bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR tuning and exhaustion of CD4+ T cells by persisting antigen
Authors: Obst Reinhard , Trefzer Anne , Pennavaria Stefanie , Wang Shu-Hung , Lober Benedikt , Asoyan Ayuna
Published: January 2023

Chronic infections and persistent antigen presentation by tumor cells functionally impair T cells over time. To address how different kinetics and dosage of antigen presentation affect CD4+ T cells, we followed TCR transgenic T cells transferred into antigen-transgenic recipients where they received transient or chronic TCR signals of varying strengths. In one of the recipient mouse lines, where antigen presentation can be easily regulated by doxycycline treatment, the quick termination of antigen presentation after priming allowed for the differentiation of memory cells with Ly6C, IL7R and cytokine production upon restimulation as their hallmarks. Persistent antigen presentation at three different levels led to different types of exhaustion that developed with different kinetics and consequences: Exhaustion markers and dysfunctionalities took longer to appear at low amounts and only at the highest antigen dose the T cells downregulated their TCRs and underwent apoptosis. We tested the Ca2+, MAPK and Akt pathways for functionality and found Ca2+ fluxes to be rather robust against persistent antigen presentation while the MAPK and Akt pathways were more easily tuned by it. These data correlate, for CD4+ T cells, several levels of exhaustion with antigen dosage in an otherwise sterile environment.
Basophil-derived IL-6 regulates TH17 cell differentiation and CD4 T cell immunity
Authors: Yuk Chae Min , Park Hyeung Ju , Kim Dong-Eon , Kim Tae Woo , Lee Seung-Hyo Lee
Published: January 2023

Basophils are rare circulating granulocytes and have been proposed to be involved in T helper (TH) type 2 immunity mainly through interleukin-4 (IL-4) secretion. In addition to IL-4, it has been known that basophils produce IL-6 and tumor necrosis factor (TNF)-α with immunoglobulin E (IgE) crosslink. TH17 cell development is achieved with the action of IL-6 and transforming growth factor (TGF)-β. Whether the capacity of basophils to produce IL-6 is involved in the differentiation of TH17 cells is unknown. The aim of this study is to determine the role of basophils on TH cell differentiation especially TH17 cell development and plasticity of TH subsets by using in vitro T cell differentiation and cholera toxin (CT)-mediated TH17 inflammation model. Bone marrow (BM) derived-basophils (BMBs) as well as splenic basophils produced large amount of IL-4 and IL-6 by stimulation with IgE crosslink or CT. BMBs have additional role of TH17 cell differentiation by supplementing the function of dendritic cells through secretion of IL-6 in vitro culture system in the presence of TGF-β. Furthermore, basophil-mediated TH17 cell differentiation effects were prominent in CT-mediated TH17 dominated lung inflammation model. Therefore, our results indicate that basophil is an important inducer of TH17 cell differentiation, which is dependent on IL-6 secretion.
An essential role for Itk in Th9 differentiation via TCR-mediated induction of IL-2 and IRF4
Authors: Gomez-Rodriguez Julio Washington , Meylan Francoise , Handon Robin , Hayes Erika , Anderson Stacie , Kirby Martha , Siegel Richard M , Schwartzberg Pamela L
Published: January 2023

T helper 9 (Th9) cells produce IL-9, a cytokine implicated in allergic asthma and autoimmunity. We show that Itk, a mediator of T-cell receptor signalling in Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation in vivo and in vitro. In a papain-induced model of allergic lung disease, Itk-deficient mice have reduced pulmonary inflammation and IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal induction of ILC2s. In vitro, naïve Itk−/− CD4+ T cells do not produce IL-9 and have reduced levels of IRF4, a critical transcription factor for effector T-cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or expression of constitutively-active STAT5, but not NFATc1. STAT5 binds the Irf4 promoter, indicating a mechanism by which IL-2 rescues weakly-activated T cells. Itk inhibition also reduced IL-9 expression by human CD4 T cells, thereby implicating ITK as a possible target to prevent Th9-mediated inflammatory diseases.
Antigen-specific CD4+ T cell differentiation in blood-stage malaria
Authors: Arroyo E Nicole , Pepper Marion
Published: January 2023

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

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

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

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 <10% within runs. Sensitivities were <1 pg/mL for many assays. All assays used the same assay diluents. Non-specific binding between assays was typically <0.1%. We measured 40 human serum and 40 EDTA plasma samples, and demonstrated good correlation with V-PLEX assays (r2 > 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

