Surveillance by Downregulation of NKG2D Ligand Expression
Authors: Rao Aparna , Zhang Xiaoran , Deibert Christopher , Sette Paola , Grandi Paola , Amankulor Nduka
Published: January 2023

Background

Diffuse gliomas are fatal primary brain tumors that are poorly immunogenic. The basis for insufficient anti-tumor immunity in diffuse gliomas is not understood. Mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma.

Methods

We analyzed the TCGA database for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligands expression levels and NK cell-mediated lysis were measured in patient-derived glioma stem cells and in genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent Decitabine as a potential NK cell sensitizing agent in IDH mutant cells.

Results

All IDH mutant glioma stem-like cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes were resistant to NK cell-mediated lysis. The hypomethylating agent Decitabine increased NKG2D ligand expression, and restored NK- mediated lysis of IDH mutant cells in an NKG2D-dependent manner.

Conclusions

IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULPB3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell-mediated immune surveillance in patients with IDH mutated diffuse gliomas.
NKG2D optimizes CD8+ T cell function while certifying them to become memory cells
Authors: Perez Cynthia , Prajapati Kushal , Burke Brianna , Plaza-Rojas Lourdes , Guevara-Patiño José A.
Published: January 2023

The ability to form memory is a major characteristic of the adaptive immune system. NKG2D, as a costimulatory molecule for CD8+ T cells, was shown to rescue memory formation in absence of CD4+ help. However, what exactly is the function of NKG2D on CD8+ T cells and how important is NKG2D for memory formation is not known. To recapitulate a full course of CD8+ T cell activation, from priming to memory recall, we immunized C57BL/6 mice with peptide-pulsed dendritic cells and followed the CD8+ T cell responses at priming, effector and memory phases. Using NKG2D deficient (NKG2D-KO) CD8+ T cells, we found that NKG2D signaling was not necessary to achieve CD8+ T cell priming. However, NKG2D-KO CD8+ T cells showed lower effector functions (reduced in vivo kill and reduced cytokine secretion upon in vitro restimulation) compared to wildtype CD8+ T cells. Blocking NKG2D signaling at the effector phase with a single injection of anti-NKG2D antibody (non-depleting) recapitulated the results observed with NKG2D-KO CD8+ T cells. Importantly, the CD8+ T cells that lacked NKG2D signaling at effector phase failed to differentiate into protective memory cells. These results indicate that NKG2D signaling certifies CD8+ T cells to become memory cells and that this certification occurs during the effector phase.