bluuebunny/crossref_metadata_2025 huggingface dataset:
A new mechanism of Treg cell dysfunction during HIV infection and propensity to oral cancer in people living with HIV
Authors: Pandiyan Pushpa
Published: January 2023

Mechanisms of immune-dysfunction associated with chronic viral infections remain nebulous. Residual systemic inflammation and mucosal immune dysfunction persist in people living with HIV despite anti-retroviral therapy, but the underlying immune processes are poorly understood. Here we show an altered immune landscape of the oral mucosa of HIV-positive patients on therapy involves increased TLR− and inflammasome signaling, localized CD4+ T cell hyperactivation, and counterintuitively, enrichment of regulatory T cells. HIV infection of oral tonsil cultures in vitro causes an increase in FOXP3+ cells expressing PD-1, IFN-γ, Amphiregulin and IL-10. These cells persist even in the presence of anti-retroviral drugs, and further expand when stimulated by TLR-2 ligands and IL-1β. Mechanistically, IL-1β upregulates PD-1 expression via AKT signaling and PD-1 stabilizes FOXP3 and Amphiregulin through a mechanism involving Asparaginyl Endopeptidase, resulting in FOXP3+ cells that are incapable of suppressing CD4+ T cells in vitro. Phenotypically similar cells are abundant in HIV-positive patients, and their presence strongly correlates with CD4+ T cell hyper-activation, suggesting diminished CD4+ T cell regulation in the oral mucosa in vivo. Taken together, this study provides unprecedented insights into the mechanism of FOXP3+ cell dysregulation and its potential role in mucosal dysfunction and cancer susceptibility in HIV patients.

Supported by NIH NIDCR R01 DE026923
Lipin-1 restrains lipid synthesis to promote proresolving macrophage function and disease resolution
Authors: Bamgbose Temitayo Taiwo , Schilke Robert M , Blackburn Cassidy M R , Woolard Matthew D
Published: January 2023

Macrophages are critical to maintaining and restoring tissue homeostasis during inflammation. Unresolved inflammation contributes to the pathophysiology of cardiometabolic diseases. The lipid metabolic state of macrophages influences their function. Lipid synthesis contributes to proinflammatory responses, while beta-oxidation is required for pro-resolving macrophage function. However, how lipid metabolism is regulated during macrophage activation is not well understood. Lipin-1 is a phosphatidic acid phosphatase with a transcriptional coregulatory activity that is proposed to act as a regulator of lipid metabolism. We have previously demonstrated that lipin-1 is atheroprotective and promotes wound healing. Within macrophages, lipin-1 is required for beta-oxidation and apoptotic cell engulfment, both key activities of pro-resolving macrophages. We investigated the contribution of lipin-1 in regulating lipid metabolism during pro-resolving macrophage responses using metabolomics, lipidomics, and Western blot analysis. IL-4 stimulation of macrophage promotes lipid catabolism; however, in lipin-1 KO bone marrow-derived macrophages, we observed the production of metabolites that contribute to lipid synthesis, decrease phosphorylation of ACC (a marker of lipid synthesis) and a build-up of free fatty acids. We also observed increased ceramides in lipin-1 KO macrophages and a decrease in pras40 phosphorylation, which is consistent with increased ceramide synthesis. These results suggest that lipin-1 restrains de novo lipid synthesis and ceramide production to promote beta-oxidation for optimal pro-resolving macrophage function.

Supported by1-R01HL131844-04 grant from NHLBI/NIH
DNA-PKcs is required for the activation and cytotoxicity of CD8+ effector T cells
Authors: Burdine Marie , Azevedo-Pouly Ana , Appell Lauren , Barker Melanie , Waldrip Zachary , Burdine Lyle
Published: January 2023

Antigen-mediated activation of naïve CD8+ T cells and their differentiation into cytotoxic cells is regulated by a series of signaling mechanisms designed to maintain T cell homeostasis and proper immune responses. Given that cytotoxic CD8+ T cells are critical to the elimination of cancerous or virally infected cells as well as mediators of autoimmune diseases, GVHD and allogenic organ rejection, it is critical that we continue to dissect these mechanisms to identify promising targets for T cell modulation in the clinic. Previously, we have shown that inhibition of the kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) reduces activity of the transcription factors NFAT, NFκB and EGR1 and production of cytokines including IL2, IL6 and IFNγ in TCR-activated T cells. Using pharmacological inhibitor and genetic approaches, we provide evidence for a novel role of DNA-PKcs in CD8+ T cell activation. Inhibition of DNA-PKcs abrogated anti-CD3/CD28 activation of CD8+ T cells as evident by reduced expression of the activation markers CD69 and CD25. Additionally, loss of DNA-PKcs activity inhibited aerobic glycolysis and proliferation of OTI-CD8+ T cells following SIINFEKL peptide stimulation. This caused reduced OTI-CD8+ T cell killing of MC38.OVA tumor cells and expression of IFNγ and the cytotoxic genes perforin and granzyme B. These results were recapitulated in DNA-PKcs-deficient CD8+ T cells isolated from an inducible Cre/DNA-PKcsfl/fl mouse model where loss of DNA-PKcs reduced expression of CD69 and CD25, IFNγ production and altered cellular metabolism. The identification of a function for DNA-PKcs in CD8+ T cell activation highlights the therapeutic potential of DNA-PKcs inhibitors on immune-related diseases.

Supported by Center for Pediatric Translational Research NIH COBRE P20GM121293
Inhibition of distinct glycolytic enzymes produces differential effects on CD4 T cell function
Authors: Godfrey Wesley H , Kornberg Michael
Published: January 2023

Background

When T cells are activated, they upregulate glycolysis and take on a requisite Warburg phenotype. In this study, we evaluated the effect of inhibiting two distinct glycolytic enzymes, GAPDH and PGAM, on CD4 T cell differentiation and effector activities in order to gain a mechanistic understanding of the relationship between glycolysis and T cell function.

