bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of DCAF1-GSTP1-ROS axis in regulating T cell senescence in immunological ageing and tumors
Authors: Guo Zengli , WAN Yisong
Published: February 2023

Immunological aging is characterized by chronic nonspecific inflammation, compromised vaccination and declined immune competence, contributing many age-associated diseases. As the central regulator of immune system, whether and how regulatory T (Treg) cells are regulated during ageing are not fully understood due to a lack of specific markers. By a combination of cellular, molecular and bioinformatic approaches, we discovered that Treg cells senesce more severely than conventional T (Tconv) cells during ageing. Treg cells from aged mice were less efficient than young Treg cells to suppress Tconv cell function in an inflammatory-bowel-disease model and to prevent Tconv cell ageing in the irradiation-induced ageing model. Further study revealed that DCAF1 (DDB1 and CUL4 associated factor 1) was downregulated in aged Treg cells and was critical to restrain Treg cell ageing via glutathione S-transferase P (GSTP1) regulated reactive-oxygen-species (ROS). Functionally, interfering with GSTP1 and ROS pathways reinvigorated the proliferation and function of aged Treg cells. Thus, the DCAF1-GSTP1 regulated ROS contributes to Treg cell senescence, which led to systemic immunological ageing.

In tumor microenvironment, ROS produced by Treg cells and myeloid-derived suppressive cells (MDSCs) is one of the major inhibitory molecules suppressing anti-tumor immunity. We found that there was significant increase of senescent T cells in tumors in a B16 melanoma model, featured with increased p16 expression, elevated senescence associated β-gal activity and increased KLRG1 expression. Further investigating the role of DCAF1-GSTP1-ROS axis in tumor infiltrating T cells will provide novel mechanistic insights to cancer immunotherapy.
Corepressors in CD4+ T cell development and homeostasis
Authors: David Natalie A , Lee Robin D , Owen David L. , Farrar Michael A.
Published: February 2023

Corepressors NCOR1 and NCOR2/SMRT serve as scaffolds for the recruitment of histone deacetylases to transcription factor-DNA complexes. These closely related proteins are functionally non-redundant. Though they were first identified by their interactions with nuclear receptors, NCOR1/2 also interact with non-nuclear receptor transcription factors, including several involved in T cell development and polarization. Previous research identified that NCOR1 promotes survival of double positive thymocytes and protects them from negative selection. We have built upon this work by examining the effect of combined NCOR1/2 deletion in developing thymocytes. NCOR1/2 deletion using a CD4-Cre model results in an 8-fold decrease in the frequency of CD4 single positive T cells, compared to a 2-fold decrease and an insignificant decrease for NCOR1 and NCOR2 deletion alone, respectively. NCOR1/2-deficient T cells demonstrate exquisite sensitivity to TCR stimulation and polarize to Th subsets at different frequencies compared to wild-type CD4+ T cells. Despite a profound loss of CD4 single positive T cells and thymic regulatory T cells (Tregs), the frequency of both Treg progenitor populations remains unchanged. In contrast, the frequency of splenic Tregs is not reduced in NCOR1/2-deficient mice. However, there is a striking increase in activation of conventional T cells, suggesting that T cell tolerance is perturbed. Using an OX40-Cre deletion model which deletes NCOR1/2 in dendritic cells (DCs), activated T cells, and Tregs, we observe a decreased CD4+/CD8+ ratio and high incidence of ulcerative dermatitis. We are currently examining how Treg or DC specific deletion of NCOR1/2 affects T cell development, homeostasis, and tolerance.
Building a Mini Biomedical Ethics Curricular Thread in an Immunology Course in Undergraduate Medical Curriculum
Authors: Zhao Yuan , Olaiya Oluwaseun
Published: February 2023

