bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of heterologous HIV-1 Tier 2 neutralizing mAbs cloned from a macaque immunized with viral quasispecies present in a human subject with neutralization breadth
Authors: Spencer David A , Malherbe Delphine C , Henderson Heidi , Pandy Shilpi , Bernat Néstor Vázquez , Àdori Monika , Goldberg Ben , Dambrauskas Nicholas , Sutton William , Sather Noah , Kobie James , Ackerman Margaret E , Karlsson Hedestam Gunilla B , Haigwood Nancy Logan , Hessell Ann Jones
Published: January 2023

Eliciting Abs that efficiently neutralize multi-clade HIV strains will likely be necessary for an effective HIV-1 vaccine. Such broadly neutralizing Abs have been cloned from a minority of chronically infected patients but have yet to be generated through pre-clinical vaccine regimens in NHPs. Here we characterize the Env-specific mAb response in an NHP with autologous Tier 2 neutralizing activity after co-immunization with plasmid gp160 DNA(x6) + gp140 trimeric protein(x2), using viral quasispecies present in a human subject immediately prior to and concurrent with the development of neutralizing breadth. In brief, 46 mAbs were cloned from single IgG+, antigen-specific, memory B cells, 12 of which neutralized heterologous Tier 1 and Tier 2 virus at IC50s < 4.2 mg/ml in a TZM-bl assay. These mAbs bound SHIVSF162P3-infected rhesus PBMCs significantly more than non-neutralizers (p < 0.001). Octet measurements and peptide mapping revealed the strongest neutralizers bound autologous F8 SOSIP with high affinity (KD ≤ 1.32×10−9), and 10 of these mAbs targeted the viral V3 region in the VH5.51 “cradle” orientation. Interestingly, non-neutralizing mAbs demonstrated high in vitroEnv binding (EC50s < 0.001 mg/ml) via ELISA, and several mediated ADCP against HIVSF162gp120 coated beads. Additionally, ADCC activity was observed with select neutralizing and non-neutralizing mAbs via KHYG-1/NKR24 SHIV-infected reporter assay. Collectively, these results suggest this immunization strategy recapitulated in part the clonal neutralization expansion and Env targeting observed in the patient and provide insights into effector functions of both neutralizing and non-neutralizing HIV-1 Env-specific mAbs elicited during vaccination.
Low weight piglets show differences in intestinal microbiota, intestinal transcriptome and immune cell profile compared to high weight piglets during the first two weeks of lactation
Authors: Blais Mylene , Morissette Bruno , Talbot Guylaine , Beaudoin Frederic , Deschene Karine , Verso Luca Lo , Bissonnette Nathalie , Lauzon Karoline , Guay Frederic , Lessard Martin
Published: January 2023

Piglets born with lower weight and showing low performances during the first weeks of lactation are associated with higher risks of gastroenteric diseases after weaning. Therefore, we investigated the developmental differences between these pigs compared to the high weight piglets. Eight litters adjusted to 12 piglets were used. In each litter, piglets showing the lowest weight gain (LWG) or the highest weight gain (HWG)in the first week of life were selected. For each piglets enrolled, ileal gene expression, immune cell populations and microbiota profiles were studied. Results obtained from microarray and Q-PCR analysis showed that, at 16 days of age, the expression of genes involved in oxidative stress and immune response was altered in LWG piglets’ ileum. Analysis of the LH-PCR data of the microbiota using non-metric multidimensional scaling (NMS) and blocked multiresponse permutation procedure (MRBP) revealed that the microbiota of the HWG and LWG piglets tended to differ in ileal mucosa (p = .097) and differed incolonic lumen (p = .024). From day 8 to 16, LWG piglets failed to show an increase of CD21+ B cells in their blood (P weight × day = 0.01), but showed an increase of CD4+CD8α-Th cells (P = 0.002), unlike the HWG piglets. Percentages of CD14+ monocytes and other MHC-II+ cells were respectively higher and lower on day 8 compared to day 16 (P < 0.01). Moreover, LPS activated PBMC from LWG piglets produced less IL-6 (P < 0.05). These results suggest that piglets low performances during lactation affect the development of their immune system and is associated with differences in intestinal gene expression profile and microbiota.
Equine bone marrow mesenchymal stromal cells inhibit reactive oxygen species production by neutrophils without affecting other important microbicidal functions
Authors: Henriquez Claudio , Espinosa Gabriel , Schenffeldt Andres , Alarcon Pablo , Gajardo Gonzalo , Plaza Anita , Caffi Valeria , Uberti Benjamin , Moran Gabriel
Published: January 2023

Neutrophils (PMN) are the largest leukocyte population in the blood of most mammals including horses, playing an important defensive role in many infectious diseases. However, the mechanisms that increase PMN as one of the main cellular subsets in the defense against pathogens, could also be involved in the pathophysiology of dysregulated inflammatory conditions. Mesenchymal stem/stromal cells (MSCs) are a heterogeneous population with a modulatory potential on the inflammatory response, and are known to interact with nearly all cells of the immune system including PMN. In this study, we investigate the in vitro modulation of equine bone marrow-derived MSCs on phagocytosis, ROS production, and NETs generation from equine PMN. We found that in co-culture with MSCs, unstimulated PMN produces less ROS (2.88% ± 1.43) than PMN in single culture (5.89% ± 2.63); interestingly, co-cultured PMN remains conditioned to produce fewer ROS after PMA stimulation in comparison to PMN not cultured with MSCs. We also found that inhibition of ROS production by co-cultured PMN occurred not only after prolonged PMN-MSC co-incubation (4 hours), but also when MSC were present in the culture at the moment of adding the stimuli (30% less than control), suggesting that MSCs don’t only modulate PMN functions prior to their arrival to the inflammatory site, but also at the inflammatory niche itself. Additionally, we found that incubation with MSC supernatant strongly inhibited ROS production (83% ± 6.35 less than control) without affecting phagocytosis or capacity for NETosis. These results support the potential use of MSCs in equine inflammatory conditions where neutrophils are the main effector inflammatory cells
Concomitant effector and LAG-3 regulatory responses of Th1 committed cells in acute influenza
Authors: Dutta Avijit , Huang Ching-Tai , Hung Chen-Yiu , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , Chen Ting-An , Huang Yu-Lin
Published: January 2023