Methods

To evaluate the effect of GAPDH inhibition on T cell function, we cultured naïve murine CD4+ T cells under Th1 or Treg polarizing conditions and treated with either the GAPDH inhibitor heptelidic acid or the PGAM inhibitor EGCG. We then assessed either IFNγ production or identified CD25+Foxp3+ cells in response to either Th1 or Treg polarization, respectively. To evaluate the impact of GAPDH inhibition in vivo, we used the MOG35–55 experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS) and compared neurological function in mice treated with vehicle or heptelidic acid.

Results

We found that GAPDH inhibition had a potent anti-inflammatory effect on T cells in vitro, drastically reducing IFNγ production at both the transcriptional and protein levels. Interestingly, PGAM inhibition had the opposite effect, potently blocking Treg differentiation. In the EAE mouse model of MS, heptelidic acid attenuated the disease course, indicating that GAPDH inhibition is anti-inflammatory in vivo.

Discussion

GAPDH inhibition was anti-inflammatory in vitro and in vivo, while PGAM inhibition had the opposite effect. These findings suggest that the functional consequences of glycolysis inhibition depend on the specific enzymes targeted, which has both mechanistic and therapeutic implications for the role of glycolysis in immune cell function.
Intestinal and tracheal microbiomes inhibit in vitro growth of Coccidioides immitis
Authors: Tejeda-Garibay Susana , Diep Anh , Hoyer Katrina K
Published: January 2023

Valley fever is a disease caused by Coccidioides, a fungal pathogen, and is frequently misdiagnosed as community acquired bacterial pneumonia and treated with several rounds of antibiotics prior to accurate diagnosis. Antibiotic treatment changes the microbiome repertoire and host immunity; the impact of these microenvironmental changes on Coccidioides invasiveness is unknown. A soil microbial antagonist related to Bacillus subtilis inhibits in vitro growth of Coccidioides immitis through a clear zone of inhibition between fungi and bacteria. Whether host microbiota also could inhibit Coccidioides is unknown. To assess host microbiota inhibition capabilities against Coccidioides, we performed two types of inhibition assays. A 50/50 inhibition assay was performed in which the host microbiota and Coccidioides were placed in direct competition simultaneously on agar plates. A spike in inhibition assay was also performed in which the host microbiota was allowed to reach ~80% confluency, to mimic an established in vivo microbiome, before spiking in Coccidioides. The area of growth was observed and measured at day 4, 7, and 11. Our in vitro data indicate that specific intestinal and tracheal host microbiome species inhibit Coccidioides growth. To assess if partial in vitro depletion of host microbiota allows greater Coccidioides growth, antibiotic disc diffusion assays were performed. Our data suggests that depleting commensal microbiomes, allows a niche for Coccidioides growth. These in vitro findings could have clinical relevance and shape the way physicians assess prescription of antibiotics and coccidioidomycosis diagnosis. Experiments to identify the inhibitory microbiota and proteomic interactions are ongoing.
Arresting microbiome development limits immune system maturation and resistance to infection
Authors: Silverman Michael A , Lubin Jean-Bernard , Denu Lidiya , Green Jamal , Duranova Tereza , Lanza Mathew , Wynosky-Dolfi Meghan , Brodsky Igor E , Planet Paul
Published: January 2023

The transition from milk to solid food is a critical period in post-natal human development. Weaning coincides with a dramatic and rapid maturation of both the microbiome and immune system. Whether these weaning induced changes in commensal communities shape immune system development and susceptibility to enteric infection remains unknown. Here we find maturation of the microbiome during weaning is required for proper immune system development and protection against enteric infection. To this end, we developed a stable gnotobiotic mouse model of the early-life microbiome composed of a representative nine-member consortium of phylogenetically diverse microbes found pre-weaning, designated as Pediatric Community (PedsCom). PedsCom efficiently colonized germfree adult mice and was vertically transmitted to offspring. Unexpectedly, the composition of PedsCom was unaffected by the transition from a milk-based to a fiber rich solid food diet. This permitted us to study adult mice with a pre-weaning intestinal microbiome. We find that arresting intestinal microbiome maturation in PedsCom mice limited immune system development. PedsCom mice displayed decreased both peripheral regulatory T cells accumulation and Immunoglobulin A production, hallmarks of microbiota-driven immune development. Consistent with defects in maturation, adult PedsCom mice retain high susceptibility to Salmonella infection characteristic of young mice and humans. Altogether, our work illustrates how the post-weaning transition in intestinal microbiome composition is essential for normal immune maturation, and protection from enteric infection.

Supported by 1R21AI146629-01A1
CSF-1 maintains pathogenic but not homeostatic myeloid cells in the central nervous system during autoimmune neuroinflammation.
Authors: Hwang Daniel , Seyedsadr Maryam S , Ishikawa Larissa Lumi Watanabe , Boehm Alexandra , Sahin Ziver , Casella Giacomo , Jang Soohwa , Gonzalez Michael V , Garifallo James P , Hakonarson Hakon , Zhang Weifeng , Xiao Dan , Rostami Abdolmohamad , Zhang Guang-Xian , Ciric Bogoljub
Published: January 2023

The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bio-activities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is upregulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing antibody halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1. Both anti-CSF-1 and BLZ945 treatment greatly reduced the number of monocyte-derived cells and microglia in the CNS. However, anti-CSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. Anti-CSF-1 treatment reduced the size of demyelinated lesions and microglial activation in the grey matter. Lastly, we found that bone marrow-derived immune cells were the major mediators of CSF-1R-dependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with ablation of CSF-1R-dependent cells.

