bluuebunny/crossref_metadata_2025 huggingface dataset:
A generalized pathway of immunocompromise following central nervous system insult: the release of large immunosuppressive molecules and thymic involution
Authors: Ayasoufi Katayoun , Pfaller Christian K , Khadka Roman , Jin Fang , Zheng Jiaying , Schuelke Matthew R , Evgin Laura , Hansen Michael , Himes Benjamin , Fain Cori , Tritz Zachariah Peter , Goddery Emma , Yokanovich Lila T , Pease Larry R , Vile Richard G. , Johnson Aaron J
Published: January 2023

Immunosuppression following damage to the CNS is a common, yet poorly understood, feature of neurological diseases as diverse as stroke, traumatic brain injury, and glioblastoma. This immunosuppression is a barrier to successful patient outcomes. We sought to define the effect of various brain insults on the thymus and T-cell responses. We tested thymic function following various neurological insults, including viral infection, brain tumor, sterile inflammation, physical injury, and seizures. All brain insults resulted in significant thymic involution that was reversible if the insult was cleared. Thymic involution did not occur following similar peripheral insults. Using parabiosis, we demonstrated that thymic involution was transferable via circulatory routes from glioma-bearing to non-tumor-bearing parabionts. Similarly, serum obtained from mice with ongoing neurological insults potently inhibited T-cell proliferation in vitro. We next fractionated the serum based on molecular weight and tested the resulting fractions’ immunosuppressive potential. Interestingly, we found that serum fractions with large molecular weights of greater than 100 kiloDaltons were responsible for the immunosuppressive properties of serum obtained from glioma-bearing mice. In short, CNS-specific insults, regardless of nature, induce immunosuppression by prompting thymic involution and systemic immunosuppression mediated through circulating factors with large molecular weight. These studies provide evidence of the mechanisms leading to immune deficiencies observed in patients following neurological injuries.
YAP is a novel regulator of CD4+ and CD8+ T cells during activation, differentiation, and anti-tumor immunity
Authors: Szeto Gregory Lee , Stampouloglou Elena , Cheng Nan , Federico Anthony , Slaby Emily , Linton Jonathan , Livingston Natalie , Schneck Jonathan P , Monti Stefano , Varelas Xaralabos
Published: January 2023

Advanced understanding of T cell regulation has fueled cancer immunotherapy, but durable responses remain confined to a minority of patients. Poor responses can be linked to either suppressed T cell function in tumors or “cold” tumors that exclude immune cells. Identifying novel T cell regulators involved in these processes is a high priority. Our studies identified the protein YAP as a novel regulator of T cells. YAP mRNA and protein were rapidly expressed after T cell activation in mouse CD4+ and CD8+ T cells. T cell-specific Yap knockout (YapKO) generated CD4+ and CD8+ T cells that express elevated levels of activation markers (CD25, CD44, CD69) compared to WT cells. YapKO increased the sensitivity of T cells to anti-CD3 dose, and increased CD4+ T cell differentiation efficiency under Th1, Th2, Th17, and Treg skewing conditions. YapKO CD8+ T cells expanded >20-fold more than WT when cultured in engineered hydrogel matrices optimized for T cell expansions and adoptive transfer. Syngeneic tumors grown in T cell YapKO mice had impaired growth and increased immune infiltration compared to tumors in WT hosts. Co-adoptive transfer of YapKO and WT CD8+ T cells showed only YapKO cells infiltrated B16F10 tumors. RNAseq of CD4+ and CD8+ T cells from tumors and draining lymph nodes showed gene expression changes including upregulation of T cell receptor and cytokine signaling pathways in YapKO hosts. T cells from tumor-draining lymph nodes were not significantly different between YapKO and WT. YapKO gene signatures correlated with immune infiltration and survival in multiple human cancers in TCGA. Our data demonstrated YAP is a novel regulator of diverse T cell functions and may be a useful target for enhancing responses to cancer immunotherapy.
A paradoxical role for classical dendritic cells and Flt3 ligand in tumor immune response
Authors: Darrasse-Jeze Guillaume , Régnier Paul , Cagnard Nicolas , Podsypanina Katrina
Published: January 2023

Flt3 ligand (FL) induces differentiation and proliferation of classical dendritic cells (cDCs) and is a promising molecule in therapies of cancer and vaccination, but has mixed effects in clinical trials. The role of cDCs in antitumor response is ambiguous as they may induce priming of anti-tumor lymphocytes, but also may favor recruitment of tumor-protecting regulatory T cells (Tregs).

To better understand the role of FL/DCs axis in cancer, we monitored tumor growth in a mouse model of melanoma in the context of low, normal or high levels of FL. Paradoxically, both genetic FL depletion and overexpression stalled tumor growth and increased animal survival as compared to control. We show that FL-deficient mice tumor growth is predominantly controlled by the adaptive immune system unleashed by a diminution of Tregs, while high FL levels favor the innate anti-tumor response through the recruitment of NK cells. Analysis of human gene expression data confirmed the paradoxical effect of FL as well as cDCs on survival in several types of cancer. Tumor samples from patients in these groups expressed Treg and NK cells signatures in correlation with the levels of cDCs, FL, IL15 and IL15RA expression.