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

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

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

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

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

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

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

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

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.
Thromboxane (TXA2) Attenuates Lung Inflammatory Responses and Th9 Cell Differentiation and Function During Allergic Lung Inflammation
Authors: Li Hong , Bradbury Alyce , Edin Matthew L , Gruzdev Artiom , Sparks Matthew , Hoopes Samantha L , DeGraff Laura M , Bortner Carl D , Coffman Thomas , Zeldin Darryl C
Published: January 2023

Thromboxane(TXA2) is a potent bronchial smooth muscle spasmogen implicated in the pathogenesis of asthma. T helper type 9 (Th9) cells, a subpopulation of CD4+ T cells, play an important role in the pathogenesis of asthma;however, it is unknown whether TXA2 regulates Th9 cell differentiation or function during allergic lung inflammation. Hence, we used an in vivo ovalbumin (OVA)-induced allergic inflammation model to study the role of TXA2 and its receptor in Th9 cell differentiation during allergic lung inflammation. Following OVA sensitization/exposure, the percentage of IL‑9+/CD4+ T cells was significantly decreased in lungs, blood and spleen of mice that were implanted with mini pumps containing thromboxane (TXA2). Also, TP−/− mice had a significantly higher percentage of Th9 cells after OVA sensitization/exposure compared to WT mice, and also exhibited increased allergic lung inflammation. In vitro experiments showed that differentiation of naïve CD4+ T cells to Th9 cells was significantly inhibited by TXA2. TXA2 also inhibited Th9 cell differentiation of OVA-sensitized CD4+ memory T cells. CD4+ T cells did not produce significant amounts of TXA2, but expressed the TP receptor during Th9 cell differentiation. TXA2 activation of TP receptors increased cAMP production and enhanced phosphorylation of p38 MAPK, ERK and PI3K in CD4+ T cells during Th9 differentiation in vitro. These effects were markedly attenuated in TP−/− cells. Importantly, TXA2 signaling suppressed activation of the Il9 promoter via a mechanism that involved binding of PBX1 and NFE2 transcription factors to their corresponding response elements. Thus, TXA2 acts through TP receptors to suppress Il9 promoter activation and Th9 cell differentiation.
DC-independent TSLP-mediated acute allergic airway inflammation via CD11c+ Interstitial Macrophages
Authors: Lai Jen-Feng , Thompson Lucas J , Ziegler Steven F
Published: January 2023

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine important for the development of type-2 inflammatory responses at mucosal surfaces. In humans, TSLP has been found to be elevated in the lungs of asthmatics, and in mouse models TSLP can promote type-2 airway inflammation. DC have been shown to respond directly to TSLP and subsequently promote the differentiation of Th2 cells. In addition, other lung-resident MHCII-expressing cells, such as Alveolar Macrophages (AM) and Interstitial Macrophages (IM), have been shown to regulate the activation of T cells. We have identified a subset of CD11c-expressing Interstitial Macrophages (CD11cIM) addition to AM and IM, which can directly induce proliferation and differentiation of CD4 T cells in a TSLP-dependent manner. Following exposure to TSLP, these cells are more potent then DC for expansion and Th2 cytokine production of CD4 T cells. More importantly, CD11cIM are necessary and sufficient for the induction of Th2 responses and downstream eosinophilic inflammation in the lungs. These findings indicate a novel unique and critical role for TSLP-signaling on CD11cIM in the development of acute Th2-dependent allergic airway inflammation in pulmonary microenvironment.
Smith-Lemli-Opitz syndrome reveals requirement for sterol biosynthesis in the innate immune response
Authors: Gabor Kristin , Madenspacher Jennifer H , Wassif Christopher A , Bushel Pierre R , Porter Forbes D , Fessler Michael B
Published: January 2023