Education in biomedical ethics has become an essential part of undergraduate medical curriculum. Integrating this topic longitudinally in the curriculum ensures early educational exposure and provides opportunities for continued advancement of students reasoning and behavior. The objective of this study is to develop a mini ethics curricular thread in the immunology course and to raise student awareness of ethical issues associated with disorders of the immune system. In our project, three ethics learning modules are designed and built in a six-week immune system course in the second semester of undergraduate medical curriculum. A mixed teaching approach using multiple teaching tools will be employed. The first module uses a movie about a well-known patient with severe combined immunodeficiency disease. The second module is delivered through a team-based learning case on human immunodeficiency virus. The third module uses small and large group discussions on blood transfusion and bone marrow transplantation. Quizzes and reflective writings are embedded in each module to assess student knowledge and attitudes. Student surveys will provide information on challenges and opportunities in teaching ethics within a biomedical science course. The qualitative analysis of student reflections will be presented in the poster. We believe this curricular design has the potential to help students build connections between science, human disease, and ethics. It will also shed light on how to integrate ethics in biomedical science courses to cultivate professionalism in the early stages of their medical training. Future direction includes the evaluation of the longitudinal impact of this training on students clinical practice.
Investigating the age and sex associated mucosal immune landscape of urinary bladder
Authors: Hamade Ali , Li Deyang , Tyryshkin Kathrin , Shakfa Noor E. , Chenard Stephen , Siemens Robert , Koti Madhuri
Published: February 2023

The incidence of BC is four times higher in males than females, however, females tend to present with a more aggressive disease, a poorer response to immunotherapy, and suffer worse clinical outcomes. Our recent novel findings demonstrated sexual dimorphism in the tumor immune microenvironment of non-muscle invasive bladder cancer, which is primarily treated with local immunotherapy. Investigating sexual dimorphism in the clinical journey of BC beyond the static local immune landscapes remains a challenge. With similarities in mucosal immune physiology between humans and mice, and the recently reported age-associated pathogenic stimuli independent inflammation observed female murine bladders, we conducted a comprehensive evaluation of the sex and age-related immune alterations in healthy murine bladders.

Bulk-RNA sequencing and multiplex immunofluorescence based spatial immune profiling of normal murine bladders from male and female mice of the ages 3, 6, 9, 12, 15, and 18 months were performed. Preliminary findings from the transcriptomic analysis showed a highly altered immune landscape that exhibited sex differences in aged bladders. Spatial profiling of using markers specific to macrophages (CD163, CD11b), T lymphocytes (CD3, CD8), B lymphocytes (Pax5), activated dendritic cells (CD208), high endothelial venules (PNAd), inflammatory cells (Ly6G) and the PD-L1 immune checkpoint, showed sex and age associated differences. Older female mice had a higher density of tertiary lymphoid structures/lymphoid aggregates compared to both young female and male equivalents. Findings from this study will allow the appropriate modeling in pre-clinical studies evaluating immunotherapeutic agents for NMIBC treatment.
CD8 T cell differentiation in cancer is comprised of two distinct phases
Authors: Prokhnevska Nataliya , Cardenas Maria , Valanparambil Rajesh , Jansen Caroline , Master Viraj , Sanda Martin , Kissick Haydn
Published: February 2023

A crucial part of the immune response to tumors are CD8 T cells, with CD8 infiltration predicting disease progression in many cancers. Recent work has shown two subsets of CD8 T cells that respond to tumors, one a stem-like CD8 T cell (TCF1+) that can give rise to a more cytotoxic terminally differentiated cell (TCF1−). To understand the CD8 T cell response to tumors it is important to study how tumor-specific CD8 T cells activate and differentiate. To study this we have made a prostate cancer model which expresses the LCMV glycoprotein (GP) and acts as a tumor-specific antigen. This model allows us to transfer LCMV GP specific P14 CD8 T cells into TRAMPC1-GP bearing mice to study how tumor-specific CD8 T cells activate. These studies have shown that tumor-specific CD8 T cells are activated in tumor draining lymph nodes (TDLN), where they retain an activated undifferentiated phenotype, upregulating CD44, PD1, while maintaining TCF1. These tumor-specific CD8 T cells only differentiate (TCF1−) once they have migrated into the tumor. This model can also be seen in human prostate cancer, with CD8 T cells in TDLNs retaining an activated undifferentiated phenotype (PD1+CD45RA-TCF1+). To determine if these cells are related to the CD8s within the tumor we have shown TCR overlap between the activated CD8s T cells in human prostate TDLNs and the CD8 T cell subsets within the tumor. These data suggest a two-step differentiation process for tumor-specific CD8 T cells where they are activated in TDLNs and differentiate further only in the tumor. This model of two distinct phases of CD8 T cell differentiation adds to the basic understanding of the immune response to cancer.
Gene expression analysis of sarcoma-infiltrating NK cells identifies signatures correlating with favorable prognosis and potential new targets for augmenting NK function
Authors: Judge Sean J , Dunai Cordelia , Razmara Aryana , Cruz Sylvia M , Canter Robert J
Published: February 2023