T-bet induced CD4+ T cells are committed to be IFN-γ competent Th1 cells. The Th1 response is essential for virus eradication, but is also a major cause of inflammation and disease aggravation in severe influenza. In our influenza hemagglutinin (HA) Ag-specific mouse experimental system, naïve CD4+ T cells respond to the infection with T-bet expression. However, they suppress cognate CD8+ T cell effector response-mediated inflammation and alleviate the disease. Here we report concomitant LAG-3 expression and IFN-γ competence in Th1 committed CD4+ T cells. LAG-3 expression increases with activation by virus as revealed by CFSE based cell division study. The Th1 committed cells with moderate LAG-3 expression produce IFN-γ and contribute to inflammation and aggravated disease. They can no longer produce IFN-γ with higher LAG-3 expression upon further activation. Th1 committed CD4+ T cells with high LAG-3 expression suppress cognate CD8+ T cells by decoupling their effector function from proliferative response, restrain lung inflammation and facilitate viral clearance. They are better than Foxp-3+HA-specific CD4+ T cells in disease amelioration. The Foxp-3+ cells suppress both effector function and proliferative response of CD8+ T cells. They moderate inflammation but impair viral clearance. Principal component analysis of genome-wide gene expression profiles further confirmed the stepwise evolution. Several regulatory function-associated genes such as Lag3, Pdcd1, Tigit, Ctla2a, Nr4a2, Klrc1, Rgs16, Sytl3, Il21, Il1r2 and Dusp4 are up-regulated graduallly as well from LAG-3Neg to LAG-3Med and LAG-3High cells.
Robust local immune response to vaccination with a live-attenuated HSV-1 strain (HSV-1 0ΔNLS) is delayed in mice vaccinated with the parental live virus
Authors: Carr Daniel J J , Gmyrek Grzegorz
Published: January 2023

We have identified a highly efficacious vaccine 0ΔNLS (deficient in the nuclear location signal of ICP0) against herpes simplex virus type 1 (HSV-1) using an experimental ocular mouse model. In this study, a side-by-side analysis of the host immune response was conducted at the site of vaccination (footpad, FP) and the draining (popliteal) lymph node (PLN) of mice vaccinated with 0ΔNLS versus the parental, wild type HSV-1. Both viruses replicated locally with near equal efficiency through day 3 post infection (PI). By day 5 PI, 0ΔNLS titers recovered from the FP of mice were significantly reduced in comparison to the parental virus and this reduction carried through to clearance by day 9 PI. Even though viral titers were equivalent early post vaccination, 0ΔNLS vaccinated mice generated a more robust localized immune response in the FP in terms of a noticeable increase in the infiltration of myeloid cells and an increase in select cytokines/chemokines including G-CSF, CXCL1, CXCL9, CXCL10, CCL2, CCL5, IL-6, and TNF-α. However, at later time points (days 3–7 PI) the levels of select cytokines/chemokines were elevated in the wild type virus-infected FP with a significant drop in levels from the 0ΔNLS-infected FP. A similar profile was found in the PLN as that reported in the FP. In addition, there were significantly more germinal center B cells (CD19+GL-7+CD95+) and B cells that had undergone isotype switching in the PLN of 0ΔNLS vaccinated mice compared to mice vaccinated with wild type virus by day 7 PI. Collectively, there are unique differences in the host innate and adaptive immune response comparing mice vaccinated with the 0ΔNLS vaccine compared to those exposed to parental wild type virus that continues to be studied in this lab.
Serum IL-6 level may differentiate immune inactive phase from other phases in patients with chronic hepatitis B virus infection
Authors: LEE HYUN WOONG , Oh In Soo , CHANG HYE YOUNG , LEE JUNG IL , LEE KWAN SIK
Published: January 2023

Background and Aims

Previous studies have found that serum level of IL-6 was increased in HBV-infected patients. Here we evaluate the impact of IL-6 levels according to the natural history of chronic hepatitis B(CHB) infection.

Method

Patients(n=71) with hepatitis B virus(HBV) were enrolled between September 2016 and Feb. 2018. Treatment-naïve chronic HBV carries were recruited. IFN-γ, TNF-α, IL-2, IL-6, and IL-17A were measured by a cytometric bead assay according to manufacturer’s instructions. Soluble PD-1 and soluble CD14 were made by the enzyme linked immunosorbent assay (ELISA) technique. The assay procedures were followed according to the manufacturer’s instructions. We studied patients with immune inactive phase (HBV DNA< 1,000 copies/ml, anti-HBe + and normal ALT, n=22), immune tolerant phase (HBV DNA > 10,000 copies/ml, HBeAg + and normal ALT, n=36), and immune active phase (elevated ALT and HBV DNA>10,000 copies/ml, n=13).

Results

Serum IL-6 levels in patients with immune inactive phase were significantly lower than patients with immune tolerant and immune active phases (267.3±245.6 pg/mL vs. 620.2±207.8 pg/mL and 1,815±803.2) (p<0.001). Serum TNF-α levels in patients with immune inactive phase was slightly lower than patients with immune tolerant and immune active phases (3.36±4.40 pg/mL vs. 8.74±12.91 pg/mL and 111.9±377.4) (p=0.079). However, other cytokines (serum IL-2, IL-17A, IFN-γ), soluble CD 14, and soluble PD-1 levels might not be significantly related to the natural history of CHB phase.