As a proof-of-principle, we showed that the combination of FL overexpression and Tregs depletion resulted in regression of established tumors in mice. We propose that FL is a master regulator of immune control of tumor growth owing to its key role in DCs function.
CD26 enzymatic activity modulates efficient migration of adoptively transferred cancer-specific T cells to solid tumors
Authors: Wyatt Megan M. , Bailey Stefanie , Knochelmann Hannah , Smith Aubrey S , Dwyer Connor , Rivera Guillermo O Rangel , Reyes Amalia Rivera , Nelson Michelle , Paulos Chrystal M
Published: January 2023

The restricted ability of adoptively transferred T cells to eradicate solid tumors limits their use in some patients. Efforts to improve ACT for solid tumors aim to identify strategies that poise T cells for migration. We have previously identified a specific subset of CD4 T cells which express high levels of the ubiquitous ectoenzyme dipeptidyl peptidase-4 (DPP-4), also known as CD26, that produce a tremendous antitumor response in solid tumors. We therefore sought to investigate the functional importance of CD26 on T cells destined for ACT. We transferred tumor specific CD26+ T cells into melanoma-bearing CD26−/− mice and blocked the CD26 enzymatic activity of the donor cells in vivo with sitagliptin, an established competitive inhibitor of CD26. Sitagliptin-treated mice eventually succumbed to tumor burden, while tumors in untreated mice were ablated, eliciting long-term cures exceeding 4 months. Additional analysis determined that tumor infiltrating donor and host T cells diminished with sitagliptin treatment. A 32-plex cytokine array of peripheral blood plasma from these mice revealed a global diminishment of cytokines and chemokines, signifying that the inflammatory response of the T cells was dampened with sitagliptin treatment. Further experiments examined how CD26+ T cells responded to tumor trafficking signals using a transwell migration assay and found that sitagliptin treatment significantly impaired their migratory capacity. However, sitagliptin did not impair the ability of T cells to mount a functional response to tumor antigen. These data reveal that the enzymatic activity of CD26 is important for the ability of T cells to migrate to malignant sites and supports an effective antitumor response.
Microbiota-derived butyrate promotes metabolism and memory potential of effector CD8+T cells
Authors: Bachem Annabell , Makhlouf Christina , Binger Katrina J , de Souza David P , Tull Dedreia , Lew Andrew M , Perdomo Carolina , Kupz Andreas , Figgett William , Mackay Fabienne , Oleshansky Moshe , Russ Brendan E , Parish Ian A , Kallies Axel , McConville Malcolm , Turner Stephen J , Gebhardt Thomas , Bedoui Sammy
Published: January 2023

Interactions with the microbiota influence many aspects of immunity, including immune cell development, differentiation and function. Here we examined the impact of microbiota on one of the key functions of CD8+T cells, the transition to long-lived and protective memory. Antigen-activated CD8+T cells transferred into germ-free mice failed to transition into long-lived memory cells with enhanced recall capacity and had transcriptional impairments in oxidative metabolism. To the contrary, the microbiota-derived short-chain fatty acid (SCFA) butyrate promoted cellular metabolism, enhanced memory potential of activated CD8+T cells and was required for optimal recall responses upon antigen re-encounter. Mechanistic experiments revealed that the SCFA butyrate increased turnover of glycolysis and oxidative phosphorylation (OXPHOS) of effector CD8+T cells but led to a partial uncoupling of the tricarboxylic acid cycle from glycolytic input. This allowed preferential fueling of oxidative phosphorylation through short-chain fatty acids. Our findings reveal a role for the microbiota in promoting CD8+T cell long-term survival as memory cells and suggest that microbial metabolites potentially guide the metabolic rewiring of activated CD8+T cells that enables this transition.
Mechanistic mathematical representation of CD8+ T cell mediated drug-induced liver injury (DILI) Part 3: translation from mouse to human amodiaquine (AQ) response
Authors: Shoda Lisl , Battista Christina , Kenz Zackary
Published: January 2023

Idiosyncratic DILI (iDILI) is poorly understood and costly to patients and drug developers. Some iDILI events appear immune-mediated. This work builds on DILIsym mouse modeling of CD8+ T cell mediated killing of OVA or AQ-expressing hepatocytes1,2 to develop a human representation of AQ DILI.

The representation was adapted to account for human AQ exposure and endogenous T cell numbers3,4.

Simulation results indicate an adult human treated with 200–700 mg AQ weekly is unlikely to present with liver injury, even if liver tolerance is broken. Simulations reflect alternate mechanisms of non-response depending on antigen levels. With low antigen, few responsive T cells of low affinity and avidity result in low level expansion without effective cytotoxicity. At high antigen levels, the emergent T cell response is dominated by dysfunctional T cells (exhaustion). However, when regulatory pathways (co-inhibition, exhaustion) are perturbed, simulated human liver injury manifests over weeks to months consistent with case reports5. Halting treatment led to simulated recovery provided sufficient liver mass remained; subsequent AQ re-challenge resulted in a more rapid ALT response, consistent with data5, mechanistically mediated by re-activation of memory T cells.

