bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antigen-specific CD4+ T cell differentiation in blood-stage malaria
Authors: Arroyo E Nicole , Pepper Marion 
Published: January 2023

Abstract
Although efforts to induce protection from malaria through vaccination have been ongoing for over sixty years, none have produced long-lasting immunity in malaria-endemic areas. This is likely due to a lack of knowledge about the immunological mechanisms required for protection. While it is clear that both CD4+ T cells and B cells are important components of the immune response to the blood stage of infection, little is known about their specific functional contributions. We have used the murine model P. yoelii to understand the lymphocyte populations that form and are required for protection. Using transgenic parasites and MHC class II tetramers to track CD4+ T cell responses, we have found that early in the response, the vast majority of CD4+ T cells are T follicular helper (Tfh)-like cells, despite the lack of a germinal center at this time point. Interestingly, in the absence of B cells, the CD4+ T cells alternatively develop a significant T effector (Teff) population, primarily Th1 cells. Interestingly, while there are stark differences in the type of CD4+ T cells generated in the presence and absence of B cells, parasitemia remains comparable in both WT and uMT mice for approximately 1.5 weeks post-infection, at which point mice deficient in B cells are unable to control the parasite growth. We are currently investigating how CD4+ T cells may contribute to the parasite control at this early stage and which aspects of the Th1 and Tfh responses are important for maintaining low parasitemia. An effective vaccine may need to stimulate the formation of antigen-specific Teff cells to manage parasite burden until Tfh cells have coordinated effective antibody responses by B cells in order to provide effective immunity against re-challenge.
CD8 T cell mediated blood-brain barrier disruption promotes vascular leakage and edema in vital brain regions during experimental cerebral malaria
Authors: Huggins Matthew A , Jin Fang , Johnson Aaron J 
Published: January 2023

Abstract
Human cerebral malaria (HCM) is a serious lethal complication of Plasmodium falciparum infection. The most severe outcomes for patients include coma, permanent neurological deficits, and death. Recently, a large-scale MRI study in humans identified brain swelling as the most prominent indicator of fatal HCM. In this study, we sought to define the mechanism causing edema during cerebral malaria by utilizing the murine experimental cerebral malaria model (ECM). We determined that areas of blood-brain barrier (BBB) permeability colocalized with the disorganization of the tight junction proteins claudin-5 and occludin. Furthermore, we put together a magnetic resonance imagine (MRI) platform to analyze the murine brain. Through gadolinium enhanced T1-weighted MRI analysis we discovered that vascular permeability is not homogeneous, but rather confined to specific regions of the brain. Our findings show BBB permeability localized within the brain stem of Plasmodium berghei ANKA (PbA) infected C57BL/6 mice. This could explain hallmark symptoms of ECM such as strained respiration and impaired balance. Concurrently with the initiation of vascular permeability, T2 weighted MRI analysis shows edema and brain swelling during PbA infection. This correlates with the human disease and solidifies the ECM model as a useful system for mechanistic insight into HCM. Interestingly, mice lacking perforin are fully protected from vascular permeability and edema. We propose that CD8 T cells initiate BBB breakdown during ECM through disruption of tight junctions. In turn, vascular leakage into the brain parenchyma causes brain swelling and edema. This results in a breakdown of homeostatic maintenance that likely contributes ECM pathology.
Distinct migratory phenotypes of luminal CD4 T cell subsets in the female genital tract
Authors: Kohlmeier Alison , Haddad Lisa , Haaland Richard , Lupo Davis , Ofotokun Igho , Hart Clyde , Kohlmeier Jacob E 
Published: January 2023

