bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell cross-reactivity during heterologous infection results in immunodomination and enhanced cytolytic capacity at the expense of virus-specific responses
Authors: Hassert Mariah , Brien James D , Pinto Amelia K
Published: February 2023

Flaviviruses constitute a genus of closely related arthropod-borne viruses including Zika virus, the four serotypes of dengue virus, and yellow fever virus, which circulate in the same geographic regions. These viruses share a substantial degree of genetic similarity and consequently, antigenic overlap has been reported by evaluating T cell and antibody responses in humans, non-human primates, and mice. However, it has not been determined how existing immunity to a heterologous flavivirus impacts functional immune responses to virus-specific and cross-reactive epitopes. We hypothesize that cross-reactive T cells from prior flavivirus exposures will expand robustly during a heterologous challenge and that these cells primed during a heterologous infection will be functionally different from cells of the same specificity primed during a homologous infection. Using various mouse models of flavivirus infection, we have identified a pan-flavivirus reactive CD8+ T cell epitope. We show in a heterologous infection model in which dengue virus exposure precedes Zika virus infection, T cell responses to the cross-reactive epitope dominate at the expense of the Zika-specific T cell responses. These cross-reactive T cells display enhanced killing capacity, in addition to other functional changes. The culmination of these features drive altered protective capacity when compared to homologously primed T cells in our mouse model of Zika infection. Our findings provide a mechanistic understanding of cross-reactive T cell control during heterologous infection and have important implications for vaccine design, as these results define the functional consequences of priming a cross-reactive T cell response for a pan-flavivirus vaccine.
High levels of endemic coronaviruses reactive CD4+ T cells in SARS-CoV-2 seronegative health care workers
Authors: Antunes Ricardo Filipe da Silva , Pallikkuth Suresh , Williams Erin , Yu Esther Dawen , Mateus Jose , Quiambao Lorenzo , Wang Eric , Rawlings Stephen , Stadlbauer Daniel , Jiang Kaijun , Amanat Fatima , Arnold David , Andrews David , Fuego Irma , Dan Jennifer , Grifoni Alba , Weiskopf Daniela , Krammer Florian , Crotty Shane , Hoffer Michael , Pahwa Savita , Sette Alessandro
Published: February 2023

Healthcare workers (HCW) due to occupational exposure to patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a population at risk during the current coronavirus disease-2019 (COVID-19) pandemic. Pre-existing immunity induced by the exposition to several pathogens, including respiratory viruses, can shape susceptibility to infections but little is known about the presence of cross-reactive memory T cells that have the potential to recognize SARS-CoV-2. Herein we measured CD4+ T cell responses against common cold corona (CCC) viruses and SARS-CoV-2 in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC reactive T cells in SARS-CoV-2 seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses shown to be consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC reactivity was consistently decreased in SARS-CoV-2 infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego.
Human herpesvirus diversity is altered in predicted HLA class I binding peptides
Authors: Palmer William H , Norman Paul J
Published: February 2023

Human Leukocyte Antigen (HLA) Class I molecules present intracellularly derived peptide fragments for discrimination of self from non-self, thereby alerting the adaptive immune response to infecting pathogens. Among these pathogens are herpesviruses, including Epstein-Barr virus (EBV), which are large DNA viruses that have coevolved with humans. Previous analyses suggest that natural selection pressures imposed by HLA recognition may affect herpesvirus genetic diversity, especially for proteins involved in the establishment of latency. Because HLA allele frequencies are highly differentiated across human populations, we tested the hypothesis that herpesvirus latency genes have also become differentiated across human populations through adaptation to the local HLA environment. We predicted the protein regions most likely to be recognized by HLA in EBV, Human cytomegalovirus (HCMV), and Varicella-zoster virus (VZV) strains from >15 representative human populations. We compared sequence diversity and population differentiation in HLA-binding and non-binding regions of lytic and latent herpesvirus proteins. We observed increased population differentiation in herpesvirus latent proteins, specifically in regions targeted by HLA. Whereas HLA recognizes conserved peptides in most viral proteins, this pattern was reversed in HLA binding sites of EBV and HCMV proteins expressed in latency. We also predicted fewer HLA-binding latent EBV peptides from higher frequency HLA alleles, consistent with EBV adapting away from HLA recognition. Together, these results suggest that EBV and HCMV diversity is altered in regions recognized by HLA, perhaps due to population-specific natural selection by local HLA alleles.
HLA allele frequencies and susceptibility analysis in 94 Florida residents with COVID-19 disease
Authors: Shen Yiran , Nguyen Cuong Q , Ostrov David A
Published: February 2023

COVID-19 is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) of suspected zoonotic origin. Although coronaviruses (CoVs) are relatively common across species, mutations can cause severe symptoms in humans. The widespread clinical symptoms encompass a large group of asymptomatic COVID-19 patients, raising a crucial question regarding genetic susceptibility, i.e. whether human leukocyte antigens (HLA) play a role in the patient symptomology. In this study, we hypothesize that “A certain HLA allele or combination of certain alleles can serve as biomarker for the severity of COVID-19”. To test this hypothesis, we genotyped HLAs of 94 patients with confirmed COVID-19 and 89 healthy individuals in Florida, USA, we found certain alleles (HLA-A*74:01, -A*30:01, DPA1*03:01, DPB1*01:01, and DQA1*05:01) were associated with disease susceptibility and some alleles (HLA-DPA1*01:03, DPB1*11:01 and DQA1*02:01) were associated with protection. Furthermore, using in-silico HLA docking modeling, we found that DPA1*01:03 is associated with reduced SARS-CoV-2 infectivity by binding to SARS-CoV-2 spike peptide with high affinity. Also, polymorphism in the antigen binding cleft of HLA-DP has the potential to inhibit binding to peptides derived from SARS-CoV-2 spike protein. The results provided a broader understanding of the genetic HLA association pertaining to the severity of COVID-19. Additionally, the results indicted the importance in performing the HLA genotyping with virus detection to identify high risk individuals.
An early decline in circulating lymphoid progenitors contributes to the age-associated reduction in ETP numbers
Authors: Srinivasan Jayashree , Vasudev Anusha , Selden Hilary J. , Encarnacion Perez Jr. , LaFleur Bonnie , Sinari Shripad , Krueger Andreas , Richie Ellen R. , Ehrlich Lauren I.
Published: February 2023