National Multiple Sclerosis Society (RG-1803-30491) National Institutes of Health T32 training grant (T32AI134646, NIAID)
alpha-synuclein peptides presented on chimeric MHC class Ib molecules prevent loss of substantia nigra neurons in an animal model for Parkinson’s disease
Authors: Wischhusen Jörg , Wu Jingjing , Ahsan Fadhil , Rhonda McFleder , Karl Ann-Kathrin , Jayaram Shriya Mamatha , Wecklein Heike , Nienaber Anna , Brünnert Daniela , Bruttel Valentin , Ip Chi Wang
Published: January 2023

Aim

An accumulating body of evidence suggests the involvement of alpha-synuclein (aSyn) specific autoreactive T cells in Parkinson’s disease (PD). Here, we investigate whether novel antigen-specific tolerance-inducing biomolecules, that present peptide antigens on MHC class Ib-related molecules, can inhibit neurodegeneration and prevent PD in vivo.

Methods

The alpha3 domain of the human MHC class Ib molecule HLA-G inhibits T cells via the human ILT-2 or the murine PIRB receptor. We fused this HLA-G alpha3 domain to MHC class I alpha1-alpha2 antigen presenting domains, antigenic peptides and beta2-microglobulin. These single-chain AutoImmunity Modifying Biologicals (AIM Bios) induce antigen-specific tolerogenic CD8+ Treg cells in human cells in vitro and in mice in vivo. We used this platform to prevent autoimmune disease symptoms in mouse models for autoimmune diseases, including an aSynA53T-AAV driven model for PD.

Results

Single-chain model AIM Bios with MHC-derived antigen-presenting domains and antigenic peptides polarize cognate CD8+ T cells towards a CD8+ CD122+ IL-10 secreting Treg phenotype. Such antigen-specific Treg can also be induced in mice in vivo. Disease-associated aSyn CD8+ epitopes were identified in aSynA53T-AAV PD mice. Corresponding AIM Bios prevented mobility impairments. Post mortem histopathological assessment confirmed the induction a favorable in-situ immune cell composition and the rescue of substantia nigra neurons. The translational potential of this approach deserves further exploration.

Supported by grants from the IZKF Wuerzburg A421 and by Aeterna Zentaris Inc.
Chronic antigen in solid tumors drives a distinct program of T cell residence
Authors: Gavil Noah Veis , Scott Milcah , Weyu Eyob , O’flanagan Stephen , Wijeyesinghe Sathi , Smith Olivia , Masopust David
Published: January 2023

Resident-memory T cells (TRM) permanently reside in tissues, surveying the local environment for cognate antigen. Surrogate markers (e.g. CD69, CD103) and transcriptional programs from infection models are used as correlates of residency. Across numerous solid tumor types, TRM-like phenotypes have been identified and correlated with improved prognosis and responsiveness to immunotherapy. However, the migration properties of CD8+ tumor infiltrating lymphocytes (TILs) have not been well described. In this study, we employed parabiosis migration assays in a mouse model of breast cancer and demonstrated that both virus-specific bystander and tumor-specific CD8+ TILs can be resident. Canonical markers of TRM, including CD69, failed to discriminate between resident cells and recent migrants. However, the expression of markers associated with chronic T cell stimulation (PD-1, CD39, Tim-3) identified a population of resident, tumor-specific cells. We further observed that after tumor entry, Tcf-1+ PD-1lo tumor-specific T cells progressively acquired the expression of inhibitory receptors, such as Tim-3, correlating with the phenotypes that represent tumor retention and residence. Thus, TRM exist within tumors, durable intratumoral residence was not informed by common markers associated with pathogen-specific TRM that have been described in healthy tissue, but tumor-specific T cells become resident upon tumor antigen recognition and the subsequent upregulation of CD39 and Tim-3.

N.G. is a student in the Medical Scientist Training Program (MD/PhD) at the University of Minnesota and is currently supported through the National Cancer Institute (1F30CA253992-01) with past support from the Dr. Warren J. Warwick and Henrietta Holm Warwick Fellowship. Research supported by the National Cancer Institute (1R01CA238439-01A1, D.M.).
Robust and prototypical immune responses towards COVID-19 BNT162b2 vaccines in Indigenous people
Authors: Zhang Wuji , Kedzierski Lukasz , Chua Brendon Y , Wheatley Adam K , Rowntree Louise , Allen Lilith , Petersen Jan , Chaurasia Priyanka , Mettelman Robert , Miller Adrian , Thomas Paul G , Rossjohn Jamie , Subbarao Kanta , Kent Stephen J , Nelson Jane , Davies Jane , Nguyen Thi H O , Kedzierska Katherine
Published: January 2023

SARS-CoV-2 has led to >270 million infections and >5 million deaths globally. Indigenous people are disproportionately affected by infectious diseases, therefore also more susceptible to the COVID-19 pandemic. There are an estimated 476 million indigenous people globally, including an estimated 798,365 Aboriginal and Torres Strait Islander in Australia. With the high vulnerability to COVID-19, this knowledge is urgently needed to better protect indigenous populations.

We evaluated a breadth of immune responses in indigenous (n=57) and non-indigenous (n=49) individuals after COVID-19 vaccination. We tested RBD antibodies, spike/RBD-probe-specific B cells, peptide stimulations with activation-induced marker (AIM) assay and intracellular cytokine staining.