Conclusion

These results demonstrate the potential to apply serum IL-6 as a biomarker for differentiating immune inactive phase from other phases in patients with chronic HBV infection.
Innate immune responses to Rift Valley fever virus in the brain
Authors: Weilhammer Dina R , Bourguet Feliza , Sanchez Kristina , Phillips Ashlee , Sebastian Aimy , Hum Nicholas
Published: January 2023

Rift Valley Fever Virus (RVFV) is a category A biodefense pathogen due to the potential for intentional spread by aerosol and the lack of any licensed vaccine or antiviral therapeutics. Despite the brain being a major target of viral replication and tissue damage following aerosol infection, little is known about innate immune responses to RVFV in the brain. Thus, we are investigating the responses of resident innate immune cells of the brain to RVFV infection. Microglia and astrocytes mount a robust response to RVFV infection that is dependent upon MAVS and independent of TLR3 and TLR7. MAVS KO mice have significantly higher viral titers in the brain following intranasal infection, but surprisingly many markers of immune activity were similarly upregulated in the brains of WT and KO mice, including infiltration of CD45+ cells, upregulation of inflammatory genes, and cytokine expression. Indeed, many inflammatory and cytokine genes were upregulated more significantly in infected KO brains than in WT. This is in contrast to the in vitro response of microglia and astrocytes, where MAVS KO cells were virtually unresponsive to RVFV infection. RNA-seq analysis of infected brain tissue revealed that key signal transduction pathways are not activated in MAVS KO animals. These results are an important step towards understanding the precise molecular pathways responsible for controlling RVFV infection in the brain, and will be critical to informing the development of vaccines and antiviral therapeutics that are successful in preventing encephalitis caused by RVFV.
IL-36γ regulates neutrophil infiltration and limits neuroinvasion in genital HSV-2 pathogenesis
Authors: Gardner Jameson K , Herbst-Kralovetz Melissa M.
Published: January 2023

Herpes simplex virus 2 is a neurotropic virus that causes a persistent, life-long infection that increases risk for other sexually transmitted infections. The vaginal epithelium is the first line of defense against HSV-2 and secretes immune mediators, including the pro-inflammatory cytokine IL-36γ, to coordinate the immune response. Previously, we showed that IL-36γ treatment promoted polymorphonuclear cell infiltration to the vaginal cavity and protected against lethal HSV-2 challenge. Herein, we extended these findings using flow cytometry to profile the cellular infiltrate. IL-36γ significantly (p < 0.0001) induced transient neutrophil infiltration, but did not impact monocyte or macrophage recruitment. Using IL-36γ−/− mice in a lethal HSV-2 challenge model, we show that neutrophils were significantly (p < 0.0001) depleted at 1- and 2-days post infection, and that mature neutrophil recruitment was impaired. In addition, IL-36γ−/− mice develop genital disease more rapidly (p < 0.0001), have significantly (p < 0.0001) reduced survival time, and display an increased incidence (p < 0.0001) of neurologic disease compared to wild type mice. IL-36γ−/− mice exhibited a significant (p < 0.05) delay in clearance of the virus from the vaginal epithelium, and analysis of virus spread to the spinal cord, bladder, and colon demonstrated that HSV-2 spread significantly (p < 0.05) more quickly in IL-36γ−/− mice. Further, we detected productive HSV-2 infection in the brainstem of IL-36γ−/− mice significantly (p < 0.0001) more frequently than in wild type mice. Ultimately, we demonstrate that IL-36γ is a key regulator of neutrophil recruitment in the vaginal microenvironment and may function to limit HSV-2 neuroinvasion.
Cytomegalovirus infection drives avidity selection of natural killer cells
Authors: Adams Nicholas M , Lumaquin Dianne , Santosa Endi K , Whitlock Benjamin M , Jackson Benjamin T , Le Luduec Jean-Benoît , Sottile Rosa , Weizman Orr-El , Huse Morgan , Hsu Katharine C , Sun Joseph C
Published: January 2023

Adaptive lymphocyte clones with the greatest receptor affinity tend to dominate primary and secondary immune responses. Strength of receptor signal and receptor abundance, along with competition for antigen and cytokines, drive preferential outgrowth of certain clones, shaping the effector and memory pool in selection processes known as affinity and avidity maturation. Natural killer (NK) cells are innate lymphocytes capable of “adaptive” responses following infectious challenges. However, whether NK cells undergo a similar selection process on the basis of their avidity for cognate ligand is not known. Here, we show that NK cells with a broad range of avidities for the mouse cytomegalovirus (MCMV) glycoprotein m157 are initially recruited after virus infection, but those with highest avidity are selected to undergo the greatest clonal expansion to comprise the memory NK cell population. Furthermore, pre-established levels of Ly49H receptor expression within the Ly49H+ NK cell pool dictated the functional contribution of a given NK cell during MCMV infection, with lower avidity NK cells possessing greater capacity for IFN-γ production, and higher avidity NK cells possessing greater capacity for cytotoxicity and adaptive responses. Moreover, we provide evidence for avidity selection also occurring in human NK cells during human CMV (HCMV) infection. These results provide a mechanistic understanding of how heterogeneity in NK cell avidity underlies the diversification of NK cell effector function during a primary antiviral immune response, and how the process of avidity selection may serve to produce the most potent memory NK cell pool.
IL-18 defines exclusive ‘memory-like’ NK cell populations
Authors: Anderko Renee R. , Rinaldo Charles R. , Mailliard Robbie B.
Published: January 2023