Success of current T-cell based immunotherapies in soft tissue sarcomas (STS) is limited while pre-clinical and clinical studies have shown evidence of NK activity in STS. To investigate the effects of the tumor microenvironment (TME) on tumor-infiltrating NK cell gene expression, we isolated circulating and tumor-infiltrating NK and CD3 T cells from sarcoma patients undergoing surgery (N=4) and performed RNA sequencing analysis. Comparing Differential Gene Expression (DGE) of flow-sorted intra-tumoral NK cells to circulating NK cells, we identified upregulation of genes involved in mitogen signaling inhibition (DUSP4) and metabolic function (SMPD3, SLC7A5), but not of genes typically associated with cytotoxic function (e.g. IFNG, GZMB). In contrast, intra-tumoral T cells showed significant upregulation of both activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. We then queried the TCGA STS dataset and stratified cases into “NK Activating TME” and “NK Suppressing TME” based on the CIBERSORT NK signature analysis program (based on high or low proportion of activated NK cells, respectively). Patients with NK Activating TME had significantly improved survival compared to NK Suppressive TME (log-rank p = 0.03). DGE identified unique tumor-associated gene ontology between NK activated versus suppressive subsets with upregulation of the retinoic acid pathway. Unlike T cells which demonstrate significant DGE between circulating and tumor-infiltrating subsets, NK cells appear to have similar gene expression between blood and tumor. Therefore, although NK cells appear to be prognostic in STS, their anti-tumor effects may be dictated by systemic rather than local factors.
Phosphoproteomics reveals GITR agonism differentially regulates inflammatory vs. regulatory T cell subsets.
Authors: Davis Andrew , Cunningham Lori , Stokes Matthew , Nelson Alissa , Abell Kathryn , Lewis Michael , Scialdone Roy , Bui Than , Alonzo Emily , Freeman Gordon J , Klein Sarah
Published: February 2023

Triggering the co-stimulatory receptor glucocorticoid-induced TNFR-related protein (GITR) on T cells has been identified as a promising cancer immunotherapeutic strategy, yet the signaling events by which GITR agonism induces antitumor immunity are not well understood. GITR is expressed on activated CD4+ and CD8+ T cells, and is constitutively expressed on regulatory T cells (Tregs). While GITR appears to act as a conventional co-stimulatory receptor in effector CD4+ and CD8+ T cells, the role of GITR on Tregs remains controversial. The goal of this research was to utilize a phosphoproteomics approach to identify and compare GITR-mediated intracellular signaling events in induced-Tregs (iTregs), CD4+ T cells, and CD8+ cytotoxic T cells. We used the GITR agonist monoclonal antibody DTA-1 to stimulate the T cell subsets and confirmed GITR signaling via NFκB pathway activation and phospho-JNK induction. The phosphoproteomics data revealed different protein phosphorylation patterns among T cell subsets. For example, we observed phospho-p38 MAPK induction in the iTreg subset but not in the CD4+ T cells or CD8+ cytotoxic T cells. We also observed additional phospho-proteins induced by GITR triggering in the p38 pathway in the iTreg subset including phospho-MKK3, phospho-MKK6, phospho-MAPKAPK-2, and phospho-ATF-2. Induction of p38 MAPK signaling is functionally important as inhibition of p38 abrogated GITR-induced proliferation of iTregs. These data demonstrate that GITR agonism induces distinct phospho-proteome profiles between T cell subsets, and this differential signaling leads to unique responses that will need to be considered when targeting GITR with immunotherapeutic strategies.
PD1 Blockade of Tumor-primed CD4 T Cells Instigates Protective Immunity Against Tumor Rechallenge.
Authors: Falo Louis D , Hao Xingxing , Chen Guo , Carey Cara D , Liu Zuqiang , Bartlett David , Storkus Walter J , Falo Louis D , You Zhaoyang
Published: February 2023