Smith-Lemli-Opitz Syndrome (SLOS) is a rare neurodevelopmental disorder caused by mutation of 7-dehydrocholesterol (7DHC) reductase (DHCR7), a terminal enzyme in cholesterol biosynthesis. SLOS patients have increased incidence of infections, but no mechanism of immunosuppression has been identified. As cholesterol is a key component of membrane raft microdomains and rafts have been implicated in Toll-like receptor (TLR) signaling, we examined whether TLR responsiveness is altered in SLOS. Primary fibroblasts from SLOS patients with varying degrees of deficit in DHCR7 activity were used to assess the TLR4 response. Upon stimulation, significantly lower NF-kB activation and IL-6 and IL-8 production were observed in SLOS cells than in controls. Across patients, a direct relation between residual DHCR7 enzyme activity and IL-6 production was observed, together with an inverse relation between clinical severity scores and IL-8 production. Microarray analysis revealed that several pro-inflammatory chemokines are less responsive to LPS in SLOS. Deficient LPS-induced cytokines were also observed in macrophages from Dhcr7 mutant mice and in RAW264.7 macrophages treated with DHCR7 inhibitors. Supporting cholesterol deficit as the cause of the TLR4 impairment, the LPS response of SLOS macrophages was partially rescued by cholesterol repletion. Mechanistically, confocal microscopy revealed that TLR4 localization to rafts is decreased in SLOS macrophages. Taken together, these findings indicate that the SLOS mutation confers an abnormal TLR4 response. Deficient innate immunity may be a clinically relevant contributor to SLOS pathogenesis, and SLOS highlights the importance of cholesterol biosynthesis to innate immunity.
Autoimmune variant PTPN22 is associated with impaired responses to influenza virus vaccination
Authors: Crabtree Juliet Nicole , He Wenqian , Guan Weihua , Miller Matthew S , Peterson Erik J
Published: January 2023

High-affinity antibody production, T cell activation, and Interferon upregulation all contribute to protective immunity that occurs in humans following influenza immunization. Hematopoietic cell-specific PTPN22 encodes Lymphoid Phosphatase (Lyp), which regulates lymphocyte antigen receptor and Pattern Recognition Receptor (PRR) signaling. A PTPN22 variant R620W (LypW) predisposes to autoimmune and infectious disease, and confers altered signaling through antigen receptors and PRRs. We tested the hypothesis that LypW-bearing humans would have diminished immune response to trivalent influenza vaccine (TIV). LypW carriers exhibited decreased induction of influenza-specific CD4 T cells expressing effector cytokines, and failed to increase antibody affinity following TIV. No differences between LypW carriers and non-carriers were observed in virus-specific CD8 T cell responses, early interferon transcriptional responses, or myeloid APC costimulatory molecule upregulation. LypW association with defects in TIV-induced CD4 T cell expansion and antibody affinity maturation suggests that LypW may predispose to diminished capacity to generate protective immunity against influenza.
Investigating mechanisms restricting responsiveness of B cells specific for foreign antigens
Authors: Steach Holly R , Taylor Justin J.
Published: January 2023

In order to prevent antibody-mediated autoimmunity, B cells reactive against self-antigens are subjected to tolerance mechanisms including induction of a state of restricted functionality termed anergy. We hypothesize that anergy also restricts responsiveness of B cells specific for foreign antigens due to B cell receptors (BCR) cross-reactivity with endogenously expressed molecules. Using a previously published antigen-specific enrichment strategy, we have shown that 60–80% of antigen-specific B cells fail to respond to antigen immunization. While some of this poor responsiveness is likely a result of low affinity, many B cells binding high levels of antigen are found within the non-responding population. A broadly-inclusive marker for self-reactivity in mice with an intact polyclonal B cell repertoire has been identified using the orphan nuclear receptor Nur77, known to be up-regulated downstream of BCR signaling. Using Nur77-eGFP reporter mice, we have found that populations of B cells specific for non-self antigens display a range of baseline Nur77 expression similar to that of total B cells, perhaps indicating a similar extent of self-antigen recognition. Coordinately, we have found that antigen-specific naive B cells expressing low levels of Nur77 are more responsive to immunization than their counterparts expressing high levels of Nur77. These data suggest that responsiveness of naive B cells specific for foreign antigens is restricted by cross-reactivity with self-antigens. Our results could lend a unique perspective to the paradigmatic definition of B cell anergy and offer an accession to immunogen design as the principle factor governing protective humoral immune responses following vaccination.
GATA2 is critical for mast cell differentiation and maintenance
Authors: Li Yapeng , Liu Bing , Lukin Kara , Finkelman Fred , Hagman James , Roers Axel , Huang Hua
Published: January 2023

The GATA binding protein 2 (GATA2) plays critical roles in hematopoietic stem cell survival and proliferation, granulocyte–monocyte progenitor differentiation, and basophil and mast cell differentiation. However, precise roles of GATA2 in mast cell differentiation and maintenance have not been delineated. We identified GATA2 is essential for pre-basophil and mast cell progenitors (pre-BMPs) to differentiate into mast cells. We found that GATA2 is critical in maintaining the expression of a wide range of genes that are important for performing mast cell functions. To determine the in vivo role of GATA2 in mast cell development, we generated connective tissue mast cell-specific Gata2 deficient mice. We found that Gata2 was required for connective tissue mast cell development. These mice failed to develop IgE-mediated anaphylaxis. GATA2 regulated the expression of Hdc gene, which is critical for IgE-mediated anaphylaxis, by binding to regulatory regions of the Hdc gene to promote its transcription. Our study reveals GATA2 is critical for mast cell differentiation and maintenance.
Defining the role of CCR8 in promoting thymocyte development and thymic epithelial homeostasis
Authors: Thyagarajan Hiran Maye , Hu Zicheng , Ehrlich Lauren I R
Published: January 2023