While NK cells are well known for their killing effector function, they also play a critical role as immune helper cells, providing innate alarm signals that shape and regulate the adaptive immune response. In chronic HIV-1 infection, an expanded population of CD56dim FcRγ deficient NK cells persists, similar to the ‘memory-like’ FcRγ− NK cell type identified in cytomegalovirus infection. The purpose of this study was to explore the phenotypic and functional relationship between these FcRγ− NK cells and the previously described IL-18-induced ‘memory-like’ NK helper cells in the setting of HIV-1 infection. We utilized HIV-1 seropositive participants of the Multicenter AIDS Cohort Study to measure baseline frequencies of peripheral blood FcRγ− NK cells by flow cytometry. We also treated NK cells for 24h with IL-18 alone, or in combination with IL-12, and assessed the phenotypic and functional impact on the FcRγ− and FcRγ+ subsets by flow cytometry. The frequencies of FcRγ− NK cells varied widely in HIV-1 infected men. IL-18 drove the differentiation of CD25+/CD83+ NK helper cells, which produced IFNγ and strongly diminished CD16 expression in response to IL-12. In contrast, FcRγ− NK cells did not respond to IL-18, illustrated by failure to express CD25 or CD83, to downregulate CD16 expression, and to produce IFNγ. FcRγ deficiency was also characterized by an attenuated capacity to express IL18Rα upon IL-18 and IL-12 co-activation. Our results indicate that ‘memory-like’ NK helper cells and FcRγ− NK cells are exclusive populations and suggest that HIV-1 drives the development of cytokine-exhausted FcRγ− NK cells that are unable to provide proper immune ‘help’ in response to innate activation signals.
Understanding the role of hnRNPA1 on T cell differentiation and function in the Gut
Authors: White Theresa L , Gable Matthew , Jin Ye , Morel Penelope A
Published: January 2023

Regulatory T cells (Treg) play a critical role in preventing autoimmunity diseases and maintaining tolerance in various tissues, particularly the gut. Peripheral derived Tregs (pTregs) are especially crucial in gut homeostasis and tolerance to commensal and food antigens. We have previously shown that knockdown of the RNA-binding protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, reduces in vitro pTreg induction upon low TCR stimulation. In addition, we have shown that, under conditions of low TCR stimulation, in which pTreg are induced, hnRNP A1 is phosphorylated by Akt. In order to explore the impact of hnRNP A1 phosphorylation by Akt on pTreg induction in vivo we have generated a mutant mouse model, hnRNPA1-S199 in which the known Akt phosphorylation site on hnRNP A1 is mutated to Alanine. Extensive characterization of immune cells in this mutant mouse revealed no abnormalities in the number or frequency of major immune cell types at steady state. However, we identified abnormalities in the number of Treg in the mesenteric lymph nodes (mLN) in mutant mice, prompting further investigation. Using the Cytex Aurora flow cytometer we developed a 19 color T cell panel to characterize T cells in the mLN, Peyer’s patches and lamina propria. Detailed characterization of the pTreg and tTreg populations in the gut of WT and mutant mice will determine whether the abnormalities observed in the mLN are reflected in different intestinal areas. In addition, we are examining the induction of pTreg using an adoptive transfer model of OT-II TCR transgenic mutant and WT CD4 T cells followed by oral OVA administration. Further studies will focus on the mechanisms by which hnRNP A1 impacts pTreg induction.
Genetics but not intestinal microbiota determines the onset of systemic autoimmune disease in BXD2 mice
Authors: Hong Huixian , Wu Qi , Yang PingAr , Duck Wayne , Morrow Casey D. , Foote Jeremy B. , Schoeb Trenton R. , Elson Charles O. , Hsu Hui-Chen , Mountz John D.
Published: January 2023

The onset and severity of autoimmunity can be attributed to both genetic predisposition and environmental triggers including intestinal microbiota dysbiosis. To determine if development of spontaneous autoimmunity is dependent on gut microbiota, we generated germ-free (GF) BXD2 lupus mice which otherwise develop spontaneous germinal centers (GCs) and high titers of serum autoantibodies. The microbiome and immune phenotypes in specific pathogen free (SPF) and GF BXD2 and normal B6 strains of mice were analyzed. The GF status was confirmed by gut bacterial culture. Surprisingly, the GF status did not affect disease onset and severity in 6-mo-old mice. There were comparable serum levels of IgG or IgM autoantibodies against lupus autoantigens (DNA, histone, and RNP) in SPF versus GF BXD2 mice. Flow cytometry analysis showed that the frequency and absolute numbers of GC B cells, CD4+ T cells, follicular T-helper (Tfh) cells in the spleen were also indistinguishable between SPF and GF BXD2 mice although GF BXD2 mice exhibited a higher number of CD19+ B cells and Tregs, compared to the SPF counterpart. Histologic examination of kidney and lung also showed similar disease scores between SPF and GF BXD2 mice. At older age (12-mo-old), however, there were diminished GC B cells and serum levels of autoantibodies in GF BXD2 mice compared to SPF mice. The results suggest a model in which genetic factors play a dominant role in determining the onset and susceptibility of autoimmunity and that endogenous self-antigens are the primary driver of autoreactive T and B cells leading to spontaneous GCs in BXD2 mice. In contrast, environmental factors such as gut microbiomes may play a modifying role in regulating the persistence of systemic autoimmunity.
Single cell RNA sequence analysis reveals novel clusters unique to post-infectious myocarditis in the mouse model of Coxsackievirus infection
Authors: Reddy Jay , Lasrado Ninaad , Arumugam Rajkumar , Jayaraja Sabarirajan , Thibivilliers Sandra , Libault Marc , Starr Timothy K
Published: January 2023