Blockade of immune checkpoint PD1 in cancer patients results in remarkable therapeutic benefits. Revealing mechanisms underlying this revolutionary therapy is critical to identifying biomarkers for predicting responses to this treatment and developing novel PD1 blockade combined therapeutic regimens for broader clinical efficacy. Rejuvenation of preexisting intratumoral ‘exhausted’ T cells and recruitment of peripheral antitumor T cells into tumors have been suggested to explain how PD1 blockade works, but mechanisms of action of anti-PD1 therapy remain to be fully delineated. In a clinically relevant mouse model of spontaneous metastatic breast cancer 4T1.2-Neu, we show that effective and durable anti-PD1 therapy depends on systemic immunity, including type 1 conventional dendritic cells (cDC1: CD8α+/CD103+DC), B cells, CD4 and CD8 T cells. Increased circulating and/or tumor-infiltrating IL12+CD103+DC and IL2+CD4/CD8 T cells are associated with the response to anti-PD1 therapy. PD1 blockade results in tumor-primed CD44+IL2+CD4T cells in the periphery regardless of cDC1. In the breast cancer model with adoptive cell transfer (ACT), PD1 blockade-driven CD44+CD4+T cells infiltrate into tumors and ignite protective immunity against new tumors in an IL2-dependent and tumor-specific manner. In a distinct ACT model using enforced IL2-expressing melanoma antigen specific TRP1-transgenic (Tg) CD4 T cells, IL2+TRP1-Tg CD4 T cells control the growth of PD1-resistant endogenous BP or B16 melanoma. These data suggest the prominent role of PD1 blockade on the effectiveness of tumor-primed CD4T cells in tumor-specific immunity.
ELISA detection of human endogenous retroviral protein HERV-WL in saliva of cancer patients and normal individuals
Authors: Tsiagbe Vincent K , Nemati-Rad Azadeh , Bortnik Volha , Fatahzadeh Mahnaz , Li Yu
Published: February 2023

Previous studies revealed that a novel Human Endogenous Retrovirus (HERV-WL) was overexpressed in tissues from patients with certain aggressive human cancers, using immunohistochemistry techniques. ELISA detection also showed overexpression of HERV-WL in saliva of patients with certain aggressive cancers. To further explore these findings, saliva samples obtained from 12 patients with various cancers (1 with breast & colon cancer, 1, with colon cancer, 1 with multiple myeloma, 1 with prostrate cancer, 1 with renal carcinoma, and 7 with squamous cell carcinoma, were compared to saliva from 5 normal individuals. The studies showed significantly higher HERV-WL protein expression in saliva samples from all the 12 cancer patients (2–157 ug/ml), while HERV-WL protein expression was undetectable or less than 0.5 μg/ml in saliva from normal individuals. These findings warrant further research into the use of HERV-WL expression in saliva as a non-invasive assessment of responsiveness of certain cancers patients to therapy.
BATF3/MYC axis drives hyper-proliferation of TET2 deficient CAR T cells
Authors: Jain Nayan , Zhao Zeguo , Iyer Archana , Lopez Michael , Feucht Judith , Koche Richard , Yang Julie , Zhan Yingqian , Sadelain Michel
Published: February 2023

A recent report described a patient presenting a dominant chimeric antigen receptor (CAR) T cell clone at the height of anti-tumor response wherein a TET2 allele was disrupted by the integration of the lentiviral 4-1BB CAR vector (Fraietta et. al. Nature 2018). The enhanced proliferative ability conferred by this chance TET2 disruption opened up the possibility of generating TET2 edited CAR T cells for an enhanced anti-tumor efficacy. We modeled and evaluated loss of TET2 in human T cells engineered to express different CAR receptors in a murine model of human acute lymphoblastic leukemia (ALL). Our results indicate that CAR design plays a critical role in determining anti-tumor efficacy and early stage T cell phenotype upon TET2 disruption. We further observe that TET2 edited CAR T cells, over time, enter a hyper-proliferative state with a near total loss of effector function. The frequency with which TET2 edited T cells achieve hyper-proliferation is dependent on the signaling properties of the CAR receptor. This state is in contrast to the canonical exhaustion state wherein loss of proliferative ability precedes loss of effector function. This hyper-proliferative state shares some transcriptional features with T cell leukemia/lymphoma with sustained upregulation of cell cycle associated factors. Exome analysis identified point mutations and chromosomal aberrations in hyper-proliferative cells, but they were not conserved across different populations. Chromatin accessibility analysis revealed that loss of TET2 sets an epigenetic state that allows for sustained elevated levels of BATF3 which in turn drives a MYC-dependent proliferative program in TET2 deficient CAR T cells.
GD2 CAR-T Cells exhibit strong cytolytic potency against glioma stem cells
Authors: Hayes Heather B , Logun Meghan T , Chvatal Stacie A , Mueller Katherine P , Piscopo Nicole J , Das Amritava , Saha Krishanu , Brat Daniel J , Karumbaiah Lohitash
Published: February 2023