Generation of functional and self-tolerant T cells in the thymus is crucial to preventing autoimmune diseases. Medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present diverse auto-antigens to developing thymocytes; strong TCR reactivity in response to self-antigens causes thymocytes to undergo negative selection, eliminating autoreactive cells from the repertoire. Chemokine receptors expressed on the surface of developing thymocytes have been shown to promote accumulation of post-positive selection thymocytes in the medulla, where they may encounter mTECs and DCs presenting auto-antigens. CCR8 is highly expressed by post-positive selection CD4 thymocytes while its ligands, CCL1 and CCL8 are expressed by mTECs. Thus, CCR8 signaling may promote medullary entry of post-positive selection thymocytes and efficient interactions with mTECs and enforcing central tolerance. Our preliminary data indicate that CCR8−/− thymi have a reduced medullary area, implicating impaired thymocyte:mTEC interactions that impacts normal proliferation and differentiation of mTECs. Furthermore, 2-photon imaging reveals that CCR8 deficiency results in a significant increase in the path straightness of CD4 SP thymocytes, which could impact the efficiency of interactions with medullary stromal cells. Strikingly, CCR8 deficiency results in increased cellularity of CD4SP OTII TCR transgenic thymocytes in a wild-type host, suggesting CCR8 is critical for efficient clonal deletion to endogenous auto-antigens. Thus, our preliminary data indicate that CCR8 contributes to the migratory properties and stromal interactions of post-positive selection thymocytes that promote efficient negative selection.
IRF8 represses GM-CSF expression in tumor cells to mediate myeloid-derived suppressor cell differentiation.
Authors: Paschall Amy V , Redd Priscilla , Zhang Ruihua , Xiong Huabao , Abrams Scott I , Liu Kebin
Published: January 2023

Myeloid-derived suppressive cells (MDSCs) are immature myeloid cells that are induced by inflammatory mediators. Massive accumulation of MDSCs is a hallmark of cancer in mice and human patients. Proinflammatory factors are believed to induce MDSC differentiation. In an early study, we showed that T cell-produced GM-CSF, a proinflammatory cytokine, induces MDSC accumulation under physiological conditions. We further determined that IRF8 represses MDSC accumulation in vivo. Tumor cells are known to produce abundant GM-CSF; we therefore sought to test the hypothesis that IRF8 regulates GM-CSF expression to mediate MDSC accumulation in the tumor-bearing host. We observed that mouse sarcoma cells rapidly induce MDSC accumulation, which is positively correlated with tumor growth kinetics. Analysis of purified MDSCs from tumor-bearing mice indicated that GM-CSF mRNA levels increase and IRF8 expression decreases as tumor sizes increase. GM-CSF and IRF8 expression kinetics are therefore inversely correlated in MDSCs in vivo. Analysis of the csf2 gene promoter DNA sequence identified an IRF8-binding consensus element, and ChIP analysis determined that IRF8 binds to this DNA element in the csf2 promoter. Analysis of human sarcoma specimens revealed that the IRF8 promoter DNA is hypermethylated. Silencing IRF8 expression using IRF8-specific shRNA increased GM-CSF expression. In summary, we determine that tumor cells use GM-CSF to induce MDSC differentiation, and IRF8 functions as a transcriptional repressor of GM-CSF expression to suppress MDSC accumulation. Human sarcoma cells may use DNA methylation to silence IRF8 and increase GM-CSF expression to promote MDSC accumulation, thereby suppressing the anti-tumor immune response.
Prognostic significance of tumor infiltrating immune cells in oral squamous cell carcinoma
Authors: wang zhi , Fang Juan , Song Jingjing , Chen yichen , Ma Da , Liu Xiangqi , cheng bin
Published: January 2023

Background

Oral squamous cell carcinoma (OSCC) is the most common malignancy in oral maxillofacial region. Although modest improvements in the survival rates have been reported, the prognosis remains unsatisfactory. Tumor infiltrating immune cells have been proved playing critical roles in tumor prognosis. We conducted this study to evaluate the predictive value of CD4+ T cells, T-bet+ Th 1 cells, CD8+ T cells, CD57+ NK cells and CD68+ macrophages for OSCC outcome.