Age-associated thymic involution is characterized by the decreased generation and export of T cells from the thymus, which in turn, is associated with increased susceptibility to infections. Elucidating the cause of thymic involution will inform strategies to restore thymopoiesis with age. T cells develop from thymus seeding progenitors that migrate from the bone marrow (BM) to the thymus. Early T-cell progenitors (ETPs) require signals from the thymic microenvironment to undergo T-lineage commitment and differentiation. In mice, we find that there is a significant decline in thymocyte cellularity as early as 3 months of age (3MO), which is mirrored by a striking reduction in ETPs. This early thymic involution could be driven by changes in hematopoietic progenitors and/or thymic stromal cells. Using a multicongenic BM transplantation approach, we determined that the number of ETP-supportive niches does not diminish with age. However, modulating the thymic stromal compartment sustains ETP cellularity, suggesting that age-associated defects in ETP niche quality may contribute to ETP decline. Notably, the number of lymphoid progenitors in the BM and blood declines dramatically by 3MO, although their cell-intrinsic capacity to seed and differentiate in the thymus is unimpaired. Together, our findings suggest that the early decline in ETP numbers is due to both hematopoietic and stromal age-associated changes.

This work was supported by P01 AG052359 from the NIH-National Institute of Aging.
Thpok promotes CD4+-lineage differentiation by recruiting the NuRD chromatin remodeling complex
Authors: Gao Yayi , Zamisch Monica , Vacchio Melanie S , Paine Elliott , Lyons Gaelyn , Ciucci Thomas , Chopp Laura , Xiao Qi , Vinson Charles , Jenkins Lisa , Bosselut Rémy
Published: February 2023

T cell precursors develop into helper CD4 or cytotoxic CD8 T cells in thymus. Thpok, a BTB/POZ family transcription factor, enforces MHC II restricted thymocytes commitment to the CD4 lineage, while Runx3 protein drives MHC I restricted thymocytes to the CD8 lineage. However, the molecular mechanisms underlying Thpok functions during CD4 T cell development are not understood. Replacing the BTB domain from Thpok with that from Bcl6, another BTB/POZ zinc finger transcription factor that recruits NCOR-family co-repressors through its BTB domain, abrogated CD4 T cell development in thymus; this suggests a unique feature associated with Thpok BTB domain. Here, using mass-spectrometry, we identified nucleosome remodeling and deacetylase (NuRD) complex as a novel Thpok cofactor. We demonstrated that the Thpok BTB domain is essential for NuRD recruitment. Moreover, we identified BTB domain amino acid residues critical for NuRD complex interaction, and showed that mutation of these Thpok residues disrupts CD4 T cell development in vivo. Reciprocally, a fusion protein reconstituting NuRD binding to a BTB-less version of Thpok (which cannot bind NuRD) restored CD4 T cells in vivo. In addition, we found that CD4-lineage thymocytes expressing that fusion protein have a transcriptome similar to that of thymocytes expressing wild type Thpok. Finally, we found that NuRD binding is required for Thpok to antagonize Runx3 expression in thymocytes. Thus, our results demonstrate that NuRD recruitment is both necessary and sufficient for the functions of the Thpok BTB domain in CD4 T cell development and it is also required to antagonize Runx3-dependent differentiation towards CD8 lineage.
Pathogenic effector TH2 cells discriminate between allergic and eosinophilic inflammation in food allergy and eosinophilic gastrointestinal disease
Authors: Makiya Michelle A , Frischmeyer-Guerrerio Pamela A , Khoury Paneez , Klion Amy D. , Kuang Fei Li
Published: February 2023

IgE-mediated food allergies (FA) and delayed food-triggered (FT) gastrointestinal tissue eosinophilia (EGID) have distinct clinical manifestations and are treated differently. Pathogenic effector TH2 (peTH2) cells are implicated in inappropriate TH2 responses in both of these diseases. Furthermore, EGID patients can have concomitant FA, and some EGID patients do not report any food triggers. To better understand the role of peTH2 cells in the context of FA and EGID and the overlap between these two conditions, peTH2 cells (CD3+CD4+CD27-CD49d+CRTH2+CD161+) from purified peripheral blood mononuclear cells (PBMC) were profiled by intracellular cytokine flow cytometry in these deeply-characterized patient cohorts: patients with FA alone (FA, n=5), EGID with FA and FT (EGID+FA+FT, n=7), EGID with FT (EGID+FT, n=7) and EGID without FA or FT (EGID, n=9). Patients with any food allergy or food trigger (FA, EGID+FA+FT, and EGID+FT) have increased peTH2 cells compared to healthy volunteers (HV). Interestingly, a greater percentage of peTH2 cells in patients with food triggers (EGID+FA+FT, EGID+FT) exhibit a trend toward IL5+IL13+ double positivity (P = 0.0556) than those from patients without food triggers (FA, EGID). In contrast, peTH2 cells from patients with FA had a greater percentage that were IL17A+ (P=0.009) or IFNγ+ (P=0.0444) than patients with eosinophilic disease (EGID+FA+FT, EGID+FT, EGID). Our studies indicate that while elevated levels of peTH2 cells are indicative of allergic inflammation of both immediate hypersensitivity and eosinophilic disease, the cytokine profile of each disorder may be distinct, where IL17A+ and IFNγ+ predominate in those without eosinophilic disease.
Plasma cells Dynamics in the Bone Marrow Niche
Authors: Jing Zhixin , Benet Zachary L , Fooksman David R
Published: February 2023