Abstract
The trafficking and retention of T cells in the genital mucosa provides a means of protection against many invading pathogens, yet this process is poorly understood. We measured six chemokine receptors and integrins important for T cell trafficking into peripheral sites on both resident (TRM: CCR7lo and CD69+) and recirculating (TRCM: CCR7hi CD69−) memory CD4 T cells localized at the luminal surface of the genital tract from healthy women to characterize migratory phenotypes under steady state conditions. Compared to memory CD4 T cells from blood, total memory CD4 T cells from the lumen expressed an increased frequency of CCR5, CCR6, CX3CR1, and α4β7, with no significant difference in CCR9 and CXCR3. Comparing luminal TRM and TRCM, we observed the TRCM subset expressed increased CCR6, CXCR3, and CX3CR1, with no difference in CCR5, α4β7, and CCR9. The increased frequency of CCR5+ cells among luminal TRM and TRCM suggested that CCR5 may regulate the trafficking on memory CD4 T cells to this site. Using a dual adoptive transfer mouse model, we observed that CCR5-deficient CD4 T cells were significantly impaired in their ability to migrate to the luminal surface of the genital tract under steady state conditions, but showed no defect in migration to other mucosal sites such as the gut and lung. These data reveal that TRCM express a broader migratory phenotype compared to luminal TRM, which may enable their ability to perform immune surveillance at multiple peripheral sites. Furthermore, the increased frequency of CCR5+ TRM and TRCM at the luminal surface and the defective trafficking of CCR5-deficient cells to this site suggests that CCR5 is required for the establishment of both TRM and TRCM CD4 subsets at the surface of the genital mucosa.
Development and analytical validation of a flexible multiplexing platform for cytokine assays.
Authors: Sandberg Eric M , Liu Pu , Ning Qiang , Rangwala Sameera , Shelburne Christopher , Kobayashi Lori E , Lu Claire , Stewart David , Tsionsky Michael , Plisova Tatiana , Glezer Eli N , Oberoi Pankaj , Wohlstadter Jacob N 
Published: January 2023

Abstract
Multiplex assays have advantages in being able to reduce time and sample volumes; however, multiplexing offers additional challenges, which include developing multiple assays that can be run with the same protocol, reagents, and sample dilution. Moreover, transferring reagents that work in one assay format to another without compromising performance and the integrity of sample measurement is difficult. The U-PLEX® platform enables flexible multiplexing of immunoassays using MSD’s MULTI-ARRAY® technology.
Antibodies used in V-PLEX® kits were transferred to the U-PLEX platform by biotinylating the capture antibodies. The assays represented a variety of analyte classes (chemokine, interleukin, interferon), antibody types (monoclonal, polyclonal), and analytical properties (sensitivity, dynamic range, concentration-response slope). During development, antibody concentration, biotin and MSD SULFO-TAG™ label ratios, and calibrator concentrations were evaluated and optimized.
Assays were readily transferred to the U-PLEX platform with calibration curves showing expected signals, sensitivity, precision, and accuracy. Controls for the assays showed CVs of &lt;10% within runs. Sensitivities were &lt;1 pg/mL for many assays. All assays used the same assay diluents. Non-specific binding between assays was typically &lt;0.1%. We measured 40 human serum and 40 EDTA plasma samples, and demonstrated good correlation with V-PLEX assays (r2 &gt; 0.9; slopes 0.8 – 1.2).
The utility and convenience of the U-PLEX platform was demonstrated by easily transferring over 40 human cytokine assays onto the platform without compromising performance. When multiplexed, these assays enable multiple measurements from small amounts of human samples.
Clinical significance of neutralizing antibodies to interferon beta in patients with Multiple Sclerosis
Authors: Zarkesh-Esfahani Sayyed Hamid , Zare Nasrin , Gharagozloo Marjan , Shaygannejad Vahid 
Published: January 2023

Abstract
Development of neutralizing antibodies (NAbs) is a major problem in relapsing-remitting Multiple Sclerosis (RRMS) patients treated with interferon-beta (IFN-b). NAbs against IFN-b are associated with a loss of bioactivity and decreased clinical efficacy of the drug. The objective of this study was to evaluate the clinical significance of NAbs to IFN-b in RRMS patients receiving three different types of this drug including: CinnoVex, Rebif, and Betaferon. The NAbs against IFN-b were measured in 31 RRMS patients receiving at least 12 months of IFN-b using a MxA gene expression assay (real-time RT-PCR). Expanded Disability Status Scale (EDSS) scores and relapse were analyzed using the results of NAbs assays. Of the 31 RRMS patients receiving IFN-b, 25 (80.6 %) showed NAbs after 12 months of treatment. There was no significant difference in the number of relapse (P = 0.1) and EDSS (P = 0.9) among three groups, but there was statistically significant difference in duration of treatment (P = 0.017) among three groups. In conclusion, samples showing NAb positivity failed to be associated with worsening of symptoms based on the EDSS of patients. It also can be concluded that development of Nabs should not be predicted by response of patients to treatment.
IL-6R signaling inhibits generation of Th3 cells and is a promising therapeutic target for enhancing oral tolerance induction
Authors: Kuhn Chantal , Rezende Rafael , Weiner Howard L 
Published: January 2023