Simulation results provide proof-of-concept that CD8+ T cell mediated cytotoxicity can explain human AQ DILI. Evaluation of additional compounds and the identification of testable experimental hypotheses are needed to demonstrate model utility in understanding iDILI.
PD-L1 on macrophages protects from severe collagen induced arthritis
Authors: Wood Megan Kay , Talor Monica V , Won Taejoon , Čiháková Daniela
Published: January 2023

Immune related adverse events linked to severe inflammatory arthritis have been identified in 10–43% of patients receiving immune checkpoint inhibitor therapy, aCTLA-4, aPD-1 and aPD-L1, indicating the importance of immune inhibitory pathways in maintaining homeostasis in the synovium. We found that immune checkpoint, PD-L1, is primarily expressed in the joint compared to other immune checkpoints. Furthermore, blocking of PD-L1 during collagen induced arthritis (CIA) resulted in more severe arthritis. Mice synovial macrophages have the highest PD-L1 expression level at homeostasis and macrophage PD-L1 expression is further increased during CIA. Similarly, in human synovial fluid, macrophages have the highest frequency of PD-L1 expression. We hypothesize that the expression of PD-L1 by macrophages is essential for synovial homeostasis and regulation of joint inflammation. We selectively deleted PD-L1 in macrophages using the cre/flox system and induced CIA analyzing disease severity using flow cytometry. LyzcrePD-L1fl/fl showed more severe CIA with a greater change in paw thickness, higher disease and histology score and a higher frequency of CD4 and CD8 T cells in the synovium. Interestingly, female LyzcrePD-L1fl/fl mice have higher arthritis severity to LyzcrePD-L1fl/fl males. This indicates the importance of sex specific PD-L1 expression by synovial macrophages during CIA. Our project has high translational potential since joint specific PD-L1 expression could be modulated for therapeutic purposes in humans with rheumatoid arthritis or check point inhibitors induced arthritis.
A novel human milk protein-derived peptide BCCY-1 exerts immunomodulatory effects on monocytes through NF-κB and MAPK signaling pathways
Authors: Cao Yan , Cai Jinyang , Li Xin , Ji Chenbo
Published: January 2023

Protective effects of breastfeeding in reducing childhood infection have been reported. Human breast milk contains diverse bioactive compounds with direct and indirect antimicrobial activities as well as molecules that modulate the immune system. Although some of the molecules involved in human milk’s beneficial effects on development of immune function have been studied, the role of specific milk component in immunomodulation is not yet clear. Milk protein-derived bioactive peptides have been identified with various health-promoting functions icluding immunoregulation. In the present study, we found that a peptide derived from human milk protein β-casein (BCCY-1) enhance the migration of monocytes without affecting cell proliferation and apoptosis. In addition, BCCY-1 induced the chemokine production via activition of NF-κB and MAPK signaling pathways, which could be blocked by specific inhibitors of NF-κB and MAPK. Moreover, the relative intensity of peptide BCCY-1 as well as a cluster of β-casein derived peptides comprising a fragment of BCCY-1 were decreased in the preterm breast milk compared with the term breast milk, indicating a potential role of BCCY-1 and the similar sequences under physiological condition. Identification of those peptides and their physiological functions are necessary to transfer their potent functional properties into food or potential applications in health-care. This study highlight the important role of β-casein derived peptides in the immunomodulatory effects of human breast milk and suggest a promising way to develop novel dietary supplements, foods and even pharmaceutical agents.
Trem2 Supports the Metabolic Program of Alternative Activated Macrophages
Authors: Gonzalez-Cotto Marieli , Palmieri Erika M , Baseler Walter A , Rice Christopher M , McVicar Daniel W
Published: January 2023

Metabolism is a key modulator of macrophage differentiation, polarization and effector responses. While M1 favors a glycolytic configuration resembling the “Warburg effect”, M2 is fueled by oxidative metabolism. Here we describe a previously unappreciated metabolic phenotype controlled by The Triggering Receptor Expressed on Myeloid Cells (TREM)-2, a key player in inflammation and innate immunity. Trem2−/− macrophages show lower TCA cycle metabolites compared to wild type, but maintenance of glycolytic intermediates. Interestingly, while we show Trem2 abrogation is beneficial in M1 and protective against MCSF withdrawal, we find Trem2−/− cells to have disrupted lipid utilization as they accumulate both long chain free fatty acids (FFA) and carnitine-conjugated lipids. Because lipid metabolism is key for M2 responses, we hypothesized that Trem2 supports a metabolic program to maintain M2 activation. Indeed, in M2 cell, Trem2 sustains mitochondrial respiratory capacity, CD36 expression and lipoprotein and FFA uptake. Lipidomics confirmed Trem2−/− M2 cells had decreased FFA and enhanced triglycerides. Moreover, Trem2−/− M2 cells show a distinct metabolic profile with enhanced glutamine utilization, suggesting compensatory mechanisms in lieu of FFA for mitochondrial respiration. Transcriptome profiling decisively shows dysregulated genes related to defense response and glycerolipid metabolism, linking the metabolic findings to effector responses. Finally, in a model of in vivo Th2 response, Trem2−/− mice failed to control infection and exhibit metabolic dysregulation. Our findings suggest Trem2 is required for reprograming of macrophages by supporting the energetic requirements for M2 function.
Lymphocyte metabolism in the islet microenvironment
Authors: Felton Jamie Lynn , Bonami Rachel , Hulbert Chrys , Conway Holly , Nelson Jen , Thomas James W
Published: January 2023