We found 22% and 34% seroconversion rates after 1st dose of BNT162b2 vaccine for Indigenous and non-indigenous individuals, respectively, which increased to 100% at 1-mth after 2nd dose for both groups. RBD-specific IgG levels in indigenous individuals at 1-mth after 2nd dose positively correlated with their body mass index. At 1-mth after the 2nd COVID-19 vaccination, CD4+ and CD8+ T cell responses via AIM expression and IFN-γ+ TNF+ production was comparable between indigenous and non-indigenous individuals. We are also going to assess the longevity of antibodies and T cells.

Therefore, COVID-19 vaccination induced similar immune responses in indigenous and non-indigenous individuals.
An inactivated SARS-CoV-2 vaccine causes enhanced type 2 inflammation in mice during coronavirus challenge.
Authors: Dillard Jake , Taft-Benz Sharon , Knight Audrey C , Anderson Elizabeth J , Sarkar Sanjay , Loome Jennifer F , Pressey Katia D , Baxter Victoria K , Heise Mark T
Published: January 2023

Inactivated vaccines administered with the adjuvant aluminum hydroxide (Alum) are currently the most widely used COVID-19 vaccines in the world and have been critical to responding to the SARS-CoV-2 pandemic. Although these vaccines are efficacious against homologous virus infection in healthy young adults, the emergence of novel SARS-CoV-2 variants has resulted in significant vaccine breakthrough. Pre-clinical studies with inactivated SARS-CoV-1 and MERS-CoV vaccines have reported enhanced immunopathology upon breakthrough infection, characterized by pulmonary eosinophilia and upregulation of type 2 cytokines. These previous reports therefore raise the concern that inactivated COVID-19 vaccines may cause enhanced immunopathology upon vaccine breakthrough. To investigate this possibility, we vaccinated female BALB/c mice with inactivated SARS-CoV-2 (iCoV2) prior to coronavirus challenge. Although iCoV2 protected mice from severe clinical disease and pulmonary pathology upon homologous challenge with pathogenic SARS-CoV-2, we observed increased pulmonary pathology and pulmonary eosinophilia upon challenge in mice vaccinated with iCoV2 and Alum (iCoV2 + Alum) compared to mice vaccinated with iCoV2 alone. Furthermore, to model vaccine breakthrough upon heterologous coronavirus infection, we challenged iCoV2-vaccinated mice with a zoonotic SARS-like coronavirus (SHC014). Upon SHC014 challenge, iCoV2 + Alum vaccination failed to control virus replication and caused enhanced pulmonary pathology, pulmonary eosinophilia and upregulation of pulmonary type 2 cytokines. Ongoing studies are investigating the mechanism of iCoV2-related immunopathology, including the role of specific iCoV2 antigens.

Supported by grants from NIH (K01 OD026529, U19 AI100625, U19 AI109680, and MSTP T32 GM008719), Infectious Disease Drug Discovery Program (ID3), Rapidly Emerging Antiviral Drug Development Initiative (READDI), NCTraCS and Emerging Challenges in Biomedical Research COVID Pilot Award, SOM Junior Investigator Development Award, Department of Pathology and Laboratory Medicine.
A Novel CD135+ Subset of Mouse Monocytes with a Distinct Differentiation Pathway and Antigen-Presenting Properties
Authors: Kamio Naoka , Yokota Asumi , Tokuda Yuichi , Ogasawara Chie , Nakano Masakazu , Nagao Miki , Tashiro Kei , Maekawa Taira , Onai Nobuyuki , Hirai Hideyo
Published: January 2023

The mononuclear phagocyte system (MPS), composed of monocytes/macrophages and dendritic cells (DCs), plays a critical role at the interface of the innate and adaptive immune systems. However, the simplicity of MPS has been challenged recently by discoveries of novel cellular components. In the current study, we identified the CD135+ subset of monocytes as a novel class of APCs in mice. CD135+ monocytes were readily found in the bone marrow, spleen, and peripheral blood at steady state, and they expressed markers specific to DCs, including MHC class II and CD209a, along with markers for monocytes/macrophages. In addition, this subset phagocytosed bacteria and activated naive T lymphocytes, fulfilling the criteria for APCs. CD135+ monocytes were derived directly from macrophage DC progenitors, not from common monocyte progenitors or other monocytes, suggesting that these are distinct from conventional monocytes. These findings facilitate our understanding of the MPS network that regulates immune responses for host defense.
Cutting Edge: mTORC2 Regulates CD8+ Effector and Memory T Cell Differentiation through Serum and Glucocorticoid Kinase 1
Authors: Patel Chirag H , Heikamp Emily B , Xu Wei , Sun Im-Hong , Oh Min-Hee , Sun Im-Meng , Wen Jiayu , Tam Ada J , Blosser Richard L , Powell Jonathan D
Published: January 2023

The mechanistic target of rapamycin is an essential regulator of T cell metabolism and differentiation. In this study, we demonstrate that serum- and glucocorticoid-regulated kinase 1 (SGK1), a downstream node of mechanistic target of rapamycin complex 2 signaling, represses memory CD8+ T cell differentiation. During acute infections, murine SGK1-deficient CD8+ T cells adopt an early memory precursor phenotype leading to more long-lived memory T cells. Thus, SGK1-deficient CD8+ T cells demonstrate an enhanced recall capacity in response to reinfection and can readily reject tumors. Mechanistically, activation of SGK1-deficient CD8+ T cells results in decreased Foxo1 phosphorylation and increased nuclear translocation of Foxo1 to promote early memory development. Overall, SGK1 might prove to be a powerful target for enhancing the efficacy of vaccines and tumor immunotherapy.
STAT6-IP–Dependent Disruption of IL-33–Mediated ILC2 Expansion and Type 2 Innate Immunity in the Murine Lung
Authors: Moarbes Vanessa , Gaudreault Véronique , Karkout Rami , Labrie Lydia , Zhao Hedi , Shan Jichuan , Fixman Elizabeth D
Published: January 2023