Abstract
Oral antigen administration results in systemic hyporesponsiveness to a subsequent challenge with the fed antigen (oral tolerance) and has been shown to inhibit autoimmune disease in multiple animal models. However, transfer of this strategy to the clinics has not yet been achieved. Our aim is to better understand mechanisms implicated in the generation of Th3 cells and oral tolerance in order to enhance oral tolerance induction for a use in the clinics. Low dose oral tolerance depends on the presence of Th3 type Tregs, characterized by surface expression of latent TGF-beta (LAP and TGF-beta) and its adapter protein GARP (LRRC32). We established an in vitro system to induce Th3 like cells that express LAP and GARP on their surface. In vitro induced CD4+LAP+ T cells suppress proliferation of naïve T cells and production of IL-17A by Th17 cells. RT- PCR showed that membrane bound TGF-beta correlated with mRNA transcription levels of GARP but not TGF-beta, suggesting that the availability of GARP directly limits the expression of membrane bound TGF-beta. We used this in vitro system to screen for molecules that modulate generation of Th3 like cells and identified IL-6 as a potent inhibitor of membrane TGF-beta. IL-6 inhibited transcription of GARP in a STAT3 dependent manner. To test the therapeutic potential of targeting IL-6R signaling additionally to oral tolerance induction we assessed the DTH (delayed-type hypersensitivity) response in wild-type or CD4CreIL6Rafl/fl mice after feeding with ovalbumin for 5 consecutive days. Importantly, oral tolerance was significantly improved in mice that lack IL-6R signaling in CD4+ T cells. This demonstrates that blocking of IL-6R signaling is a promising approach to enhance oral tolerance induction.
Allogeneic skin transplantation triggers unique miRNA signature profiles in CD4+ T cells with potential therapeutic implications.
Authors: Becker William J , Nagarkatti Prakash S , Nagarkatti Mitzi 
Published: January 2023

Abstract
Solid organ transplantation is a life-saving last resort for many people undergoing end stage organ failure. Currently, immunosuppressive drugs are employed to combat rejection; however, it is at the expense of the host’s immune system, often exposing the patient to a host of maladies, with opportunistic infection and malignancy being the most dire. Thus, it is imperative to find novel ways to induce tolerance in transplant recipients to allografts while maintaining homeostatic immune responses. MicroRNAs (miRNAs) are a class of evolutionarily conserved small non-coding RNAs that regulate gene expression by blocking translation or promoting mRNA degradation. Using a murine model of skin transplantation, microarray analysis was used to evaluate miRNA expression profiles in draining CD4+ T cells from allogenic (allo) and syngenic (syn) grafts. We identified several distinct miRNAs (e.g. miR-27a-5p, 15a-5p and several members of the 466 family) that were increased in response to alloantigen. Using Ingenuity Pathway Analysis software, these miRNAs were found to target several genes in the SMAD and TGFβ pathway that control the differentiation into immuno-suppressive T regulatory (Treg) cells. The current study suggests that targeting miRNA may constitute a novel therapeutic modality to induce Treg generation and potentially program the host’s own cells to restore immunological tolerance, while safely prolonging the health of the transplanted organ.
Functional complement-fixing antibodies are generated against a Campylobacter jejuni capsule conjugate vaccine in non-human primates
Authors: Schumack Nina M , Rimmer Joanna E. , Guerry Patricia , Laird Renee M 
Published: January 2023

Abstract
Campylobacter jejuni is among the most common causes of diarrheal disease worldwide and efforts to develop protective measures against the pathogen are ongoing. One of the few defined virulence factors targeted for vaccine development is the capsule polysaccharide (CPS). We have developed a capsule conjugate vaccine against a prototype CPS, type HS23/36, that confers 100% protection against diarrhea caused by a homologous strain of C. jejuni in a non-human primate (NHP) model; however the mechanisms of protection remain unknown. Other licensed capsule conjugate vaccines against encapsulated gram-negative organisms use bactericidal antibody titers as a correlate of protection. We developed a flow cytometry-based serum bactericidal assay (SBA) to determine if increased levels of complement-fixing antibodies correlate with protection against C. jejuni. Sera from NHPs immunized with HS23/36 CPS-CRM197 vaccine showed significantly higher SBA titers when compared with control NHPs. Results were similar to those of a bacteriological-based SBA. All NHPs, irrespective of immune status, that did not develop diarrhea, had higher SBA titers compared with NHPs that developed diarrhea. Importantly, a strong correlation was found between serum anti-CPS IgG and SBA titers in NHPs and similar studies will be conducted in upcoming human clinical trials. This flow cytometry-based SBA assay is much simpler than bacteriological-based assays and should facilitate analyses of functional antibodies against multivalent C. jejuni capsule conjugate vaccines.
DENV-specific CD4 T-cells dominantly recognize capsid-derived epitopes and display a cytotoxic phenotype
Authors: Weiskopf Daniela , Angelo Michael , Zapardiel Jose , Seumois Gregory , de Silva Aravinda , de Silva Aruna D , Vijay Pandurangan , Peters Bjoern , Sette Alessandro 
Published: January 2023