Glioblastoma (GBM) is an aggressive brain cancer without effective treatments. CAR-T cells targeted to tumor-associated antigens offer promise for treating GBM. Here, we used cellular impedance assays to compare the cytolytic potency and kinetics of conventional viral vs non-viral CRISPR engineered GD2 CAR-T cells against glioma stem cells (GSC), a subpopulation of glioblastoma cells.

Patient-derived N08 GSCs were plated at 50k cells/well on 96-well plates, and impedance was continuously monitored on the Maestro Z impedance platform (Axion BioSystems). GD2 CAR-T cells were engineered using either retroviral transduction (RV) or non-viral CRISPR editing (NV). At 48 hours, GD2 CAR-T cells were added at Effector:Target ratios of 0.1:1, 1:1, and 10:1. Comparisons were made to mCherry T cells (mCh) as a control. Impedance and cytolysis were monitored up to 7 days.

RV and NV GD2 CAR-T cells caused decreases in impedance consistent with T cell-mediated lysis of GSCs, whereas mCh T cells induced little change. NV CAR-T cells exhibited faster killing kinetics compared to RV CAR-T cells. The time to 50% cytolysis (KT50) was significantly shorter for NV vs RV CAR-T cells at 1:1 and 10:1 E:T ratios.

Cytotoxic function was validated with flow cytometry and cytokine analysis at 7 days. All T cells exhibited chronic activation measured by CD69 and CD137 upregulation. Importantly, NV CAR-T cells exhibited less exhaustion, as measured by PD1 and LAG3 expression.

Both RV and NV GD2 CAR-T cells effectively cytolyzed GSCs, with NV CAR-T cells exhibiting more potent and efficient killing. The high potency, fast kinetics, and reduced exhaustion of NV CRISPR GD2 CAR-T cells offer great clinical promise for treating GBM.
The Nanobody JK36 circumvents the epitope overlapping commonly observed between therapeutic anti-CD38 antibodies and commercially available analytical mouse antibodies
Authors: Lacaille Vashti , Kapinsky Michael
Published: February 2023

Therapeutic anti-CD38 antibodies have been demonstrated to elicit immune-mediated elimination of plasma cells and other CD38-expressing cells. Epitope overlapping with commercially available analytical monoclonal mouse anti-CD38 antibodies limits the possibility to study surface CD38 expression in the presence of these therapeutic antibodies. Nanobodies, derived from heavy-chain antibodies that naturally occur in llamas and other Camelids, can circumvent these undesired interferences. Nanobodies are single variable domain antibody fragments (VHH) that often expose a long complementarity-determining region 3 (CDR3). This feature allows them to recognize hidden epitopes, e.g. in molecular cavities, that are inaccessible to the CDRs in conventional analytical and therapeutic antibodies. In a model of anti-CD38 occupation with a therapeutic antibody, we have evaluated JK36, an anti-CD38 nanobody recognizing a cryptic epitope. Normal whole blood samples were pre-incubated with Daratumumab, a therapeutic humanized anti-CD38 mouse antibody. Binding of the Rabbit-JK36 (Rb-JK36) construct was compared to the binding observed with the conventional mouse antibodies, clones LS198-4-3 and T16. LS198-4-3 and T16 antibodies partially or completely failed to label the CD38 epitope, in case of being masked with Daratumumab. In contrast, the Rb-JK36 staining pattern and intensity remained unchanged, confirming that Rb-JK36 binds to an epitope which is not masked by Daratumumab. This study confirms that JK36 is a well-suited probe for research studies on CD38 expression in the presence of therapeutic or commonly used analytical anti-CD38 antibodies, opening new methodical approaches in plasma cell immunotherapy research.
Blockade of checkpoint ILT3/LILRB4/gp49B binding to fibronectin ameliorates autoimmune disease in BXSB/Yaa mice
Authors: Su Mei-Tzu , Inui Masanori , Endo Shota , Takai Toshiyuki
Published: February 2023