Type 1 diabetes (T1D) results in immune-mediated destruction of insulin-producing beta cells in the pancreas. Immune intervention to prevent or reverse T1D is an appealing therapeutic approach; however, selective targeting of autoreactive lymphocytes remains an elusive goal. Recently, cellular metabolism has emerged as a potent modulator of immune cell function. The metabolic state of lymphocytic infiltrate within the highly vascularized islet, termed insulitis, is not known. To test the hypothesis that pathogenic, islet-infiltrating lymphocytes have a distinct metabolic phenotype that has the potential to be selectively targeted, we compared the metabolic signatures of islet infiltrating lymphocytes in two transgenic mouse models. VH125SD.NOD mice develop accelerated diabetes and robust insulitis. BDC2.5.NOD develop insulitis but are protected from T1D. These models were used as examples of pathogenic and protective insulitis, respectively. Mitochondrial polarity was significantly increased in VH125SD.NOD islet-infiltrating B cells compared to BDC2.5.NOD islet-infiltrating B cells. Mitochondrial polarity was also increased in insulin-binding B cells compared to non-insulin-binding B cells in the pancreas, but not in the spleen or pancreatic lymph nodes. Assessment of hypoxia in islet-infiltrating lymphocytes revealed that the oxygen-sensing transcription factor, hypoxia-inducible factor 1α was increased in islet-infiltrating B cells specifically. Together, these findings suggest that tissue specific changes drive metabolic reprogramming at the site of autoimmune attack in the islet and that these metabolic pathways have the potential to be targeted for selective therapeutic interventions in T1D.
Thrombin fine tunes innate immune cell function
Authors: Wilkinson Hannah , Chen Daxin , Dorling Anthony
Published: January 2023

Thrombin is the main effector protease in the coagulation cascade. It can also signal via protease activating receptors (PAR). The presence of these receptors on the surface of innate immune cells has been well reported but the functional consequence of activation has yet to be fully defined. This study aims to investigate the role thrombin has on innate immune cell function using murine bone marrow macrophages (BMM).

In Vitro, stimulating mature MCSF-cultured BMM with thrombin did not affect gross markers of macrophage polarisation (iNOS or CD206). The stimulated cell supernatants contained increased amounts of Interferon gamma (IFNγ) but reduced IL10. Thrombin increased IFNγ receptor expression at the cell surface. Thrombin-treated cells had increased lipid rich microdomains by Cholera Toxin B staining and increased co-localisation of the LPS receptor within the lipid rafts. Compared to untreated cells, thrombin stimulated cells were highly sensitive to low dose M1 polarising stimuli, as evidenced by iNOS expression. The thrombin treated cells down regulated surface ABCA1 expression, but this was prevented by transfecting cells with siRNA against Cullin 3. This preserved ABCA1 expression and prevented the increase in lipid rich microdomains after thrombin stimulation and was associated with the loss of heightened sensitivity to low dose M1 stimuli.

Taken together this shows a clear pro inflammatory signal on the thrombin treated cells and to date the first description of ABCA1’s key role in thrombin mediated inflammatory signalling.
Subsets of thymic resident NKT cells show differential requirements for CD28 co-stimulation during antigen activation
Authors: Shissler Susannah Claire , Singh Nevil J , Webb Tonya J
Published: January 2023

Natural killer T (NKT) cells rapidly respond to antigenic stimulation with cytokine production and direct cytotoxicity. These innate-like characteristics arise from their differentiation into mature effector cells during thymic development. A subset of mature NKT cells remain thymic resident, but their activation and function remain poorly understood. We examined the roles of CD28 and CTLA-4 in driving activation of these thymic resident NKT cells. Unlike previous studies with peripheral NKT cells, the proliferation of thymic NKT cells was significantly impaired when CD28 engagement was blocked - but unaffected with CTLA-4 activation or blockade. Within NKT cell subsets, however, stage 3 NKT cells marked by higher NK1.1 expression were significantly more sensitive to the loss of CD28 signals compared to NK1.1 negative stage 2 NKT cells. In concordance with this, CD28 blockade suppressed all NKT cell cytokines, lowering the ratio of IFN-γ:IL-4 production by NK1.1 positive NKT cells. Intriguingly, the activation-dependent upregulation of the master transcription factor PLZF did not require CD28 co-stimulation in either NK1.1 positive or negative thymic NKT subsets – underlining a dichotomy between requirements for early activation and the subsequent proliferation and effector function of these cells. Collectively, our studies demonstrate the ability of CD28 co-stimulation to fine tune subset-specific responses by thymic resident NKT cells and contextually shape the milieu in this primary lymphoid organ.
Regulatory T cells in skin coordinate responses to epidermal injury by initiating anti-microbial immunity while delaying tissue repair
Authors: Moreau Joshua Michael , Boda Devi P , Rosenblum Michael D
Published: January 2023

When barrier tissues are breached, two coordinate responses need to occur: 1) clearance of pathogens and 2) repair of damaged epithelium. Regulatory T cells (Tregs) play a major role in skin barrier repair; however, the mechanisms by which they modulate local tissue are unclear. To identify molecular pathways underpinning Treg interactions with their tissue environment, we performed single cell RNA sequencing on Tregs isolated from the skin. Our analysis revealed elevated TGFβ and integrin signaling. Specifically, we found high expression of the latent TGFβ activating integrin, αvβ8. To ascertain the physiological significance of this expression we crossed Foxp3CreERT2 and Itgb8f/fmice to generate an inducible and lineage specific system for deleting Itgb8 in Tregs. Deletion of Itgb8 led to improved epidermal healing following sterile injury. Mechanistically, Treg activation of TGFβ directly induced keratinocytes to produce the myeloid chemoattractant CXCL5, leading to neutrophilic inflammation and delayed epidermal healing. We hypothesized that during barrier injury Tregs harness integrin αvβ8 to initiate innate immune responses that temporarily override tissue reparative signals. To interrogate this hypothesis we epicutaneously infected Treg Itgb8 deleted mice with the bacterial pathogen Staphylococcus aureus. Deleted mice developed an uncontrolled infection consistent with impaired immune responses. These data indicate that activation of TGFβ by skin Tregs in response to tissue injury initiates local anti-microbial immunity. Altogether, our study uncovers a unique ability of skin Tregs to leverage TGFβ signaling to coordinate epithelial repair and innate immunity for optimized tissue healing.
Cellular dynamics of immune evasion during Leishmania major infection
Authors: Zayats Romaniya , Mou Zhirong , Yazdanpanah Atta , Koh Wan , Lopez Paul , Uzonna Jude E , Murooka Thomas
Published: January 2023