Plasma cells (PCs) can become long-lived with age to maintain prophylactic antibody titer for decades in the bone marrow (BM) microenvironment that are enriched in supporting chemokines and pro-survival cytokines. It has been that PCs are sessile in the BM to receive various pro-survival signals, but how they access these signals in the dynamic BM microenvironment remains unknown. Here by establishing long-term intravital imaging of the mouse tibial BM, we show that PCs are overall motile within the BM parenchyma with unique intermittent and heterogenous motility over time. Their motility is reduced when they are in clusters or as they enter a cluster. PC cluster formation and motility requires the pro-survival cytokine APRIL. PCs motility also requires the chemokine receptor CXCR4 and its ligand CXCL12 that are known important for PC homing to BM. CXCR4/CXCL12 signaling-triggered integrin VLA-4 activation negatively regulates PC motility in the BM. Notably, not only do PCs migrate within the BM parenchyma, but they can also egress and recirculate under steady state condition or CXCR4 and VLA4 inhibition. PC motility, clustering, and recirculation are all increased with mouse age. Thus, PC motility and cluster formation underlie a dynamic BM survival niche, contributing to PC longevity and function.
Plasmacytoid Dendritic Cells Regulate Th17 Activation in Diabetic Wound CD4+ T-cells
Authors: Audu Christopher O , Wolf-Fortune Sonya , Melvin William James , Davis Frank , Sharma Sriganesh B. , Mangum Kevin , Barrett Emily , Joshi Amrita , Obi Andrea T. , Moore Bethany B , Gallagher Katherine A.
Published: February 2023

Introduction: CD4+ T-cell activation is vital for normal wound repair but the factors that control T-cell activation in wounds in vivo are not clear. Our group and others have found increased Th17 activation in diabetic wounds resulting in increased IL-17a and pathologic inflammation that prevents tissue repair. Plasmacytoid dendritic cells (pDC) are antigen presenting cells that are present in early diabetic wound tissue and may play a key role in modulating CD4+ T-cell phenotype. Hence, we hypothesized that diabetic pDCs may influence wound CD4+ T-cells towards Th17 T-cell expansion.

Methods:

Wild type C57BL/6 mice were fed normal chow diet (13.5% kcal fat; LabDiet) or high fat diet chow (60% kcal fat; Research Diets) for 12–14 weeks to generate the diet-induced obesity (DIO) model of glucose intolerance/insulin resistance. These mice were subsequently wounded, and wound plasmacytoid dendritic cells harvested on day 1 and day 3. These cells were co-cultured with naïve CD4+ T-cells for 3 days, after which T-cell phenotype was determined by flow cytometry. Additionally, pDC in wounds 1-day post wounding were examined by quantitative PCR for cytokine production.

Results:

Following exposure to DIO pDCs, wild type activated CD4+ T-cells were activated towards a Th17 phenotype via significant increases in TGFb CD4+ T-cell activation and may act to increase inflammation in diabetic wounds.
BCG-Infected Dendritic Cells Induce TNF-alpha Dependent Cell Cluster Formation That Damage theBlood Brain Barrier
Authors: Gilpin Trey E , Walter Fruzsina , Herbath Melinda , Sandor Matyas , Fabry Zsuzsanna
Published: February 2023

Central nervous system tuberculosis (CNSTB) is the most serious manifestation of extrapulmonary tuberculosis infections representing 5–10% of all cases. How Mycobacterium tuberculosis (Mtb) crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. We applied a novel in vitro model of BBB model that consists of primary brain astrocytes and microvessel endothelial cells to test the mechanisms how mycobacterium-infected DCs interact with peripheral blood mononuclear cells (PBMC) and affect the BBB. We show that mycobacterium hinders DC migration, and infected DCs adhere to brain microvessel endothelial cells and induce PBMC cell cluster formations. Infected DC-induced cell clusters contain 24% CD11c+, 57%CD11b+, and 16% CD4+ cells. PBMC and infected DC clusters strongly adhered to the in vitro BBB and over time induced cluster associated damage (CAD). CAD correlated with mitochondrial disorganization and the degradation of tight junction proteins ZO-1 and claudin-5, and upregulation of cellular adhesion molecules VCAM-1 and ICAM-1 on endothelial cells. Anti-TNF-a inhibition reduced cell cluster formation as well as ICAM-1 expression on brain microvessel endothelial cells and mitigated CAD. This research leads to better knowledge in understanding how mycobacteria infected cells affect the BBB that could lead to better therapeutic treatments for mycobacterial infection of the CNS.
Astrocyte TAK1-mediated production of chemotactic cues are necessary for the localization of CD4+T cell in gray matter in an EAE model with severe neurodegeneration.
Authors: Khaw Yee Ming , Steelman Andrew J. , Inoue Makoto
Published: February 2023

Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system that is driven by peripheral immune cell infiltration and glial cell activation. The pathological hallmark of MS is primary demyelination, but recent evidence suggests gray matter neuronal damage is a major component of the irreversible nature of the disease. While T cells are found in both gray and white matter of MS tissue, they are typically confined to the white matter of the most commonly used mouse model of MS, experimental autoimmune encephalitis (EAE). Thus, genesis of T cells in the gray matter during MS pathogenesis is poorly understood. In this study, we used a modified EAE model (Type-B EAE) that displays severe neuronal damage without disease remission to investigate the role of T cells in neurodegeneration. Using immunohistochemistry and confocal imaging, we show that CD4+ T cells migrate to the spinal cord gray matter with selective preference for the ventral horns. Compared to CD4+ T cells in white matter, gray matter-infiltrated CD4+ T cells were mostly immobile (indicative of cell–cell interactions) and can be seen interacting directly with neuronal bodies. T cell-specific deletion of CXCR2 significantly decreased CD4+ T cell infiltration into gray matter in Type-B EAE mice. Inhibition of astrocyte chemokine production such as CXCL1, which was heightened in gray matter of Type-B EAE mice, by astrocyte-specific deletion of TAK1 successfully prevented T cell migration into spinal cord gray matter and rescued neuronal damage and motor dysfunction in Type-B EAE mice. This is the first report report that identifies the mechanism by which encephalomyelitic CD4+ T cells migrate to spinal cord gray matter, which correlates with neuronal damage.
Hypoxia inducible factor-1a drives metabolic adaptation of natural killer cells during pathogen infection
Authors: Victorino Francisco , Bigley Tarin , Park Eugene , Yao Conghui , Benoit Jeanne , Yang Li-Ping , Plougastel-Douglas Beatrice , Piersma Sytse , Lauron Elvin , Davidson Rebecca , Patti Gary , Yokoyama Wayne
Published: February 2023

Natural killer (NK) cells provide an early response to pathogen infection, limiting host morbidity and lethality. This response requires NK cells to adapt due to the energy demands of activation and biosynthesis. We found that NK cells upregulate the metabolic adaptor hypoxia inducible factor-1a (HIF-1a) during murine cytomegalovirus (MCMV) infection in vivo. Specific deletion of HIF-1a in NK cells impaired their ability to reduce viral load and protect against morbidity. While HIF-1a-deficient NK cells had no defects in effector functions, their numbers were significantly reduced during early responses and there was blunted expansion of Ly49H+ NK cells. However, NK cell proliferation per se was not impaired during in vivo infection and in vitro cytokine stimulation. Instead, we found that the absence of HIF-1a impaired glucose metabolism and up-regulated the pro-apoptotic protein Bim during cytokine stimulation in vitro. Furthermore, during MCMV infection in vivo HIF1a-deficient NK cells upregulated Bim and had increased caspase activity. Thus, HIF-1a normally drives glucose metabolism and represses Bim expression during pathogen infection to sustain NK cell numbers for an optimal response.
Cell death effectors are recruited to Toxoplasma gondii parasite vacuoles targeted by interferon-inducible GTPases in infected dendritic cells
Authors: Zhao Xiaoyu , Yin Bocheng , Ewald Sarah E.
Published: February 2023

Toxoplasma gondii is an obligate intracellular pathogen whose ability to grow and evade cell autonomous immunity depends on the parasitophorous vacuole membrane (PVM). The PVM forms from the host plasma membrane during invasion and is maintained by parasite effectors. Interferon-inducible GTPases (IIGs) are central in parasite clearance, however, the precise mechanism linking IIG attack of the PVM, parasite killing and inflammatory host cell death is not clear. To identify host and parasite proteins recruited to the PVM, we used Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP), a novel discovery technique that uses a confocal microscope to visualize and photo-label proteins for identification by LC-MS. Using IFNγ to induce IIG attack of the PVM and the TLR2 ligand Pam3CSK4 to upregulate inflammasome components we examined protein recruitment to the PVM in BMDCs. AutoSTOMP identified over 300 proteins differentially enriched near the PVM of unprimed, TLR2, IFNγ and IFNγ+TLR2 treated BMDCs. To identify candidate regulators of parasite clearance, we selectively purified proteins on the PVMs coated with IIG protein GBP2. We identified the inflammasome components caspase-1 and ASC as well as the apoptotic caspases-3 and -6 on GBP2-coated PVM. This was consistent with the observation that IFNγ priming was sufficient for host cell death and parasite restriction. BMDC death was partially dependent on the Aim2, ASC, Caspase1/11 and gasdermin D, however, blocking the inflammasome didn’t impair parasite restriction. By contrast, iNOS inhibitor, was sufficient to partially rescue parasite growth. In summary, we report a new tool to study the PVM proteome and identify regulators of parasite clearance and host cell death.
Mechanistic investigations of Granzyme A-mediated TB protective effects
Authors: Rasi Valerio , Xia Mei , Wood David , Eickhoff Christopher , Hoft Daniel F
Published: February 2023