Extracellular matrix (ECM) not only provides a supporting framework for each tissue, but also elicits cellular responses by providing an inductive substrate. Fibronectin (FN), a major ECM macromolecule, is composed of multiple modules that interact with ECM-associated proteins, plasma proteins, and cell-surface proteins to ensure its modulatory roles. Here we revealed that FN is identified as a physiological ligand of the immunoglobulin (Ig)-like transcript 3/leukocyte Ig-like receptor B4 (ILT3/LILRB4) which serves as an immune checkpoint for autoimmune and cancer diseases. Human LILRB4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major LILRB4-binding site in FN was located at the N-terminal 30-kDa module (FN30). Blockade of B4–FN binding with gp49 antibody or a recombinant FN30-Fc fusion protein reduced the secretion of pathogenic IgG autoantibodies, thereby reducing PAS-positive glomerular deposition in lupus-prone BXSB/Yaa mice. Compared with the control group, activation-induced cytidine deaminase (AID) expression in splenic activated B cells of FN30-Fc-treated BXSB/Yaa mice was decreased, indicating gp49B-FN30 signaling contributes in the class switch recombination (CSR) of Ig genes. These results suggested that gp49B–FN interplay is critical for pathogenic autoantibodies-mediated glomerulonephritis in the BXSB/Yaa lupus mouse model.
Neutrophil-specific Socs3 deficiency induces brain-targeted experimental autoimmune encephalomyelitis with increased cerebellar neutrophils
Authors: Turbitt William J , Yan Zhaoqi , Yang Wei , Buckley Jessica , Zhou Lianna , Qin Hongwei , Benveniste Etty
Published: February 2023

Multiple sclerosis (MS) is associated with a dysregulated JAK/STAT signaling pathway. Suppressors of cytokine signaling (SOCS) negatively regulate the JAK/STAT pathway. MS patients with disease affecting the cerebellum display rapid progression and poor prognosis. Previous data from our laboratory demonstrates a severe, brain-targeted form of experimental autoimmune encephalomyelitis (EAE) in mice lacking Socs3 in myeloid cells (Socs3ΔLysM), with increased cerebellar infiltration of primed neutrophils. Here, we induced EAE in mice lacking Socs3 specifically in neutrophils (Socs3ΔLy6G; n=26) and Ly6G−/+ control mice (n=13), and examined disease progression, neutrophil populations and CD4+ T-cell polarization at disease peak. Ly6G−/+ mice exhibited clinical signs of classical EAE, whereas Socs3ΔLy6G mice exhibited clinical signs of brain-targeted EAE, similar to Socs3ΔLysM mice. An analysis of the cerebellar cell infiltrate in Socs3ΔLy6G mice demonstrated an increased cellularity (p=0.005), which was comprised of both an increase in the percentage (p=0.020) and total number (p<0.001) of cerebellar neutrophils. Neutrophils from Socs3ΔLy6G mice exhibited a primed phenotype with increased surface expression of CD11b (p=0.084) and reduced expression of CD62L (p<0.001). Additionally, Socs3ΔLy6G mice exhibited an increase in the percent of cerebellar CD4+ T-cells (p=0.018) and a shift toward IFN-γ-producing Th1 CD4+ T-cells (p=0.012). These data conclusively document that neutrophil-specific deficiency of Socs3 is sufficient to induce brain-targeted EAE. Future studies will identify targets to dampen the role of neutrophils in brain-targeted autoimmune neuroinflammation.
METABOLIC AND STRUCTURAL MUSCULAR CHANGES DRIVEN BY CHRONIC DISEASE-LIKE IFN-G EXPOSURE
Authors: Fenimore John Maxwell , Springer Danielle , Valencia Julio C , Edmonston Elijah , Young Howard A
Published: February 2023