Myocarditis is a common occurrence in children and adolescents that can lead to dilated cardiomyopathy. Since it is difficult to identify the triggers of this complex disease, mechanistic understanding of DCM pathogenesis may create avenues to develop new treatment strategies. We have been engaged in delineating the autoimmune pathways underlying the development of DCM using various mouse models of myocarditis that resemble human disease. One such model is Coxsackievirus B3 (CVB)-induced myocarditis in A/J mice that show two distinct phases of disease, but occur in continuum. While, the acute, infectious phase (up to ~10 days post-infection) is marked by viremia and infiltrations in the hearts, infectious virions become undetected during the chronic phase (~beyond 18 days post-infection), and animals develop DCM. The latter phenotype can occur over a period of ~60 to 90 days, and hitherto, it has been a challenge to determine the underlying mechanisms. To dissect this complex nature of disease, we recently performed single cell RNA sequence analysis on heart infiltrates obtained from CVB-infected mice. Using panels of 15 to 30 marker genes for various immune and non-immune cells, we identified 18 and 26 clusters respectively in healthy and infected hearts that also include few unknown clusters unique for each group. Likewise, transcriptome analyses led us to identify a total of 14,051 genes to be differentially expressed, and of these, 206 and 345 transcripts were unique to healthy and CVB-infected groups, respectively. The data suggest that uniquely expressed genes in the CVB-infected hearts may have a role in the development of post-infectious myocarditis leading to DCM.
Cardiac myosin-specific TCR transgenic mouse models for studying dilated cardiomyopathy
Authors: Reddy Jay , Massilamany Chandirasegaran , Arumugam Rajkumar , Lasrado Ninaad , Gangaplara Arunkumar , Jayaraja Sabarirajan , Steffen David , Gurumurthy Channabasavaiah
Published: January 2023

We previously reported that the cardiac myosin heavy chain-alpha (Myhc) 334–352 encompasses epitopes for both CD4 and CD8 T cells that can induce autoimmune myocarditis/dilated cardiomyopathy (DCM) in A/J mice expressing the IAk and IEk molecules. More recently, we made an unexpected observation that, Myhc 334–352 although by being a poor binder of IAb molecule in C57Bl/6 mice, could induce strong T cell responses, but myocarditogenicity was severely dented. In our efforts to delineate the roles of Myhc-specific, CD4 and CD8 T cells in the DCM pathogenesis, we have now created the T cell receptor (TCR) transgenic (tg) mice on C57Bl/6 background for Myhc 334–352, and demonstrate that the Myhc 334–352-specific TCR was found common to both CD4 and CD8 T cells. While, the naïve tg T cells do not respond, antigen-primed CD4 and CD8 T cells from immunized mice respond to Myhc 334–352 that produce predominantly, interferon-γ. Furthermore, although mild, the immunized tg mice developed heart lesions containing the mononuclear cells, and such a phenotype was more apparent in females than males. As the tg mice become available on both resistant (C57Bl/6) and susceptible (A/J) backgrounds, our TCR tg system can be used as a helpful tool to dissect the complexities of antigen-specific CD4 and CD8 T cells in the causation of DCM in various experimental settings.
Genetic and pharmaceutical targeting of OCA-B/Pou2af1 blocks T1D in mouse models
Authors: Kim Heejoo , Shakya Arvind , Perovanovic Jelena , Ibarra Andrea , Evavold Brian , Chou Danny H.-C. , He Xiao , Jensen Peter E , Tantin Dean R
Published: January 2023

Treatments for autoimmunity aim to inhibit autoreactivity while preserving normal immune function. The transcriptional coregulator OCA-B (gene symbol Pou2af1) is induced in stimulated naïve CD4+ T cells, where docks with transcription factor Oct1 to regulate genes such as Il2 and Ifng. Oct1 and OCA-B only regulate their targets in cases of persistent antigen exposure, a common feature of autoimmunity. We hypothesized that T cell-specific OCA-B deletion would protect mice from autoimmune diabetes, and that pharmacologic OCA-B inhibition would similarly protect diabetes-prone mice. We generated an Ocab (Pou2af1) conditional allele onto a NOD/ShiLtJ strain background. OCA-B loss in T cells had no effect on the naïve T cell receptor repertoire or gene expression in the pancreatic lymph nodes, but protected NOD mice from both mice spontaneous and induced T1D. In scRNA-seq data with pancreatic leukocytes, dominant CD8 TCR clones known to participate in disease failed to emerge in the islets of OCA-B deficient mice. Protection was also associated with reduced islet neutrophil and macrophage infiltration and reduced neutrophil activation. The protective effect of OCA-B loss was diminished, or even eliminated, using monoclonal TCR models with high affinity to artificial or neoantigens. Finally, we developed rationally-designed membrane-penetrating peptide inhibitors of the interaction between OCA-B and its downstream target, Jmjd1a. Pharmacologic OCA-B inhibition normalized glucose levels, and reduced T cell infiltration and proinflammatory cytokine expression, in newly-diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.
Lupus susceptibility gene Pbx1 promotes atherosclerosis via dysfunctional T cells in a mouse model
Authors: Li Wei , Elshikha Ahmed , Teng Xiangyu , Abboud Georges , Brown Josephine Michelle , Choi Seung-Chul , Fredenburg Kristianna , Major Amy S , Morel Laurence
Published: January 2023