Concomitant immunity against intracellular parasites is dictated by the immune response and parasite escape mechanisms that prevent clearance. Infection by Leishmania major generates a strong T cell response, yet parasite clearance is incomplete and leaves a small pool of persistently infected cells. T cell behaviours are augmented by the biochemical and spatial conditions of their immediate environments, and a gap in knowledge is the cellular and molecular mechanisms that prevent complete clearance of pathogen-infected cells in the skin.

We combined MP-IVM with a novel T-cell receptor transgenic mouse model, where T cells recognize the immunodominant Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK) peptide, to provide insights into the spatiotemporal dynamics of anti-Leishmania T cell responses during active disease and persistent infection. We show that monocyte-derived macrophage:T cell interaction dynamics were transient at steady-state, but prolonged upon antigen recognition. PEPCK-specific T cells produced high levels of IFNg in response to peptide or infection. We also show that in vitro expanded Treg cells can suppress effector T cell functions, and that greater suppression was observed by PEPCK-specific Treg cells. Ongoing studies that address the cellular and molecular mechanisms of Treg-mediated response blunting will be presented. Intravital microscopy studies characterizing PEPCK-specific T cell migration dynamics and tissue localization within skin lesions directly in live mice will also be presented. The goal of our study is to identify barriers to overcome in order to achieve complete parasite clearance and to better describe the dynamic aspects of concomitant immunity generation.
B7H3 modulate tumor micro-environment via Th1 subset response
Authors: Lee Jun Sik , Kim Mi Eun
Published: January 2023

B7H3 is a new molecule that can regulate T cells-mediated immune responses. Several studies suggested a costimulatory function for B7-H3 in murine models of cancer. In contrast, clinical association of B7-H3 expression suggested a coinhibitory role. The physiological role of B7-H3 is still unclear. To investigate the B7-H3 functions on T cell activation, we used the B7-H3 peptide, B7-H3 negative and overexpressed tumor cells. To establish the T cell inhibitory gene database by B7-H3, we performed the microarray. In the results, B7-H3 inhibited the T cell proliferation and production of cytokines including IL-2 and IFN-γ. We identified the 12 significant effective genes, and confirm the mRNA expression by real-time PCR. These results suggest that B7-H3 showed the co-inhibitory role on T cell functions. And further study is needed to identify the receptor and mechanism of B7-H3 on T cell function.
IL-23 Regulates TH17 Cell Function through Activation of the Unfolded Protein Response Pathway
Authors: Yang Xuexian O , Wu Dandan , Zhang Xing , Liu Meilian
Published: January 2023

IL-23 plays an essential role in TH17 cell-mediated cellular immunity and inflammation. However, the detailed mechanism remains elusive. In the current study, we found IL-23 activated the unfolded protein response (UPR) and promoted TH17 cell function. IL-23 induced UPR factor XBP1s and promotes its downstream genes in TH17 cells, leading to increased expression of TH17 cell cytokines. Induction of XBP1s required IRE1 activation by IL-23; pharmaceutical blockade of IRE1 largely diminished the effects of IL-23 on induction of TH17 cell effector cytokines. Taken together, our results suggest that IL-23 promotes TH17 cell function through activation of the UPR pathway. Targeting the IL-23-UPR axis may provide therapeutic benefit for TH17 cell-driven inflammatory and autoimmune diseases.
Bcl6 is the nexus transcription factor of T follicular helper cell (TFH) differentiation via a set of repressor-of-repressor gene circuits
Authors: Choi Jinyong , Diao Huitian , Faliti Caterina E. , Truong Jacquelyn , Rossi Meghan , Bélanger Simon , Yu Bingfei , Goldrath Ananda W. , Pipkin Matthew E. , Crotty Shane
Published: January 2023

T follicular helper (TFH) cells are a distinct type of CD4 T cells that are essential for most antibody and B lymphocyte responses. TFH regulation and dysregulation is involved in a range of diseases. Bcl6 is the lineage defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl6 controls TFH biology has largely remained unclear, at least in part due to intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches. We ruled out the model that TFH is a default pathway of CD4 T cell by utilizing Bcl6 and Blimp1 double deficient mice in acute virus infection and protein immunization. We showed that Bcl6 has a strong negative autoregulatory loop in TFH cells, by generating mice containing a deletion mutation of an 8-nucleotide Bcl6 binding site in the Bcl6 promoter. To determine putative Bcl6 target TFs, we developed an integrated analytic approach using RNA-seq, BCL6 ChIP-seq, and ATAC-seq data and Bcl6 and Blimp1 double KO mice. Using this approach, Runx2, Runx3, and Klf2 were identified as Bcl6-targeted repressors (Bcl6-r TFs) that regulate important TFH genes. Taken together, Bcl6 controls pleiotropic attributes of TFH differentiation and function (migration, costimulation, inhibitory receptors, and cytokines) via multiple repressor-of-repressor gene circuits.
ITK regulates IL-10 production by CD8+ T cells and lung immunopathology during influenza infection
Authors: McGee Michael C , Solouki Sabrina , Limper Candice B , Ye Kaixiong , Nidetz Natalie F. , August Avery , Huang Weishan
Published: January 2023