Methods

78 OSCC patients were included and followed up for at least 3 years. Clinical data and Paraffin-embedded samples were collected. CD4, CD8, T-bet, CD57 and CD68 expressions were examined by immunohistochemistry. The Kaplan –Meier method, log-rank test and Cox proportional hazards model were used to estimate the prognostic factors for Overall survival. ROC and AUC were used to evaluate the prognostic value.

RESULTS

Multivariate analysis revealed that no lymph node involvement, lower CD4 (+) T cells and high CD57 (+) NK cells infiltration were all significantly related with longer survival (P < 0.01). The respective predictive accuracy of CD4+ T cells, CD8+ T cells and CD57+ NK cells were all superior to TNM staging. CD57 expression provided the highest accuracy (AUC=0.759; 95% CI, 0.652–0.886).

Conclusion

Our study highlighted the predominant role of innate immune system in tumor immunity. Adaptive immunity seemed to be the major victim of immune tolerance. When compared to TNM staging, CD4, CD8 and CD57 expression significantly improves mortality prediction for OSCC patients. This study will provide new strategies to patient management of OSCC.
Modeling immune system-tumor interactions using humanized mice
Authors: Aryee Ken-Edwin , Brehm Michael A , Shultz Leonard D , Jurczyk Agata
Published: January 2023

The interplay between tumor cells, the non-malignant stoma and hematolymphoid cells (of both innate and adoptive lineages) determines the kinetics of cancer progression. However, the study of tumor-immune system interactions has primarily been restricted to mouse models. The implant of tumors into humanized mice that are engrafted with human immune systems provides a unique opportunity to interrogate the interactions between human immune cells and malignancies, and to test novel anti-tumor immunotherapies. Here we show the use of the BLT (bone marrow/liver/thymus) humanized mouse model to study the interactions between the human immune system and melanoma. The BLT mouse supports high levels of human immune system development including HLA-restricted T cells, B cells and innate immune cells. For these studies, we used NOD-scid IL2rnull (NSG) transgenically expressing SCF, GM-CSF and IL-3 (NSG-SGM3), which enhances human innate immune cell development and function. Following implant of patient derived melanomas into NSG-SGM3 BLT mice, we observed the infiltration of human T cells, mast cells, NK cells and macrophages into the tumor microenviroment. Moreover the recovery of high levels of FoxP3+ regulatory T cells, M2 macrophage and conventional CD4 and CD8 T cells expressing PD-1 and CTLA4 from the tumor suggests that the melanoma growing within NSG-SGM3 BLT mice creates an immune regulatory environment similar to that seen in patients. These results indicate that tumors implanted into NSG-SGM3 BLT mice can be used as an effective model for understanding tumor-immune system interactions.
Neuromodulatory activities of CD4+CD25+Foxp3+ regulatory T cells in 3xTg-AD Alzheimer’s disease model
Authors: Bae Hyunsu , Baek Hyunjung , Ye Minsook , Kang Geun-Hyung , Lee Chanju , Lee Gihyun , Lee Sujin
Published: January 2023

Alzheimer’s disease (AD) is the most prevalent cause of dementia marked by progressive cognitive and functional impairment and memory loss. AD patients display neuropathological lesions including deposition of extracellular amyloid-beta (Aβ) peptide and the formation of neurofibrillary tangles. Although the mechanisms causing brain atrophy and neuronal death are largely unknown, increasing evidence suggests that immunity plays a critical role in the pathogenesis of AD. In the present study, we investigated the effect of regulatory T cells (Tregs) on AD mouse model. First, we transiently depleted Treg population using anti-CD25 antibody. Depletion of Tregs for 4 months resulted in a marked aggravation of the spatial learning deficits of 6-month-old 3xTg-AD mice. Importantly, deposition of Aβ plaques and CD11b+ microglia/macrophage were significantly increased in the hippocampal CA1 and CA3 regions of the Treg depleted 3xTg-AD compared to the vehicle-treated 3xTg-AD group. Next, we explored the effect of Teffs (effector T cells; CD4+CD25−) and Treg (CD4+CD25+) in adoptive transfer model. Systemic transplantation of purified Tregs into 3xTg-AD mice improved cognitive function and reduced deposition of Aβ plaques. In contrast, adoptive transfer of Teffs showed increased Aβ plaques burden and diminished behavioral function. Our finding strongly suggested that systemic Treg administration ameliorates disease progression and could be an effective AD treatment.