The incidence of atherosclerosis is higher among patients with systemic lupus erythematosus (SLE). An imbalance between regulatory T cells (Treg) and follicular helper T cells (Tfh) occurs in the development and progression in both diseases. However, the mechanisms by which lupus T cells may aggravate atherosclerosis remain unclear. Pre-B-cell leukemia transcription factor 1 isoform d (Pbx1-d) is a lupus susceptibility gene and transgenic (Tg) Pbx1-d in CD4+ T cells expands Tfh and impairs Treg. Here, we investigated the role of CD4-Pbx1-d-Tg T cells in the low-density lipoprotein receptor-deficient (Ldlr−/−) mice, an experimental model for atherosclerosis. Pbx1-d T cells exacerbated atherosclerosis in Ldlr−/− mice as compared to B6 T cells. The enhanced atherosclerosis was associated with increased body weight, increased autoantibody production, reduced Treg in the aorta, as well as increased splenic Tfh frequency and impaired Treg cell suppressive markers. Dyslipidemia inhibited the differentiation and function of Treg cells in vitro. Moreover, Pbx1-d Treg cells were less suppressive than B6 control Treg cells in dyslipidemia conditions. Thus, our results showed that Pbx1-d-Tg T cells exacerbate atherosclerosis due to the dysregulated Treg and Tfh cells, briging autoimmunity and cardiovascular pathology.
Investigating the antigen-specific T cell response to MHC variant peptide 45D
Authors: Faust Michael A , Kolawole Elizabeth , Evavold Brian
Published: January 2023

We have previously generated a variant of the myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide by mutation of the P6 MHC anchor residue from an S to a D amino acid. Here, we confirm previous findings that the 45D MHC variant peptide is incapable of inducing experimental autoimmune encephalomyelitis (EAE) despite its CD4 epitope bearing the same TCR contact residues as wildtype MOG epitope (wtMOG). Using the micropipette adhesion frequency assay (MP), we show that induction with 45D generates reduced levels of CD4 T cells specific for MOG in the CNS but not the periphery of asymptomatic mice. Importantly, 45D induction results in a loss of high affinity CD4 T cells, which could be responsible for initiation of disease. We also found changes in MOG-reactivity within the peripheral Treg compartment of 45D-induced mice. Together, the variant peptide alters the MOG-specific Teff and Treg response. Although the 45D mutation was designed to target the CD4 MOG epitope, it also changes the antigen responsible for expanding MOG-reactive CD8 T cells. We find that priming with 45D expands CD8 T cells with heightened affinity for MOG. Taken together, our data demonstrate that 45D may enhance peripheral tolerance mechanisms that ultimately aid in the protection from autoimmune disease.
Differential effects of Flavipin on Ahr and Arid5a: A new therapeutic strategy for autoimmune encephalomyelitis
Authors: Hanieh Hamza N , Alzahrani Abdulllah
Published: January 2023

Multiple sclerosis (MS) is a World-wide spread autoimmune disease that includes episode of demyelination in the central nervous system (CNS). Accumulating evidence has demonstrated that ligand-activated aryl hydrocarbon receptor (Ahr) improves pathological outcomes in experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. Recent evidences have shown that AT-rich interactive domain-containing protein 5a (Arid5a), an RNA-binding protein, is required for development of EAE via stabilizing the mRNAs of interleukin-6 (Il6) and Ox40. To date, there are no reports studying both Ahr and Arid5a as a potential therapeutic strategy for MS. Thus, current work applied in silico, in vitro and in vivo approaches to identify a molecule that interacts with Ahr and Arid5a. The results identified Flavipin (3,4,5-trihydroxy-6-methylphthalaldehyde) as a new Ahr agonist that induces the expression of downstream genes in mice CD4+ T cells and CD11b+ macrophages. Interestingly, Flavipin inhibited the stabilizing function of Arid5a, and impaired its counteracting effects on RNase function of Regnase-1 on 3′ untranslated region (3′UTR) of Il6, Ox40 and signal transducer and activator of transcription 3 (stat3). In EAE, Flavipin ameliorated disease symptoms concomitant with inducing acetylcholinesterase (AChE)-targeting miR-132. It reduced the expression of Ox40 in CD4+ T cells and the number infiltrated CD4+CD45+ T cells in the CNS. Further analysis identified Il23a mRNA as a new target for Arid5a. Taken together, our findings provide first evidences on differential effects of Flavipin on Ahr and Arid5a and pave an avenue for a new therapeutic strategy for autoimmune diseases such as MS.
TNF-α orchestrates CD4+ T cell-dependent inflammation via TNFR2 signaling
Authors: Alam Muhammad S
Published: January 2023

CD4+ T helper subsets, in particular Th1 and Th17, produce cytokines that play critical roles in inflammatory diseases. TNF-α, produced largely by innate immune cells, is also potently pro-inflammatory. Here we asked if TNF-α contributes to effector CD4+ Th generation, and found that in the presence of TGF-β TNF-α signals via its non-death receptor TNFR2 to generate Th17 cells expressing inflammatory mediators such as GM-CSF, CXCL3, and IFN-γ. Single cell RNA-Seq of CNS-infiltrating CD4+ T cells in EAE identified an IL-17-positive cluster with an inflammatory gene signature that required TNFR2 expression. Notably, TNFR2-deficient CD4+ T cells produced fewer inflammatory mediators and were relatively nonpathogenic in EAE and colitis. Thus, TNF-α is not just an effector but also an initiator of Th differentiation, raising the possibility that in diseases in which both TNF-α and IL-17 contribute, targeting TNF-α may diminish the generation of pathogenic Th17 cells.
Aberrant expression of β5t in the periphery induces a self-reactive T-cell response
Authors: Tomaru Utano , Kimoto Rikuto , Ishizu Akihiro , Kasahara Masanori
Published: January 2023