Influenza (flu) infections cause 250,000 deaths and 3–5 million cases of severe illness during the average flu season. Severe influenza infections are associated with a combination of strong pro-inflammatory and weak anti-inflammatory immune responses. Production of the anti-inflammatory cytokine IL-10 by T cells restricts immunopathology during flu infections, however our knowledge of the signaling pathways regulating IL-10 induction 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 the development of IL-10-producing CD8+ T cells during influenza A infection. Compared to wild type (WT) mice, Itk−/− mice displayed increased morbidity and mortality after influenza infection, accompanied by a significant reduction of IL-10 producing CD8+ T cells in the airways. Using the model antigen ovalbumin (OVA) and transgenic TCR specific for OVA in CD8+ T cells (OTI), along with an allele sensitive mutation in the ITK kinase domain, we determine that ITK regulates IL-10 production in antigen-specific CD8+ T cells in a kinase dependent manner. RNA sequencing and multiparametric flow cytometric analyses revealed that ITK differentially regulates the expression of cell surface markers and transcription factors that are involved in regulating T cell differentiation, effector and memory phenotypes. Together, our data suggests that ITK is a critical regulator of IL-10 production by CD8+ T cells and regulate immunopathology during influenza infection. Modulating ITK signaling may be a strategy for regulating immunopathology due to viral infections.
Dissecting Human Tissue Resident Memory T cell Heterogeneity on the Single Cell Level
Authors: Szabo Peter Anthony , Dogra Pranay , Thapa Puspa , Poon Maya M.L. , Matsumoto Rei , Kubota Masaru , Bush Erin , Sims Peter , Farber Donna L
Published: January 2023

The majority of T cells throughout the human body persist as non-circulating tissue-resident memory T cells (TRM) in lymphoid and mucosal sites, and are vital for orchestrating protective immune responses. However, given the difficulty of sampling healthy human tissues, a full understanding of TRM identity and function across tissue sites remains lacking. Here, we utilize a unique human tissue resource where tissues are obtained from organ donors to dissect CD4+ and CD8+ TRM heterogeneity using high-dimensional flow cytometry and single cell RNA-sequencing (scRNA-seq). We identify remarkable heterogeneity in expression of key surface markers that define TRM, namely CD103, CD101, CD49a, PD-1 and CXCR6, particularly in the lung and the intestines. These phenotypic groups of TRM denote different functional subsets after activation: CD103+ and CD101+ TRM preferentially produce IL-17A, CD49a+ and CXCR6+ TRM show enhanced IFNγ production, and PD-1+ TRM produce granzyme-B. To investigate the transcriptional programming underlying TRM, we profile ~20,000 resting and activated T cells from the lungs and intestines of human organ donors with scRNA-seq and identify TRM based on our previously defined tissue signature. In both tissues, we detect multiple clusters of TRM enriched for expression of individual or a combination of TRM marker genes. Importantly, these clusters show variable expression of genes coding for cytokines (IFNG, IL23A, IL26), transcription factors (TBX21, GATA3, BATF), and cytotoxic molecules (GNLY, GZMs), suggesting differentially regulated TRM subsets. Our results therefore highlight the heterogeneity of TRM across human tissue sites and define the transcriptional programming underlying TRM function.
CD70 contributes to age-associated T cell defects and overwhelming inflammatory responses
Authors: Kong Yaxian , Wang Di , Du Juan , Song Yangzi , Wang Beibei , Song Rui , Zhao Hongxin , Zeng Hui
Published: January 2023

Aging is associated with immune dysregulation, especially T cell disorders, which result in increased susceptibility to various diseases. Previous studies have shown that loss of co-stimulatory receptors or accumulation of co-inhibitory receptors play important roles in T cell senescence. In the present study, CD70, which was generally regarded as a costimulatory molecule, was found to be upregulated on CD4+and CD8+ T cells of elderly individuals. Consistent with its contradictory role in T cell activation, which was reported by recent studies, the findings of this study highlighted an important role of CD70 in T cell senescence. CD70+ T cells from elderly individuals displayed phenotypic features of exhaustion by expressing enhanced levels of numerous inhibitory receptors and elevated susceptibility to apoptosis. In contrast, aged CD70+ T cells also produced higher levels of pro-inflammatory cytokines and expressed more intracellular Granzyme B and perforin, which was consistent with an important feature of senescent cells termed the senescence-associated secretory phenotype (SASP). Importantly, their functional dysregulation associated with aging was reversed by blocking CD70. To the best of our knowledge, this is the first evidence for the involvement of CD70 in immunosenescence. Collectively, this study demonstrated CD70 as a prominent regulator involved in immunosenescence, which led to defects and overwhelming inflammatory responses of T cells during aging. These findings provide a strong rationale for targeting CD70 to prevent dysregulation related to immunosenescence.
Epigenomics of T Cell Activation and Memory
Authors: Barski Artem
Published: January 2023