Abstract
While DENV-specific CD8+ T cell responses have been extensively studied, the breadth and specificity of CD4+ T cell responses remains to be defined. Here we map CD4 T cell responses in individuals previously exposed to dengue virus. To identify HLA class II candidates, we utilized a panel of algorithms for sixteen common HLA DR molecules representative of the main DR super types. These efforts led to the identification of 365 epitopes derived from all 10 DENV proteins. In contrast to CD8 T cell targets, the highest number of epitopes was associated with the structural capsid protein (C), followed by nonstructural NS3, NS2A, NS5 and envelope proteins (E). Phenotyping of the responding CD4+ T cell subset revealed a DENV specific T cell subset, specifically expanded in donors carrying an allele associated with protection from severe disease and which is absent in DENV negative donors. This subset of DENV specific CD4 T cells is associated with markers of cytotoxic T cells such as Granzyme B, Perforin, CD107a, T bet, Eomes and co-expression of CD8α. We are currently establishing specific gene expression signatures associated with these characteristic phenotypes, as a function of different HLA restricting alleles. Detailed knowledge of phenotype and function of DENV-specific T cells will aid in the establishment of correlates of protection against severe disease.
Detection of islet antigen expanded CD4 T cell TCR pairs and transcriptomes by single cell RNAseq
Authors: Cerosaletti Karen M , Yang Junbao , Whitman Fari , Kwok William , Linsley Peter S 
Published: January 2023

Abstract
Islet-reactive CD4 T cells contribute to the development and progression of type 1 diabetes (T1D), and may provide a window into β-cell destruction. To identify unique characteristics of islet reactive T cells from T1D subjects, we coupled CD154 enrichment of antigen specific T cells with novel single-cell RNAseq methods. PBMC from three DR4 T1D subjects and three matched healthy controls (HC) were stimulated overnight with a pool of DR4 restricted islet peptides. The following day, CD154+ cells were isolated by magnetic bead enrichment and CD154+CD69+ activated memory CD4 T cells were directly sorted into a Fluidigm C1 Single-Cell chip. Nextera sequencing libraries were sequenced on an Illumina HiSeq sequencer. RNAseq reads were aligned to the human reference genome, mapped to ENSEMBLE gene models, and tabulated to enumerate transcript levels. To identify TCR clonotypes, we assembled reads into de novo contigs using Trinity and identified contigs encoding productively re-arranged TCR V, (D), J and CDR3 regions using IMGT/HIGHV-QUEST. We detected private TCR clonotypes (α/β pairs) in islet reactive CD4 T cells from all subjects. However, T1D subjects as a group expressed identically rearranged CDR3 junctions in multiple cells (expanded clonotypes), compared with HC cells which expressed more diverse clonotypes. T1D cells expressing expanded TCR clonotypes had a gene expression profile that was distinct from T1D cells with non-expanded TCRs and HC cells, characterized by a unique subset of T cell activation/differentiation genes. Our results illuminate clonotype/phenotype relationships of islet-reactive CD4 T cells that may impact the immunopathology of subjects with T1D, and serve as biomarkers and therapeutic targets.
Comparison of serum protein concentrations to clinical observations during lipopolysaccharide (LPS) induced inflammation in swine.
Authors: Deaver Christine M , Smith Emily R. , Messenheimer Janis R. , Lewandowski Anne J , Groesbeck Crystal , Zhang Wei , Swain Trevon , Chiesa O. Alberto , Myers Michael J 
Published: January 2023