The thymoproteasome subunit β5t is specifically expressed in cortical thymic epithelial cells (cTECs) and generates unique peptides to support positive selection. However, in the context of systemic immune homeostasis, it remains unclear why the expression of β5t-containing proteasomes is restricted to the thymus. In this study, using a mouse model ubiquitously expressing β5t (β5i−/−β5t-Tg), we showed that the aberrant peripheral expression of self-peptides generated by β5t affects the peripheral naïve pool of CD8+ T cells. In β5i−/−β5t-Tg mice, the percentage of naïve CD44lowCD122lowwas reduced, and the percentage of memory CD44highCD122high CD8+ T cells was significantly elevated. When naïve CD44lowCD122low CD8+ T cells from EGFP mice, which exhibited the wild type (WT) phenotype and underwent thymic selection by β5t-expressing cTECs, were transferred into β5i−/−β5t-Tg, transferred cells changed their phenotype from CD44lowCD122low to CD44highCD122high. The transferred EGFP+ CD8+ T cells with a CD44highCD122high phenotype showed increased expression of PD-1 and Tim-3, and these alterations were affected by the condition of peripheral antigen-presenting cells in β5i−/−β5t-Tg recipient mice. The transferred CD8+ T cells from WT mice caused chronic inflammation in β5i−/−β5t-Tg recipient mice. These results demonstrate that the aberrant peripheral expression of β5t disrupts the maintenance of a naïve pool of CD8+ T cells and induces autoreactive T-cell responses. The self-peptides generated by β5t are required to confine in the thymus to avoid autoimmunity in the peripheral tissues, and self-peptides mimicking β5t-producing peptides in the periphery may cause the failure of immunological regulation and tolerance.
More roles of IL-4 than being as an antagonist of IFN-γ in experimental autoimmune uveitis
Authors: Shen Chia-Rui , Yang Wei-Cheng , Hwang Yih-Shiou , Hong Wei-Hsin , Liu Chao-Lin
Published: January 2023

IL-4 appears to be beneficial for some autoimmune diseases. Despite its role in antagonizing pathogenic Th1 response, our recent work has demonstrated IL-4 possesses functions affecting the regulatory T (Treg) cells. Here we aimed to identify the role of IL-4 in the development of experimental autoimmune uveitis (EAU). EAU was induced in WT and IL-4 KO mice via active interphotoreceptor retinoid binding protein (IRBP) immunization, and the disease development was monitored by funduscopic and histology analysis as well as the IRBP-specific immune responses by flow cytometric analysis and ELISA. It showed that the onset of disease, if the clinical score equals to 1, was day 14 and 20 in IL-4KO and WT mice, respectively. The fundoscopic images demonstrated the more and bigger size of lesions, increased leukocyte infiltration, vasculitis and neovascularization in IL-4KO mice. The histological analysis also confirmed the mononuclear cell infiltration. Moreover, Th1 effector is known as the dominant pathogenic mediators, whereas Treg cells play an important and indispensable role in control of EAU progression and disease remission. The splenic T cells from IL-4 KO EAU animals produced significantly elevated level of IFN-γ in responding to IRBP stimulation. However, there seems no significant but some alternation in the presence of Treg cells (Foxp3+) among CD4+CD25+ T cells in the spleens and lymph nodes of IL-4 KO and WT mice. These results demonstrate the lack of IL-4 disrupting the induction of EAU and leading to an increased disease severity through not only antagonizing IFN-γ but also involved mechanisms of immune-regulation. Therefore, IL-4 might play more roles than being just an antagonist of IFN-γ in autoimmune uveitis.
A Direct Link between IL-17 and Regulatory T cells in Autoimmune Colitis
Authors: Pitmon Elise V , Chen Ju , Ye Xiaoyang , Wang Kepeng
Published: January 2023

Inflammatory bowel diseases are rising in prevalence globally. While current treatments aim to achieve remission, there remains no cure for this disease. IL-17 family cytokines are important players in inflammation, but clinical trials using anti-IL-17A or IL-17RA blocking antibodies have failed for Crohn’s disease, due to exacerbated inflammation in some patients. While anti-IL-17 antibodies have shown efficacy in the treatment of other autoimmune diseases like psoriasis, patients can often develop Crohn’s disease as a side effect of these drugs. These data suggest a protective role of IL-17 in gut inflammation. The proper activation and function of regulatory T cells is essential for the protection against intestinal autoimmune inflammation. Using the naïve T cell transfer model of colitis in mice, we found that IL-17RA-null iTregs, transferred after the establishment of colitis, were less effective at controlling the colitis compared to WT iTregs. Naïve T cells cultured ex vivo in iTreg polarizing conditions had an increase in complete iTreg differentiation when stimulated with IL-17 cytokines. Our results indicate that IL-17 directly signals to Tregs and assists their anti-inflammatory role during autoimmune gut inflammation. Although the underlying mechanism remains under study, this finding suggests an inhibitory loop between IL-17 and Tregs for the control of overt inflammation.
Melanocyte-specific CD8+ T cells demonstrate tolerance characterized by an impaired ability to differentiate into a highly proliferative population
Authors: Truckenbrod Emily Nestor , Renkema Kristin R , Burrack Kristina S , da Silva Henrique Borges , Jameson Stephen C
Published: January 2023

The mechanisms governing non-deletional CD8+ T cell tolerance are poorly understood. We are using a physiologically-relevant mouse model to better understand the regulation of self-specific CD8+ T cells, which impact both autoimmune disease and cancer. We find a substantial number of polyclonal cells specific for an epitope from the melanocyte/melanoma-associated enzyme tyrosinase-related protein 2 (Trp2) in both wild-type (WT) C57BL/6 and Trp2-deficient (Trp2 KO) mice. In pre-immune mice, these cells have a naïve phenotype in both strains, and they respond similarly for the first several days after in vivo stimulation with Trp2 peptide alone or in combination with adjuvants (TriVax). However, significantly fewer WT Trp2-specific cells are present at an effector timepoint after TriVax or recombinant Trp2-expressing Listeria monocytogenes relative to KO Trp2-specific cells. WT Trp2-specific cells also show a deficiency in mediating vitiligo compared with KO cells when TriVax-generated effectors are transferred into recipient WT mice primed with TriVax and dinitrofluorobenzene (DNFB). Single-cell RNA sequencing at day three after stimulation reveals that KO Trp2-specific cells efficiently differentiate into a proliferative population, whereas few WT Trp2-specific cells demonstrate this phenotype. As a population, WT Trp2-specific CD8+ T cells exhibit covert anergy: despite a naïve appearance at steady state and initial acquisition of activation markers after stimulation, these self-specific cells are unable to expand productively or to efficiently mediate functional anti-melanocyte activity. These findings have implications for recently-described non-deletional CD8+ T cell tolerance in humans.
Somatostatin receptor subtype 4 protects against indomethacin-induced gastrointestinal mucosal injury in mouse
Authors: Cseko Kata , Kormos Viktória , Horváth Ádám István , Kecskés Angéla , Gaszner Balázs , Sudalina Maria , Iarushkina Natalia , Zelena Dóra , Filaretova Ludmila , Helyes Zsuzsanna
Published: January 2023