A characteristic feature of adaptive immunity is the rapid recall response of memory T cells, the ability of memory T cells to produce cytokines in response to pathogen much more rapidly than naïve T cells. We have recently found that this rapid recall ability is associated with epigenetic gene poising. Here, we aim to uncover the mechanism that underlie chromatin remodeling during initial activation of T cells and, thus, contribute to the establishment of memory phenotype. Upon encountering an antigen, naïve T helper cells become activated, differentiate into several lineages and contribute to immune response. Activation of T cells is dependent on organized and timely opening and closing of chromatin. Herein, we identify AP-1 as the transcription factor that directs most of this remodeling. Chromatin accessibility profiling showed quick opening of closed chromatin in naïve T cells within hours of activation. These newly open regions were strongly enriched for the AP-1 motif, and indeed, ChIP-Seq demonstrated AP-1 binding at more than 70% of them. Broad inhibition of AP-1 activity prevented chromatin opening at AP-1 sites and reduced expression of nearby genes. Similarly, induction of anergy in the absence of co-stimulation during activation, was associated with reduced induction of AP-1 and a failure of proper chromatin remodeling. The translational relevance of these findings was highlighted by the substantial overlap of AP-1–dependent elements with risk loci for multiple immune diseases, most notably multiple sclerosis, IBD and allergy. Our findings define AP-1 as the key link between T cell activation and chromatin remodeling.
Pericellular hyaluronan modulates IL-2 responsiveness through CD44
Authors: Martinez Hunter , Marshall Payton L , Kaber Gernot , Nagy Nadine , Bollyky Paul L
Published: January 2023

CD44 is ubiquitously expressed on human T cells. Stimulation through TCR results in upregulation of CD44. Through interactions with an extracellular matrix ligand, hyaluronan, CD44 classically mediates cell-cell contact for promoting adherence and migration. Here we report a distinct function of CD44 in regulating cellular production and responsiveness to the T cell growth factor IL-2. Overexpression of CD44 in IL-2 responsive cell lines results in a decreased responsiveness to IL-2. Upon CD44 cross-linking, we observe an increased sensitivity to IL-2. The enhanced response to IL-2 is accompanied by diminished Socs1 expression. These data suggest interactions between CD44 and pericellular hyaluronan function to alter cellular responses to IL-2. We propose that this is an underappreciated way inflammation, in the form of an extracellular matrix product, contributes to clonal T cell expansion.
Kennedy pathway regulation by miR-16-1/15a cluster family during T cell activation
Authors: Urena Frank R , Horgen F. David , Ma Chi , Hoffmann Fukun W. , Hoffmann Peter R.
Published: January 2023

The Kennedy pathway in the endoplasmic reticulum is vital for the synthesis of phosphatidylethanolamine (PE) that is incorporated into cell membranes and lipid-based signaling molecules. The three steps of the Kennedy pathway are catalyzed by ethanolamine kinase, CTP-phosphoethanolamine Cytidyltransferase, and selenoprotein I (SELENOI). Our data suggest that two of the three enzymes (ethanolamine kinase and SELENOI) included in this pathway are upregulated during T cell activation as a mechanism for increasing PE, to support increased membrane synthesis and lipidated signaling proteins. Moreover, we have analyzed micro-RNAs (miRs) and found that miR-16-1 and miR-15a, which are linked in the genome and are co-expressed, have sequences predicted to target ethanolamine kinase and SELENOI. Thus, we hypothesized that levels of miR-16-1/15a decrease upon T cell activation to allow increased expression of ethanolamine kinase and SELENOI, thereby driving the increased synthesis of PE. Using real-time PCR and western blot analyses, we found that both miR-16-1 and miR-15a decreased, while levels of ethanolamine kinase and SELENOI increased in mouse primary T cells upon T cell receptor (TCR) activation. This coincided with increased levels of different PE species as measured by LC-MS. Overall, these data support the model of miR-16-1/15a regulation of the Kennedy pathway, in which miR-16-1/15a levels are higher in naïve T cells as a means to keep PE synthesis low. Upon TCR engagement, these miRs are reduced and all three enzymes are upregulated, thereby allowing increased PE synthesis required by activated T cells.
Electrophilic nitro-fatty acids suppress psoriasiform dermatitis through the inhibition of STAT3
Authors: Wang Peng , Killeen Meaghan , Freeman Bruce , Falo Louis D. , Schopfer Francisco , Mathers Alicia R
Published: January 2023

Psoriasis is a systemic, immune-mediated disorder, characterized by chronic inflammatory skin and joint manifestations. Electrophilic nitro-fatty acids (NO2-FAs) have been reported to exert potent anti-inflammatory effects in several in vivo murine models of inflammatory diseases, such as chronic kidney disease and cardiovascular disease. To study the therapeutic potential of NO2-FAs on psoriasis, we employed both acute and chronic murine experimental models of psoriasis. Our studies demonstrate that oral treatments of nitro-oleic acid (OA-NO2), prior to imiquimod (IMQ) or rIL-23 treatments, significantly inhibited the induction of psoriasis-like inflammation and inflammatory cytokine production in the skin. Moreover, utilizing transgenic murine models of psoriasis, we determined that OA-NO2 had both a preventative and therapeutic effect on psoriasiform inflammation. Mechanistic in vitro experiments demonstrated that OA-NO2 decreased IL-17A-induced IL-6 expression in normal human dermal fibroblasts through the inhibition of NF-κB phosphorylation. Importantly, OA-NO2 also impaired signal transducers and activators of transcription 3 (STAT3) phosphorylation and translocation to the nucleus. Further mechanistic results indicated that OA-NO2 regulates STAT3 activation by post-translational modification of STAT3 via nitroalkylation. Overall, our results confirm the critical role of STAT3 in psoriasiform dermatitis and highlight the potential of NO2-FAs as therapeutic agents for the treatment of cutaneous inflammatory diseases, such as psoriasis.
Regulation of IDO1 expression and the kynurenine pathway in synovial fibroblasts
Authors: Municio Cristina , Ergueta Sandra , Usategui Alicia , Del Rey Manuel J , Pablos José L , Criado Gabriel
Published: January 2023