One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb), the pathogen that causes Tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. To elucidate the mechanism(s) involved in GzmA-mediated mycobacterial pathogen inhibition using a relevant in vitro human system. This GzmA was utilized to treat mycobacteria-infected primary monocytes from multiple volunteers, and their cell lysates were analyzed using 2D-DIGE global proteomics. A catalytically inactive GzmA variant was produced to study the enzymatic role in the Mycobacterial Growth Inhibition Assay (MGIA). In parallel, confocal microscopy experiments looked at GzmA internalization. We have evidence that Granzyme A’s intact active site is not necessary to inhibit the intracellular replication of mycobacteria as the S195A variant inhibits mycobacterial infection in MGIA as Wild Type (WT) GzmA. The proteomic approach identified two pathways: ER-stress response and purinergic channel receptor activation are important for GzmA-mediated intracellular mycobacterial inhibition. These results were validated in separate volunteers by western blot. Confocal microscopy experiments show that GzmA is internalized within the infected cell and clears the infection. Modulation of key proteins in the discovered pathways are being targeted to confirm our proteomic results and to test potential novel therapies that emulate GzmA. It will also be investigated if GzmA co-localizes in these cellular compartments. These studies will lead to a better understanding of GzmA biology during infection and to the development of novel host-directed therapeutics for control of Mtb infection.
Combination of dmLT and MPL-A adjuvants synergistically and differentially activates the canonical inflammasome in DCs and macrophages in the absence of pyroptosis
Authors: Bauer David Laurence , Immethun Vicki E , McLachlan James B
Published: February 2023

The initiation of innate immune responses is critical for the formation of a protective adaptive immune response following vaccination. Innate immune cells, such as dendritic cells (DCs) and macrophages, phagocytose and display antigen, produce immune modulators and migrate to lymphoid tissues, leading to the activation of naïve T cells. These professional antigen-presenting cells are activated in response to pathogen or danger associated molecular patterns, a process that is normally not robust with vaccine antigen alone. Adjuvants are able to increase the immunogenicity of vaccine antigens by influencing the activation status of these innate immune cells. Only a few vaccine adjuvants are currently approved for use in the US and, along with the discovery of novel adjuvants, combination adjuvants are likely to have a major impact on future vaccine development. Here, we investigated the ability of both the double mutant Escherichia coli heat labile toxin (dmLT) and the TLR4 agonist monophosphoryl lipid A (MPL-A) to drive innate and adaptative immune responses independently and in combination. We found that the combination adjuvant induces more robust activation of DCs and macrophages than either adjuvant alone, leading to the formation of the NLRP3 inflammasome complex and GSDMD independent secretion of IL-1b in the absence of pyroptosis. Furthermore, RNA sequencing and ingenuity pathway analysis support that the combination adjuvant may allow these DCs to polarize a multi-faceted Th1/2/17 CD4 T cell response. Because macrophages and DCs have distinct roles in the interface between innate and adaptive immunity, understanding the cellular and molecular actions of adjuvants on these cells is critical when developing new vaccines.
Expression and functional characterization of protease-activated receptor 2 (PAR2) and PAR1 on human skin mast cells
Authors: Fukuoka Yoshihiro , Sanchez-Munoz Laura B , Dellinger Anthony L , Schwartz Lawrence B
Published: February 2023

Mast cells play an important role in the pathophysiology of asthma and allergic disease by releasing proinflammatory mediators and cytokines. Activation of protease-activated receptors (PARs) may also play a role in inflammatory response. We investigated the expression of PAR1, 2, 3 and 4 as well as the function of PAR1 and 2 in cultured human skin-derived mast cells (SMCs). Using a combination of flow cytometric analysis, reverse transcriptase-polymerase chain reaction and Western blotting, we demonstrate that human SMCs express PAR1, 2, 3 and 4. Confocal microscopy indicates PAR2 partially colocalize with tryptase within SMCs. Exposure to PAR2 agonist, trypsin and activating peptide (AP) 2-Furoyl-LIGRLO-NH2 induce Ca2+ mobilization in SMCs. The PAR1 APs, SFLLRN-NH2 and TFLLR-NH2 cause degranulation and induce GM-CSF and IL-8 synthesis. Similarly, the PAR2 APs, SLIGKV-NH2, 2-Furoyl-LIGRLO-NH2 also cause degranulation and induce GM-CSF and IL-8 synthesis in SMCs. Both PAR1 APs and PAR2 APs induce rapid phosphorylation of ERK and pertussis toxin (PTX) robustly inhibits phosphorylation of ERK by both PAR1 APs and PAR2 APs. These studies indicate that human SMCs can be activated by both PAR1 and PAR2 which may contribute to MC-mediated inflammatory process.
MicroRNA-155 regulates MAIT cell development, effector subsets differentiation and function
Authors: Liu Tingting , Wang Jie , Subedi Kalpana , Yi Qijun , Mi Qing-Sheng , Zhou Li
Published: February 2023

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that develop in the thymus through sequential maturation stages and differentiate into MAIT1 (T-bet+) and MAIT17 (RORγt+) subsets. MAIT cells have emerged in recent years as crucial players in the effective mucosal immunity and the association of a broad spectrum of disease development. However, detailed molecular mechanisms in regulating MAIT cell development and function remain unclear. Recent studies indicate the involvement of microRNAs in MAIT cell regulation, but few individual miRNAs have been identified, that play the role. To investigate the role of microRNA-155 (miR155) in the development of MAIT cells, we used both gain- and loss-of-function strategies. Using thymus-specific miR-155 knock-in mouse model, it was identified that overexpression of miR-155 significantly reduced numbers and frequencies of MAIT cells in thymus and all peripheral lymphoid and mucosal tissues. Furthermore, MAIT cells with miR-155 overexpression showed interrupted maturation with down regulated PLZF expression and reversed CD8+/CD4−CD8− MAIT subsets distribution. Strikingly, miR-155 overexpression perturbed MAIT17 cell differentiation, and transiently induced miR-155 overexpression inhibited peripheral MAIT cells IL-17 production. Using conventional miR-155 deletion mice model, we found that germline miR-155 deletion did not significantly interrupting MAIT cell development and peripheral tissue MAIT cell differentiation and function. Overall, our results indicate that adequate miR-155 expression is required for MAIT cell development, effector subsets differentiation and function.
Microglial autophagy-mediated clearance of amyloid-beta plaques is dysfunctional in Alzheimer’s Disease mice
Authors: Daily Kylene P , Amer Amal O
Published: February 2023