Despite recent advancements in the pharmacologic and endoscopic management of gastrointestinal ulceration, it is still associated with high mortality. Therefore, it is important to understand mucosal protective mechanisms. Somatostatin, present in the endocrine cells & sensory nerves, and its analogues reduce mucosal blood flow, pepsin and gastric acid secretion. Its receptor subtype 4 (sst4) mediates anti-inflammatory & analgesic actions, as discovered by our group. Therefore, our aim was to investigate its role in gastrointestinal injury.

We treated sst4 gene-deleted (sst4−/−) and wildtype mice with indomethacine (IDM; 35 mg/kg) or vehicle s.c.. Gastric & small intestine lesions were evaluated after 4 & 48 h macroscopically & microscopically. Plasma protein extravasation & myeloperoxidase (MPO) activity were assessed by ex vivo imaging, TNFα, IL-1, COX2, sst4 & somatostatin mRNA levels by qPCR.

Gastric (4h) and small intestinal macroscopic (24 h) lesions, MPO activity (24, 48 h), and plasma extravasation (30 min) were significantly greater in IDM-treated sst4−/− mice, while histopathological scores & small intestinal length did not show strain difference. TNFα, IL-1, COX2 significantly upregulated after 4h in the stomach of sst4−/− mice, and the same pattern was observable in wildtype mice at later timepoint (48 h).

These results provide evidence for the protective role of the sst4 receptor against IDM-induced gastrointestinal mucosal injury. This suggests the safety of sst4 agonist drug candidates in the gastrointestinal tract, and even their potential use for gastroprotective indication.

Supported by: GINOP-2.3.2-15-2016-00050 – PEPSYS, GINOP-2.3.2 STAY ALIVE, EFOP-3.6.2-16-2017-00008, ÚNKP-19-3-III-PTE-211
Increased neuroinflammation and atherosclerosis in sleep fragmented mice
Authors: Moriarty Alina K , Keeter W. Coles , Waseem Tayab , Ma Shelby , Gauronskas Phillip Joseph , Butcher Matthew J , Wellman Laurie , Sanford Larry , Galkina Elena
Published: January 2023

There are roughly 70 million Americans who report insufficient sleep due to work responsibilities, sleep apnea, or lifestyle choices. Altered duration and poor sleep quality are associated with an increase in brain inflammation, high rates of cardiovascular disease and other inflammatory diseases in humans. It is known that psychological stress is associated with increased atherogenesis, it is also known that sleep fragmentation (SF) can exacerbate and be induced by SF. However, the exact relationship between SF, neuroinflammation, and atherogenesis is not well known. We found that chronic SF led to increased plaque formation in the aortas HFD fed Apoe−/− mice. SF also resulted in alterations in the immune composition of the bone marrow (BM), blood, aortas, and lymph nodes in the HFD fed Apoe−/− mice. Importantly we detected an increased content of myeloid cells in the blood of SF and HFD fed female Apoe−/− mice. There was an increased percentage of CD68+ macrophages in SF Apoe−/− aortas. Additionally, we detected an increase in CD11b expression and inflammatory markers in the brains of mice that were sleep fragmented when compared to control mice. Our data suggest that SF induces elevated myelopoeisis resulting in monocytosis, neutrophilia, and the altered immune composition of the aortas of atherosclerosis-prone mice, resulting in accelerated atherosclerotic plaque burden.
Progressive immunosenescence in diabetic chronic kidney disease
Authors: Hong Ruo-Wei , Shu Kai-Hsiang , Chen I-Yu , Lay Fang-Yun , Chuang Yi-Fang , Chiu Yen-Ling
Published: January 2023

Type 2 diabetes is an important challenge given the worldwide epidemic and is the most important cause of end-stage renal disease in developed countries. It is known that patients with end-stage renal disease and advanced renal failure suffer from immunosenescence and premature T cell aging, but whether such changes develop in patients with less severe chronic kidney disease (CKD) is unclear. We investigated level of immunosenescence in 770 Patients with type 2 diabetes with different levels of renal function. Consistent with previous observations in the general population, both T and monocyte immunosenescence in diabetic patients positively correlate with age. Compared to patients with eGFR>60 ml/min, patients with more severe CKD showed a progressive decrease in total CD3+ T cells, but not in monocyte numbers. Immunosenescence, as defined by multiple phenotypic markers, was significantly upregulated in both CKD stage 3 and stage 4/5 patients, especially of CD8+ T cells. In age and sex-adjusted regression models, stage 3 CKD patients exhibited significantly elevated percentages of CD28−, CD127−, and CD57+ cells among CD8+ T cells when compared to patients with eGFR>60 ml/min. In contrast, no change was detected in monocyte subpopulations as renal function declined. Our study indicates that therapeutic approaches to prevent the emergence of immunosenescence in renal failure patients should be implanted before stage 3 CKD.