Abstract
The serum kinetic profile of the proteins MCP1 (CCL-2), C - reactive protein (CRP), and Serum Amyloid A (SAA) were compared to clinical observations in swine given a single i.v. administration of lipopolysaccharide (LPS; 2 μg/kg). Blood samples were collected at 0, 1, 3, 6, 8, & 24 hr after LPS administration using an in-dwelling catheter inserted prior to LPS administration. Serum protein concentrations were determined using species-specific ELISA. Clinical observations include temperature, respiration rate (RR), skin redness (SR), lethargy, and respiratory character (RC). LPS administration failed to result in elevated CRP and SAA levels. MCP-1 was induced within 1 h of LPS administration, and remained elevated through 8 h, returning to baseline values by 24 h post-LPS. The kinetics of MCP1 protein differ from that of MCP1 mRNA, which exhibits elevated levels through 48 h post LPS (Peters et al, Vet Immol Immunopath, 2014, 148: 236–242). The clinical results suggest that the administered challenge resulted in toxicity beyond the expected systemic inflammatory response. However the results also suggest that MCP1 may be a serum protein biomarker of inflammation associated with severe endotoxicity and RR, RC, SR and lethargy may be clinical measures that can be used to qualify analytical measures such as MCP1 as surrogate endpoints. These results further suggest the model needs to be refined. The discordant regulation of MCP1 protein and mRNA levels suggest additional molecular controls regulating MCP1 protein production.
BACE1: a novel player in the pathogenesis of Th17 cells in EAE
Authors: Mir Gerard Hernandez , Henkel Matthew A , Maggio Maria J , Ramani Kritika , Stolz Donna B , Biswas Partha S , McGeachy Mandy J 
Published: January 2023

Abstract
Th17 cells are implicated in autoimmune disease, including attack of the central nervous system (CNS) in multiple sclerosis. β-site APP-cleaving enzyme 1 (BACE1) is a membrane protease expressed in neurons and astrocytes. BACE1 is most well known for its role in promoting neurodegeneration in Alzheimer’s disease by cleaving amyloid precursor protein, although it also plays a role in central nervous system myelination. BACE1 has also been reported to contribute to lesion severity following brain injury, as has IL-17, although these two molecules have not previously been linked. We observed that BACE1 is upregulated in experimental autoimmune encephalomyelitis (EAE) lesions, a mouse model of multiple sclerosis, suggesting a role in autoimmune CNS damage. Surprisingly, we discovered that Bace1-deficient MOG-reactive T cells were defective in IL-17A production. In vitro differentiated Th17 cells were similarly defective in IL-17A. Conversely, overexpression of Bace1 increased IL-17A production. Transfer experiments demonstrated that BACE1 deficiency in T cells impairs their pathogenic function and confers resistance in EAE. Although the impact on IL-17A production was dramatic, there was only a mild defect in expression of RORγt, IL-23R and other prototypic Th17-associated molecules such as IL-17F. This was confirmed by RNA-Seq, which revealed other potential Bace1 targets outside the classic Th17 armory. Therefore, we propose that Bace1 is a novel modulator of the effector function of Th17 cells, particularly IL-17A production.
Overexpression of sirtuin 6 suppresses allergic airway inflammation through the deacetylation of GATA3
Authors: Jang Hyun-Young , Woo Seong-Ji , Choi Ji-In , Lee Sang-Myeong , Park Byung-Hyun 
Published: January 2023

Abstract
Sirtuin 6, an NAD+-dependent deacetylase and mono-ADP-ribosyl transferase, is known to have an anti-inflammatory function. It is unclear, however, whether sirtuin 6 also negatively contributes to the development of allergic airway inflammation (AAI). To elucidate the role of sirtuin 6 in AAI pathogenesis and its underlying mechanism. An adenovirus expressing Sirt6 (AdSirt6) and a catalytically inactive Sirt6-H133Y were used to deliver sirtuin 6. We analyzed the features of AAI in BALB/c mice after sensitization with two different allergens, ovalbumin (OVA) and house dust mite (HDM). Sirtuin 6 level increased in lung tissues of OVA-challenged mice. AdSirt6 injection before allergen challenge reduced all cardinal features of AAI; eosinophilic airway inflammation, mucus hypersecretion, airway hyperresponsiveness, and Th2 immune responses were markedly suppressed. Furthermore, the differentiation of CD4+ T cells into Th2 cells was also decreased in the draining lymph nodes of AdSirt6-injected mice. Finally, in vitro overexpression studies and asthmatic lung tissues demonstrated that sirtuin 6 interacts and deacetylates GATA3, a master transcriptional regulator of Th2 immune responses. Our results showed that local overexpression of sirtuin 6 was effective in preventing AAI through the deacetylation of GATA3.