Recent interest has focused on innate-type cytokines as promoters of type 2 immunity and targets for drug development in asthma. IL-33 induces production of IL-4 and/or IL-13, which is associated with STAT6-dependent responses in innate cells, including group 2 innate lymphoid cells (ILC2s), macrophages, and eosinophils. Our published data show that STAT6-immunomodulatory peptide (STAT6-IP), an immunomodulatory peptide designed to inhibit the STAT6 transcription factor, reduces induction of Th2 adaptive immunity in respiratory syncytial virus infection and asthma models. Nevertheless, the mechanism of STAT6-IP–dependent inhibition has remained obscure. In this study, we demonstrate that STAT6-IP reduced IL-33–induced type 2 innate lung inflammation. Specifically, our data show that STAT6-IP reduced recruitment and activation of eosinophils as well as polarization of alternatively activated macrophages. Decreases in these cells correlated with reduced levels of IL-5 and IL-13 as well as several type 2 chemokines in the bronchoalveolar lavage fluid. STAT6-IP effectively inhibited expansion of ILC2s as well as the number of IL-5– and IL-13–producing ILC2s. Our data suggest that STAT6-IP effectively disrupts IL-13–dependent positive feedback loops, initiated by ILC2 activation, to suppress IL-33–induced type 2 innate immunity in the murine lung.
A Bcl6 Intronic Element Regulates T Follicular Helper Cell Differentiation
Authors: Lai Chen-Yen , Marcel Nimi , Yaldiko Allen W , Delpoux Arnaud , Hedrick Stephen M
Published: January 2023

In response to an intracellular infectious agent, the immune system produces a specific cellular response as well as a T cell–dependent Ab response. Precursor T cells differentiate into effector T cells, including Th1 cells, and T follicular helper (TFH) cells. The latter cooperate with B cells to form germinal centers and induce the formation of Ab-forming plasmacytes. One major focal point for control of T cell differentiation is the transcription factor BCL6. In this study, we demonstrated that the Bcl6 gene is regulated by FOXO1-binding, cis-acting sequences located in a highly conserved region of the first Bcl6 intron. In both mouse and human T cells, deletion of the tandem FOXO1 binding sites increased the expression of BCL6 and enhanced the proportion of TFH cells. These results reveal a fundamental control point for cellular versus humoral immunity.
CCL17 Promotes Colitis-Associated Tumorigenesis Dependent on the Microbiota
Authors: Metzger Rebecca , Winter Lis , Bouznad Nassim , Garzetti Debora , von Armansperg Benedikt , Rokavec Matjaz , Lutz Konstantin , Schäfer Yvonne , Krebs Sabrina , Winheim Elena , Friedrich Verena , Matzek Dana , Öllinger Rupert , Rad Roland , Stecher Bärbel , Hermeking Heiko , Brocker Thomas , Krug Anne B
Published: January 2023

Colorectal cancer is one of the most common cancers and a major cause of mortality. Proinflammatory and antitumor immune responses play critical roles in colitis-associated colon cancer. CCL17, a chemokine of the C-C family and ligand for CCR4, is expressed by intestinal dendritic cells in the steady state and is upregulated during colitis in mouse models and inflammatory bowel disease patients. In this study, we investigated the expression pattern and functional relevance of CCL17 for colitis-associated colon tumor development using CCL17–enhanced GFP-knockin mice. CCL17 was highly expressed by dendritic cells but also upregulated in macrophages and intermediary monocytes in colon tumors induced by exposure to azoxymethane and dextran sodium sulfate. Despite a similar degree of inflammation in the colon, CCL17-deficient mice developed fewer tumors than did CCL17-competent mice. This protective effect was abrogated by cohousing, indicating a dependency on the microbiota. Changes in microbiota diversity and composition were detected in separately housed CCL17-deficient mice, and these mice were more susceptible to azoxymethane-induced early apoptosis in the colon affecting tumor initiation. Immune cell infiltration in colitis-induced colon tumors was not affected by the lack of CCL17. Taken together, our results indicate that CCL17 promotes colitis-associated tumorigenesis by influencing the composition of the intestinal microbiome and reducing apoptosis during tumor initiation.
IL-6 Signaling Attenuates TNF-α Production by Plasmacytoid Dendritic Cells in Rheumatoid Arthritis
Authors: Papadaki Garyfalia , Goutakoli Panagiota , Tiniakou Ioanna , Grün Joachim R , Grützkau Andreas , Pavlopoulos Georgios A , Iliopoulos Ioannis , Bertsias George , Boumpas Dimitrios , Ospelt Caroline , Reizis Boris , Sidiropoulos Prodromos , Verginis Panayotis
Published: January 2023

Rheumatoid arthritis (RA) is characterized by autoimmune joint destruction with debilitating consequences. Despite treatment advancements with biologic therapies, a significant proportion of RA patients show an inadequate clinical response, and restoration of immune self-tolerance represents an unmet therapeutic need. We have previously described a tolerogenic phenotype of plasmacytoid dendritic cells (pDCs) in RA patients responding to anti–TNF-α agents. However, the molecular mechanisms involved in tolerogenic reprogramming of pDCs in RA remain elusive. In this study, guided by transcriptomic analysis of CD303+CD123+ pDCs from RA patients in remission, we revealed enhanced expression of IL-6R and its downstream signaling compared with healthy pDCs. Functional assessment demonstrated that IL-6R engagement resulted in marked reduction of TNF-α secretion by pDCs whereas intracellular TNF-α was significantly increased. Accordingly, pharmacologic inhibition of IL-6R signaling restored TNF-α secretion levels by pDCs. Mechanistic analysis demonstrated impaired activity and decreased lysosomal degradation of ADAM17 (a disintegrin and metalloproteinase 17) sheddase in pDCs, which is essential for TNF-α cleavage. Importantly, reduction of TNF-α secretion by IL-6–treated pDCs attenuated the inflammatory potential of RA patient–derived synovial fibroblasts. Collectively, these findings position pDCs as an important source of TNF-α in RA pathogenesis and unravel an anti-inflammatory mechanism of IL-6 by limiting the pDC-derived TNF-α secretion.
Affinity-Driven Site-Specific High Mannose Modification Determines the Structural Polymerization and Function of Tetrameric IgM in a Primitive Vertebrate
Authors: Yin Xiaoxue , Li Xiaoyu , Mu Liangliang , Bai Hao , Yang Yanjian , Chen Nuo , Wu Liting , Fu Shengli , Li Jun , Ying Wantao , Ye Jianmin
Published: January 2023