Alzheimer’s Disease (AD) is a neurodegenerative disorder with no preventative methods or cure. Deposition of aggregated amyloid-beta (Aβ) in the brain is hypothesized to initiate a vicious cascade, leading to inflammation and tissue damage. Microglia are brain-resident immune cells which help maintain homeostasis and clear protein aggregates, including Aβ. However, in AD, microglia produce inflammatory mediators and do not adequately clear Aβ. This project seeks to determine how healthy microglia clear Aβ from the brain and why diseased AD microglia do not. Autophagy is a homeostatic process providing the cell with energy during stress. Autophagy can act as a defense mechanism, by clearing foreign particles and defective proteins. Previous research on AD established that autophagy is dysfunctional in neurons, but autophagy is not well-characterized in AD microglia. Functional autophagy requires expression of multiple autophagy proteins. We hypothesized that AD microglia fail to clear Aβ due to reduced expression of autophagy proteins and that restoring expression of these proteins will enhance autophagy activity. Utilizing microglia from adult AD model mice and various autophagy mice, we demonstrated that AD microglia do not degrade Aβ due to reduced expression of autophagy proteins, which are necessary for Aβ degradation. We identified an upregulated cluster of microRNA in microglia from mouse and human AD brain tissue samples. Inhibiting these microRNA improves expression of specific autophagy proteins and restores Aβ degradation in AD mouse microglia. Our findings indicate that autophagy is dysfunctional in AD microglia and establish a mechanism behind dysfunctional autophagy which may be therapeutically targeted.
Butyrate mediates the amelioration of Staphylococcal Enterotoxin B-induced Acute Respiratory Distress Syndrome by downregulating the expression of Histone Deacetylases
Authors: Sultan Muthanna Ali , Nagarkatti Prakash , Nagarkatti Mitzi
Published: February 2023

Despite rigorous global containment and quarantine efforts that have been applied globally to control SARS-CoV-2, the spreading of this contagious disease has accelerated all over the world. In our study, we used staphylococcal enterotoxin B (SEB), which is a superantigen can lead to toxic shock syndrome in C57BL/6 murine model and thus, mimics COVID19. In the current study, we investigated the suppressive effect of sodium butyrate (BUT) on a murine model of SEB-induced Acute Respiratory Distress Syndrome (ARDS). The data shows that supplementation with BUT at a dose of 200 mg/kg orally reduced overall ARDS severity. Plethysmography analysis data indicated that sodium butyrate improves the clinical functions of the lungs. In addition, our flow cytometry data indicated that BUT mediated the suppression of lung mononuclear cells such as CD4+ T cells, CD8+ T cells, VB8+ T cells and NK-T cells while at the same time increasing T regulatory cells. Cytokine storm is one of the most important challenge in COVID19 cases, in our study, we have found that the proinflammatory cytokines such as IL-2, TNFα and IFNγ in broncho-alveolar lavage fluid (BALF) significantly decreased in SEB+BUT compared to SEB+VEH as detected by ELISA. Additionally, histopathological analysis of H & E stained lungs indicated that BUT decreased the level of infiltration of inflammatory cells in the lungs of SEB+BUT compared to SEB+VEH mice. Additionally, we investigated the role of BUT on the expression of histone deacetylases (HDACs). Collectively, our data suggested that BUT ameliorated SEB-induced ARDS by downregulating the expression of class I and Class II HDACs which mediate the increase in T regulatory cells that suppress the inflammatory response in SEB-induced ARDS.
ITK regulates IL-10 production by CD8+ T cells and lung immunopathology during influenza infection
Authors: McGee Michael C , Solouki Sarbina , Limper Candice Bree , Ye Kaixiong , Nidetz Natalie , Islam Rezwanul , Zhang Tianyi , Magazine Nick , Huang Weishan , August Avery
Published: February 2023