Background

Regulation of tryptophan metabolism along the kynurenine pathway by Indoleamine 2,3-dioxygenase (IDO) has been implicated in immune modulation. The purpose of this study was to investigate the mechanisms underlying IDO1 expression and its consequences on the pro-inflammatory and senescent phenotype of human synovial fibroblasts (SF).

Methods

Human SF were acutely treated with TNFα or IL6/sIL6R. Expression of genes implicated in tryptophan metabolism (IDO1, IDO2, KYNU and WARS) and the senescence associated secretory phenotype (SASP) (IL6, IL8, MCP1 y MMP3) were quantified by RT-PCR. IDO1 was silenced by lentiviral vectors. Expression of IDO1 protein was determinated by Western Blot. Production of kynurenine, a surrogate marker of IDO1 activity, was quantified by ELISA. Quantitative data were statistically analyzed by Pearson or Spearman correlation coefficient or Mann-Whitney test where appropriate.

Results

Basal expression of genes involved in Trp metabolism along the kynurenine pathway was comparable in SF regardless of their origin (normal, rheumatoid arthritis and osteoarthritis). Both TNFα and IL6/sIL6R induced Trp degradation and the expression of IDO1, KYNU and WARS, with IL6/sIL6R being more potent, but only IL6/sIL6R was able to induce expression of IDO2. IDO1 silencing specifically promoted an increase in basal IL6 mRNA levels and protein secretion without affecting other SASP components (IL8, MCP1 and MMP3).

Conclusion

Inflammatory cytokines TNFα and IL6/sIL6R induce the expression of enzymes implicated in the kynurenine pathway in SF. IDO1 negatively regulates IL6 production by SF.
Elucidating Topological Regulation of Antigen Receptor Gene Assembly
Authors: Allyn Brittney , Hayer Katharina , Nganga Vincent , Oltz Eugene M. , Bassing Craig
Published: January 2023

The compaction of antigen receptor (AgR) loci is thought to be important for efficient assembly of broad AgR gene repertoires through recombination of V gene segments with distal D and/or J gene segments of the recombination center (RC). Compaction of mammalian genomes occurs through CTCF protein-mediated chromosome looping, as well as by compartmentalization of chromatin based on transcriptional state. The large AgR loci generally have many CBEs strewn throughout their V segment regions positioned in convergent orientation with a few CBEs flanking their RC. The field hypothesizes that distal V CBEs promote recombination of nearby V gene segments by bringing them into proximity with D and J segments through looping to the convergent RC CBEs. To test this hypothesis, we have mutated CBEs within the mouse TCRβ locus. We find that deletion of the Trbv1 CBE, as well as deletion or inversion of the Trbv14 CBE, lowers, but does not ablate, contact and rearrangement of flanking Vβ gene segments with the distal RC. Unexpectedly, deletion of all convergent CBEs of the RC alters Vβ repertoire but has no obvious effect on overall levels of Vβ recombination, implying that compartmentalization might be the dominant mechanism for compaction and recombination of TCRβ loci. Consistent with this scenario, we show that deletion of the Trbv1 promoter inactivates Trbv1 chromatin and ablates interactions between Trbv1 and the RC. Our ongoing work seeks to determine relative roles of chromosome looping and compartmentalization in promoting and shaping AgR gene assembly.
Characterizing Zap70 signaling mutants in primary T cells using CRISPR/Cas9
Authors: Frie Meredith , Ill Riley , Lillemeier Bjorn
Published: January 2023

T cell activation is controlled by a complex series of signaling events that originate from the T cell receptor (TCR) at the plasma membrane. These signaling events are both spatially and temporally regulated to ensure that T cells activate only in a correct and specific context. A key aspect of T cell activation is the ability of the TCR to recognize peptides with specific affinities while simultaneously discounting a large proportion of MHC-TCR interactions with lower affinities. Our lab has identified Zap70 interaction dynamics with phosphorylated motifs in the TCR as a key mediator of T cell activation thresholds. However, studying these signaling events within primary T cells remains challenging because manipulating the signaling threshold through germline mutations will cause abnormal T cell development and selection in the thymus.

To investigate the role of Zap70 in regulating the activation threshold in mature, peripheral T cells, we have developed a CRISPR/Cas9 expression system in primary murine T cells. Using a retroviral construct, we can simultaneously knock down endogenous Zap70 and replace its expression with ectopic Zap70 mutants with differing TCR interaction dynamics and signal transduction. Ectopic Zap70 is expressed up to 41 days after infection and 33 days after adoptive transfer to Rag1−/− mice. In addition, the primary cells are sensitive to antigenic stimulation comparable to uninfected primary T cells. Using this system, we can both dissect the mechanisms by which Zap70-TCR dynamics regulate signal transduction and determine whether manipulating Zap70-TCR dynamics can affect T cell activity under various pathological conditions.