Teleost tetramer IgM is the predominant Ig in the immune system and plays essential roles in host defense against microbial infection. Due to variable disulfide polymerization of the monomeric subunits, tetrameric IgM possesses considerable structural diversity. Previous work indicated that the teleost IgM H chain was fully occupied with complex-type N-glycans. However, after challenge with trinitrophenyl (TNP) Ag, the complex N-glycans in the Asn-509 site of Oreochromis niloticus IgM H chain transformed into high mannose. This study, therefore, was conducted to examine the functional roles of the affinity-related high-mannose modification in tilapia IgM. The TNP-specific IgM Ab affinity maturation was revealed in tilapia over the response. A positive correlation between TNP-specific IgM affinity and its disulfide polymerization level of isomeric structure was demonstrated. Mass spectrometric analysis indicated that the relationship between IgM affinity and disulfide polymerization was associated with the Asn-509 site-specific high-mannose modification. Furthermore, the increase of high mannose content promoted the combination of IgM and mannose receptor (MR) on the surface of phagocytes. Moreover, the increased interaction of IgM and MR amplified the phagocytic ability of phagocytes to Streptococcus agalactiae. To our knowledge, this study demonstrates that site-specific high-mannose modification associates with IgM Ab affinity and its structural disulfide polymerization and amplifies the phagocytosis of phagocytes by the combination of IgM and MR. The present study provides evidence for understanding the association of IgM structure and function during the evolution of the immune system.
Pazopanib Is a Potential Treatment for Coronavirus-Induced Lung Injuries
Authors: Luan Yi , Yuan Qianying , Wang Qijun , Compton Susan , Wu Dianqing , Tang Wenwen
Published: January 2023

Severe acute respiratory syndrome coronavirus 2, responsible for the severe acute respiratory syndrome known as COVID-19, has rapidly spread in almost every country and devastated the global economy and health care system. Lung injury is an early disease manifestation believed to be a major contributor to short- and long-term pathological consequences of COVID-19, and thus drug discovery aiming to ameliorate lung injury could be a potential strategy to treat COVID-19 patients. By inducing a severe acute respiratory syndrome–like pulmonary disease model through infecting A/J mice with murine hepatitis virus strain 1 (MHV-1), we show that i.v. administration of pazopanib ameliorates acute lung injuries without affecting MHV-1 replication. Pazopanib reduces cell apoptosis in MHV-1–infected lungs. Furthermore, we also identified that pazopanib has to be given no later than 48 h after the virus infection without compromising the therapeutic effect. Our study provides a potential treatment for coronavirus-induced lung injuries and support for further evaluation of pazopanib in COVID-19 patients.
Zebrafish sirt5 Negatively Regulates Antiviral Innate Immunity by Attenuating Phosphorylation and Ubiquitination of mavs
Authors: Chen Xiaoyun , Fan Sijia , Zhu Chunchun , Liao Qian , Tang Jinhua , Yu Guangqing , Cai Xiaolian , Ouyang Gang , Xiao Wuhan , Liu Xing
Published: January 2023

The signaling adaptor MAVS is a critical determinant in retinoic acid–inducible gene 1–like receptor signaling, and its activation is tightly controlled by multiple mechanisms in response to viral infection, including phosphorylation and ubiquitination. In this article, we demonstrate that zebrafish sirt5, one of the sirtuin family proteins, negatively regulates mavs-mediated antiviral innate immunity. Sirt5 is induced by spring viremia of carp virus (SVCV) infection and binds to mavs, resulting in attenuating phosphorylation and ubiquitination of mavs. Disruption of sirt5 in zebrafish promotes survival ratio after challenge with SVCV. Consistently, the antiviral responsive genes are enhanced, and the replication of SVCV is diminished in sirt5-dificient zebrafish. Therefore, we reveal a function of zebrafish sirt5 in the negative regulation of antiviral innate immunity by targeting mavs.
Insights into the Antibacterial Properties of Complement Peptides C3a, C4a, and C5a across Vertebrates
Authors: Zhang Xu-Jie , Zhong Ya-Qin , Ma Zi-You , Hu Ya-Zhen , Su Jian-Guo , Zhang Yong-An
Published: January 2023