Respiratory viral infections are a major cause of human morbidity and mortality. Influenza (flu) infections cause 250,000 deaths and 3–5 million cases of severe illness during the average flu season. Severe flu infections are associated with a combination of strong pro-inflammatory and weak anti-inflammatory immune responses. Production of the immunomodulatory cytokine IL-10 by T cells restricts immunopathology during flu infections, however our knowledge of the signaling pathways regulating IL-10 induction during flu is limited. Using IL-10GFP reporter mouse models, we found that Interleukin-2 inducible T cell kinase (ITK), a critical component in T cell receptor (TCR) signaling, regulates immunopathology and the development of IL-10-producing CD8+ T cells in the airways during influenza A infection. Utilizing alternative coculture, the model antigen ovalbumin (OVA), and transgenic TCR specific for OVA in CD8+ T cells (OTI) adoptive transfer, we determined that ITK regulates flu antigen-specific IL-10+CD8+ T cell differentiation in a CD8+ T cell intrinsic and extrinsic manner. Exogenous IL-2 rescued IL-10 production by Itk−/− CD8+ T cells, in vitro and in vivo, suggesting that ITK may play a role in IL-2-producing T helper cells that promote IL-10 production in CD8+ T cells. The absence of ITK impaired the expression of transcription factors (TFs) IRF4 and Blimp-1, and retroviral transduction enabling the reintroduction of these TFs could partially rescue IL-10 production in Itk−/− CD8+ T cells. Our data suggests that ITK is a critical regulator of IL-10 production by CD8+ T cells and flu-induced pulmonary immunopathology. Modulating ITK signaling may be a strategy for regulating immunopathology due to viral infections.
A role for the transcription factor STAT5 in antagonizing CD8+ T cell exhaustion
Authors: Beltra Jean-Christophe , Manne Sasikanth , Abdel-Hakeem Mohamed S , Nzingha Kito , Zhang Zhen , Huang Hua , Kurachi Makoto , Muroyama Yuki , Huang Yinghui Jane , Su Leon L , Picton Lora , Decaluwe Helene , Huang Alexander C , Berger Shelley L , Garcia Christopher K , Wherry E. John
Published: February 2023

Exhaustion gradually establishes in chronically stimulated CD8s and this process is not reverted by current therapeutic approaches due to establishment of a stable epigenetic program. Recent advances have informed on the developmental process of exhaustion and highlighted TOX as a key lineage-defining TF in the process. Yet, little remains known on molecular pathways capable of antagonizing the TOX-dependent exhaustion program. By depicting transcriptional changes at key developmental steps of exhaustion, we demonstrate an antagonistic role for the TF STAT5 in the development of CD8 T cell exhaustion. STAT5 transcriptional network is heavily silenced upon chronic antigenic stimulation in a TOX-dependent manner which allows initiation of the exhaustion lineage. Increasing STAT5 activity abrogates establishment of the exhaustion lineage leading to the development of effector-like CD8s that acquire a unique transcriptional identity, distinct from exhausted cells, persist throughout chronicity and demonstrate higher protective capacity. Using temporal loss and gain of function approaches, we show that STAT5 triggers loss of progenitor identity by exhausted CD8s (Tex) and subsequent differentiation into the recently identified effector-like intermediate Tex subset. Temporal increase in STAT5 activity also robustly synergizes with PD-L1 blockade by further fostering intermediate Tex cells accumulation. Together, we show that modulating STAT5 activity may counteract the exhaustion process and favor instigation of effector-like characteristic in Tex cells suitable for optimal therapeutic efficacy.

This work is supported by the Parker Institute for Cancer Immunotherapy (PICI). JC-Beltra is a PICI scholar awardee.
An AGS associated mutation causes cellular RNA editing deficiency in mouse brain which lead to IFN pathway activation, mimicking the interferonopathy of patient brains
Authors: Wang Qingde , Guo Xinfeng , Sheng Yi , Wang Tony , Steinman Richard , Billiar Timothy R
Published: February 2023

Objective:

To determine whether and how a gene mutation found in Aicardi-Goutières syndrome (AGS), a severe autoimmune encephalopathy, is sufficient to activate the interferon signaling pathway in the brain.

Background:

Genetic mutations in six coding genes were found associated with AGS patients, however, none of them was demonstrated able to cause IFN pathway activation in the brain tissue, the most critical pathogenetic change of AGS. How an AGS-associated mutation activates the IFN pathway in the brain that leads to brain injury remains to be revealed.

Methods:

A knock-In mouse model was established through CRISPR/Cas9 gene editing approach, in which a single G>T nucleotide replacement was introduced into mouse genome, that encodes the change for p.K948N in mouse ADAR1 at its catalytic domain, equivalent to the p.K999N mutation found in AGS patients. We analyzed the enzyme activity of ADAR1 on the neuron specific mRNA substrates in the KI mice through RNA sequencing, the activities of IFN signaling pathway by measuring expressions of IFN stimulated inflammatory cytokines. Brain injury was observed by pathology studies.

Results:

The genetic mutation was confirmed. RNA transcription and protein translation were tested not affected by the mutation. The cellular RNA process in neurons was altered due to decreased enzyme activity of mutant ADAR1 on certain mRNA substrates. ISG expression were significantly increased at RNA and protein levels. Cell death in the brain was observed in the KI mice.

Conclusion:

A mouse model was successfully established recapitulating the genetic and innate immunologic features of AGS. An ADAR1 gene mutation is sufficient to cause IFN pathway activation in the brain through altering cellular RNA process.
USP13 enhances the function of p62/SQSTM1 by increasing its stability and aggregate formation
Authors: Lee Bin , Kim Young Hun , Lee Woori , Kwak Man Sup , Shin Jeon Soo
Published: February 2023

p62/SQSTM1 is a multifunctional protein that serves as a receptor protein for selective autophagy and a scaffold protein. In selective autophagy, p62 functions as a bridge between poly-ubiquitinated proteins and the autophagosome. In addition, p62 acts as a signaling hub for many cellular pathways, including mTORC1, NF-κB, and Keap1-Nrf2. These functions are regulated by post-translational modification of p62. In particular, changes in the binding partner or intracellular function of p62 by phosphorylation or ubiquitination are well studied. However, the opposite process of the p62 ubiquitination, the deubiquitination mechanism is still unclear. It was hypothesized that the unknown pathway of p62 deubiquitination might regulate its stability and functions. In this study, it was found that ubiquitin-specific protease 13 (USP13), one of the USP family, directly bound to p62 and removed ubiquitination at its PB1 domain. The deubiquitinated p62 by USP13 was enhanced the stability and the aggregate formation. Furthermore, increased p62 aggregation by USP13 activated autophagy and inhibited Keap1 from functioning as a negative regulator of Nrf2. Consequently, activated Nrf2 increased its migration to nucleus and acted as a transcription factor, increasing the expression of the antioxidant response gene. Taken together, USP13 enhanced the stability and aggregate formation of p62, which activated autophagy and antioxidant response.
A dysbiotic gut microbiome suppresses antibody-mediated protection against Vibrio cholerae
Authors: Macbeth John , Liu Rui , Alavi Salma , Hsiao Ansel
Published: February 2023

Vibrio cholerae is the etiologic agent of cholera, a severe diarrheal disease that represents a significant burden on global health and productivity. Given the need for more effective prophylactics, more research is needed to ascertain the interplay of protective immune responses and the resident gut microbiome, which in cholera-endemic areas is strongly and repeatedly modulated by malnutrition, cholera, and non-cholera infectious diarrhea. Initially, we conducted fecal transplants from healthy human donors into germ-free mice in order to characterize differential immune response activity. As we observed distinctive responses in our initial sampling, we moved to a more tractable antibiotic-treated mouse model where we assemble representative model communities of either human gut microbes resembling those of healthy individuals or those of individuals recovering from diarrhea or malnutrition. We establish these communities in a murine infection model, and show that the dysbiotic gut microbiome, commonly present in areas where malnutrition and diarrhea are common, suppresses the immune response against Vibrio cholerae through the action of CD4+ cells. Our findings suggest that the composition of the gut microbiome at time of infection may be pivotal for providing robust mucosal immunity, suggesting a target for the improvement of responses to oral cholera vaccines.
Plasmodium falciparum-specific B cell responses in malaria-susceptible children and malaria-immune adults
Authors: Gonzales Stephen Jacob , Reyes Raphael , Batugedara Gayani , Ssewanyana Isaac , Rek John , Lavinder Jason J , Ippolito Gregory C , Bol Sebastiaan , Greenhouse Bryan , Bunnik Evelien
Published: February 2023

Malaria is a vector-borne disease caused by parasites of the Plasmodium genus that infect millions of people annually, with P. falciparum being the most common and deadliest species. Development of immunological protection against clinical malaria requires chronic or repetitive parasite exposure, is mediated by IgG, and is correlated with antibodies against proteins expressed by merozoites, the free form of the parasite that invades erythrocytes. The goal of our study was to better understand the development of this protective antibody response, using merozoite surface protein 1 (MSP1) as a model antigen. MSP1 is highly abundant on the merozoite surface, highly immunogenic, and highly variable between parasite strains. Using B cell tetramers, we isolated MSP1-specific IgM+ and IgG+ memory B cells (MBCs) from immune adults and non-immune children living in Tororo, Uganda, a region of high transmission. In children, MSP1-specific B cells were mainly unswitched (IgM+) classical MBCs (cMBCs; CD21+ CD27+), while adults showed enrichment for class-switched (IgG+) cMBCs. Sequence analysis of the heavy chain variable regions revealed that MSP1-specific MBCs from adults carried higher rates of somatic hypermutation and showed extensive clonal expansion compared to those from children. When expressed as IgG, antibodies from IgM+ MBCs showed low avidity to MSP1, while those from IgG+ MBCs displayed strong reactivity with recombinant MSP1 and whole merozoites and inhibited parasite growth. These results suggest that extensive evolution of B cell responses takes place as a result of life-long P. falciparum exposure, which may be essential for the development of protection against malaria.
A transmissible γδ intraepithelial lymphocyte hyperproliferative phenotype is associated with the intestinal microbiota
Authors: Jia Luo , Alonso Sara , Wu Guojun , Lam Yan , Zhao Liping , Edelblum Karen L
Published: February 2023

Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor provide a first line of defense at the intestinal barrier, yet how the intestinal microbiota influences this sentinel population remains unclear. Since commensal-induced tonic type I interferon (IFN) signaling promotes lamina propria lymphocyte homeostasis, we hypothesized that IFNα/β receptor (IFNAR) signaling may contribute to maintenance of the γδ IEL compartment. Morphometric analysis revealed a 2-fold increase in the number of GFP+ γδ IELs in IFNAR KO mice compared to WT housed in a standard barrier facility (SBF). γδ IEL proliferation was enhanced 50% in SBF IFNAR KO mice relative to SBF WT. This increase in γδ IEL number was not observed in IFNAR KO mice rederived into a cleaner, enhanced barrier facility (EBF) indicating that the γδ IEL hyperproliferative (γδHYP) phenotype occurs independently of IFNAR signaling. The transfer of dirty bedding from SBF IFNAR KO mice to EBF IFNAR KO breeding cages was sufficient to induce the γδHYP phenotype in both the breeders and their offspring, and antibiotic treatment prevented this transmission. SBF WT and IFNAR KO mice were crossed to generate F2 littermates, which all exhibited the γδHYP phenotype regardless of genotype. Evidence of horizontal and vertical transmission of the γδHYP phenotype led us to analyze the fecal microbiota. Greater microbial diversity was observed in mice with the γδHYP phenotype relative to those that did not (Shannon Index, p<0.01). Moreover, 6 amplicon sequence variants (ASV) were strongly associated with the γδHYP phenotype (P=1.075e-05). Thus, we have serendipitously discovered a specific microbiota that is both necessary and sufficient to promote the expansion of the γδ IEL compartment.