Complement peptides C3a, C4a, and C5a are important components of innate immunity in vertebrates. Although they diverged from a common ancestor, only C3a and C4a can act as antibacterial peptides in Homo sapiens, suggesting that C5a has evolved into a purely chemotactic molecule; however, the antibacterial properties of C3a, C4a, and C5a across vertebrates still require elucidation. In this article, we show that, unlike those in H. sapiens, Mus musculus C3a, C4a, and C5a all possess antibacterial activities, implying that the antibacterial properties of C3a, C4a, and C5a have evolved divergently in vertebrates. The extremely different net charge, a key factor determining the antibacterial activities of cationic antimicrobial peptides, of vertebrate C3a, C4a, and C5a supports this speculation. Moreover, the antibacterial activity of overlapping peptides covering vertebrate C3a, C4a, and C5a further strongly supports the speculation, because their activity is positively correlated with the net charge of source molecules. Notably, the structures of C3a, C4a, and C5a are conserved in vertebrates, and the inactive overlapping peptides can become antibacterial peptides if mutated to possess enough net positive charges, indicating that net charge is the only factor determining the antibacterial properties of vertebrate C3a, C4a, and C5a. More importantly, many vertebrate C3a-, C4a-, and C5a-derived peptides possess high antibacterial activities yet exhibit no hemolytic activities, suggesting the application potential in anti-infective therapy. Taken together, our findings reveal that vertebrate C3a, C4a, and C5a are all sources of antibacterial peptides that will facilitate the design of excellent peptide antibiotics.
IL-7 Receptor Drives Early T Lineage Progenitor Expansion
Authors: Paiva Rafael A , Ramos Camila V , Leiria Gonçalo , Martins Vera C
Published: January 2023

IL-7 and IL-7R are essential for T lymphocyte differentiation by driving proliferation and survival of specific developmental stages. Although early T lineage progenitors (ETPs), the most immature thymocyte population known, have a history of IL-7R expression, it is unclear whether IL-7R is required at this stage. In this study, we show that mice lacking IL-7 or IL-7R have a marked loss of ETPs that results mostly from a cell-autonomous defect in proliferation and survival, although no changes were detected in Bcl2 protein levels. Furthermore, a fraction of ETPs responded to IL-7 stimulation ex vivo by phosphorylating Stat5, and IL-7R was enriched in the most immature Flt3+Ccr9+ ETPs. Consistently, IL-7 promoted the expansion of Flt3+ but not Flt3− ETPs on OP9-DLL4 cocultures, without affecting differentiation at either stage. Taken together, our data show that IL-7/IL-7R is necessary following thymus seeding by promoting proliferation and survival of the most immature thymocytes.
Peripheral CD23hi/IgE+ Plasmablasts Secrete IgE and Correlate with Allergic Disease Severity
Authors: Simonin Elisabeth M , Babasyan Susanna , Wagner Bettina
Published: January 2023

Production and secretion of IgE by B cells, plasmablasts, and plasma cells is a central step in the development and maintenance of allergic diseases. IgE can bind to one of its receptors, the low-affinity IgE receptor CD23, which is expressed on activated B cells. As a result, most B cells bind IgE through CD23 on their surface. This makes the identification of IgE producing cells challenging. In this study, we report an approach to clearly identify live IgE+ plasmablasts in peripheral blood for application by both flow cytometry analysis and in vitro assay. These IgE+ plasmablasts readily secrete IgE, upregulate specific mRNA transcripts (BLIMP-1 IRF4, XBP1, CD138, and TACI), and exhibit highly differentiated morphology all consistent with plasmablast differentiation. Most notably, we compared the presence of IgE+ plasmablasts in peripheral blood of allergic and healthy individuals using a horse model of naturally occurring seasonal allergy, Culicoides hypersensitivity. The model allows the comparison of immune cells both during periods of clinical allergy and when in remission and clinically healthy. Allergic horses had significantly higher percentages of IgE+ plasmablasts and IgE secretion while experiencing clinical allergy compared with healthy horses. Allergy severity and IgE secretion were both positively correlated to the frequency of IgE+ plasmablasts in peripheral blood. These results provide strong evidence for the identification and quantification of peripheral IgE-secreting plasmablasts and provide a missing cellular link in the mechanism of IgE secretion and upregulation during allergy.
An Engineered Monovalent Anti–TNF-α Antibody with pH-Sensitive Binding Abrogates Immunogenicity in Mice following a Single Intravenous Dose
Authors: Watkins J Monty , Watkins Jeffry D
Published: January 2023

Therapeutic Abs directed toward TNF-α display significant immunogenicity in humans, frequently leading to lower serum concentrations of the Ab that are associated with lower treatment efficacy. The enhanced incidence of immunogenicity observed with this class of therapeutics may be mediated by the expression of TNF-α as a homotrimer, both as a soluble serum protein and as a membrane-associated protein (mTNF-α) on the surface of dendritic cells. The TNF-α homotrimer enables the formation of polyvalent Ab–TNF-α immune complexes (ICs) that enhance binding to FcR and neonatal FcR. Polyvalent ICs and Ab bound to mTNF-α on the surface of dendritic cells can internalize, traffic to the lysosomes, and be processed for presentation by MHC molecules. To diminish immunogenicity caused by trafficking of ICs and mTNF-α to the lysosomes, we engineered a monovalent format of adalimumab with pH-sensitive binding to TNF-α. The engineered variant, termed AF-M2637, did not cross-link TNF-α trimers and consequently formed small, nonprecipitating ICs only. AF-M2637 bound TNF-α with high affinity at pH 7.4 (EC50 = 1.1 nM) and displayed a significantly faster dissociation rate than adalimumab at pH 6.0. No immune response to AF-M2637 was detected in mice following a single i.v. dose. In contrast, rapid immunization was detected following the injection of a single i.v. dose of adalimumab, monovalent adalimumab, or the bivalent form of the pH-sensitive variant. These data suggest that ICs and mTNF-α both contribute to the immunogenicity of adalimumab in mice and provide a general strategy for engineering less immunogenic therapeutic TNF-α Abs.
Identification of Cell Types and Transcriptome Landscapes of Porcine Epidemic Diarrhea Virus–Infected Porcine Small Intestine Using Single-Cell RNA Sequencing
Authors: Fan Baochao , Zhou Jinzhu , Zhao Yongxiang , Zhu Xuejiao , Zhu Mingjun , Peng Qi , Li Jizong , Chang Xinjian , Shi Danyi , Yin Jie , Guo Rongli , Li Yunchuan , He Kongwang , Fan Huiying , Li Bin
Published: January 2023

Swine coronavirus–porcine epidemic diarrhea virus (PEDV) with specific susceptibility to pigs has existed for decades, and recurrent epidemics caused by mutant strains have swept the world again since 2010. In this study, single-cell RNA sequencing was used to perform for the first time, to our knowledge, a systematic analysis of pig jejunum infected with PEDV. Pig intestinal cell types were identified by representative markers and identified a new tuft cell marker, DNAH11. Excepting enterocyte cells, the goblet and tuft cells confirmed susceptibility to PEDV. Enrichment analyses showed that PEDV infection resulted in upregulation of cell apoptosis, junctions, and the MAPK signaling pathway and downregulation of oxidative phosphorylation in intestinal epithelial cell types. The T cell differentiation and IgA production were decreased in T and B cells, respectively. Cytokine gene analyses revealed that PEDV infection downregulated CXCL8, CXCL16, and IL34 in tuft cells and upregulated IL22 in Th17 cells. Further studies found that infection of goblet cells with PEDV decreased the expression of MUC2, as well as other mucin components. Moreover, the antimicrobial peptide REG3G was obviously upregulated through the IL33-STAT3 signaling pathway in enterocyte cells in the PEDV-infected group, and REG3G inhibited the PEDV replication. Finally, enterocyte cells expressed almost all coronavirus entry factors, and PEDV infection caused significant upregulation of the coronavirus receptor ACE2 in enterocyte cells. In summary, this study systematically investigated the responses of different cell types in the jejunum of piglets after PEDV infection, which deepened the understanding of viral pathogenesis.
PPP2R2D Suppresses Effector T Cell Exhaustion and Regulatory T Cell Expansion and Inhibits Tumor Growth in Melanoma
Authors: Pan Wenliang , Scherlinger Marc , Yoshida Nobuya , Tsokos Maria G , Tsokos George C
Published: January 2023

We had shown previously that the protein phosphatase 2A regulatory subunit PPP2R2D suppresses IL-2 production, and PPP2R2D deficiency in T cells potentiates the suppressive function of regulatory T (Treg) cells and alleviates imiquimod-induced lupus-like pathology. In this study, in a melanoma xenograft model, we noted that the tumor grew in larger sizes in mice lacking PPP2R2D in T cells (LckCreR2Dfl/fl) compared with wild type (R2Dfl/fl) mice. The numbers of intratumoral T cells in LckCreR2Dfl/fl mice were reduced compared with R2Dfl/fl mice, and they expressed a PD-1+CD3+CD44+ exhaustion phenotype. In vitro experiments confirmed that the chromatin of exhaustion markers PD-1, LAG3, TIM3, and CTLA4 remained open in LckCreR2Dfl/fl CD4 T conventional compared with R2Dfl/fl T conventional cells. Moreover, the percentage of Treg cells (CD3+CD4+Foxp3+CD25hi) was significantly increased in the xenografted tumor of LckCreR2Dfl/fl mice compared with R2Dfl/fl mice probably because of the increase in the percentage of IL-2–producing LckCreR2Dfl/fl T cells. Moreover, using adoptive T cell transfer in mice xenografted with melanoma, we demonstrated that PPP2R2D deficiency in T cells enhanced the inhibitory effect of Treg cells in antitumor immunity. At the translational level, analysis of publicly available data from 418 patients with melanoma revealed that PPP2R2D expression levels correlated positively with tumor-infiltration level of CD4 and CD8 T cells. The data demonstrate that PPP2R2D is a negative regulator of immune checkpoint receptors, and its absence exacerbates effector T cell exhaustion and promotes Treg cell expansion. We conclude that PPP2R2D protects against melanoma growth, and PPP2R2D-promoting regimens can have therapeutic value in patients with melanoma.
Mechanism of CD79A and CD79B Support for IgM+ B Cell Fitness through B Cell Receptor Surface Expression
Authors: Huse Kanutte , Bai Baoyan , Hilden Vera I , Bollum Lise K , Våtsveen Thea K , Munthe Ludvig A , Smeland Erlend B , Irish Jonathan M , Wälchli Sébastien , Myklebust June H
Published: January 2023

The BCR consists of surface-bound Ig and a heterodimeric signaling unit comprised of CD79A and CD79B. Upon cognate Ag recognition, the receptor initiates important signals for B cell development and function. The receptor also conveys Ag-independent survival signals termed tonic signaling. Although the requirement of a CD79A/CD79B heterodimer for BCR complex assembly and surface expression is well established based on mice models, few studies have investigated this in human mature B cells. In this study, we found that human tonsillar B cells with high surface expression of IgM or IgG had potentiated BCR signaling compared with BCRlow cells, and high IgM expression in germinal center B cells was associated with reduced apoptosis. We explored the mechanism for IgM surface expression by CRISPR/Cas9-induced deletion of CD79A or CD79B in four B lymphoma cell lines. Deletion of either CD79 protein caused loss of surface IgM in all cell lines and reduced fitness in three. From two cell lines, we generated stable CD79A or CD79B knockout clones and demonstrated that loss of CD79A or CD79B caused a block in N-glycan maturation and accumulation of immature proteins, compatible with retention of BCR components in the endoplasmic reticulum. Rescue experiments with CD79B wild-type restored surface expression of CD79A and IgM with mature glycosylation, whereas a naturally occurring CD79B G137S mutant disrupting CD79A/CD79B heterodimerization did not. Our study highlights that CD79A and CD79B are required for surface IgM expression in human B cells and illuminates the importance of the IgM expression level for signaling and fitness.