Cardiovascular myopathies and fatigue have been associated with chronic inflammatory responses and activated cell-mediated immunity. We report aberrant cardiovascular activity in the form of reduced ejection fraction in vivo in male mice with persistent low-level expression of IFN-g (ARE mice). ARE mice have a replacement with random nucleotides in the 3 prime UTR of the IFN-g gene that serves to remove a regulatory element (AU Rich) that is integral at controlling the stability of IFN-g mRNA. ARE mice had increased cellular and mineral, infiltrates into heart tissue and mitochondria linked metabolic changes that were concomitantly observed following a stress-exacerbated decrease in heart function, with or without exposure to IFN-a. Our data also demonstrates an increase in glucose expenditure and increased general fatigue of ARE mice as well as structural and ultrastructural changes in the heart in male mice. We also observe a decrease in the transcripts and a reversible decrease in function of aerobic respiratory components in the muscle of male mice exposed to IFN-g. Notably, we observe a decrease in X chromosome linked pyruvate dehydrogenase, a key component in pyruvate facilitated entry to the TCA cycle, and other downstream mitochondrial genes. Furthermore, the in vivo data revealed an increase in factors associated with fatigue, such as lactic acid production and changes in expression of genes associated with an increase inanaerobic respiration in cardiac musculature. These results indicate that the chronic expression of IFN-g results in a model for male biased heart failure understress in an autoimmune environment, like the idiopathic cardiomyopathy found in male Systemic Lupus Erythematosus patients.
Aberrant CD39 levels and regulation mark COVID-19 late stage
Authors: Longhi Maria Serena , Wang Na , Kalbasi Ahmadreza , Vuerich Marta , Manickas-Hill Zachary J. , Hecht Jonathan , Shaefi Shahzad , Robson Simon C.
Published: February 2023

Previous studies have reported immune cell exhaustion and perturbed T-cell immunity in novel-coronavirus-disease-19 (COVID-19). Heightened levels of CD39, an ectonucleotidase key to immunoregulation, are detected in CD4 and CD8 T-cells during viral infection where they correlate with worse disease outcome.

We performed a comprehensive transcriptome analysis of T-cell related gene pathways in PBMCs from 18 COVID-19 patients (6 convalescent, 6 with moderate disease and 6 with severe disease) and 6 healthy controls (HC); and in formalin-fixed autoptic tissue, obtained from the spleen, lung, kidney, liver and heart of 5 COVID-19 cases and 2 controls. Analysis was performed using nCounter CAR-T characterization panel (NanoString).

Our data show that in COVID-19 tissues there is a marked decrease in TCR diversity that is also present in PBMCs of severe patients, when compared with patients with moderate disease, convalescents and HC. A similar pattern is also noted when analyzing PBMCs for TCR signaling, Th1, Th17 and Treg-related transcripts. Heightened CD39 transcripts are detected in lung, liver and spleen of COVID-19 patients as well as in PBMCs obtained from patients with severe disease. A similar trend is observed for STAT-3, and HIF-1alpha, transcription factors that regulate CD39. Notably, CD39-AS RNA, a lncRNA that regulates CD39 at the post-transcriptional level, is markedly reduced in severe COVID-19 patients.

In conclusion, our data indicate systemic and local alterations in T-cell immunity in COVID-19. Aberrantly high levels of CD39 and related transcription factors and concomitant decreases in CD39-AS RNA in the late stage of disease suggest a role for CD39 and purinergic signaling in COVID-19 progression.
A role for PD1-PDL1/2 regulatory axis in suppressing T cell independent memory B cell generation and activation.
Authors: Spurrier M. Ariel , Jennings-Gee Jamie E , Daly Christina A , Haas Karen M
Published: February 2023

T cell-independent type 2 antigens (TI-2 Ags), including polysaccharides present on the surface of encapsulated bacteria, are limited in their capacity to generate a recall response. The failure of native polysaccharide-based vaccine-elicited antibody responses to boost following either revaccination or natural infection is a barrier to establishing protection against infectious diseases and is a poorly understood facet of humoral immunity. Previous work has indicated a role for antigen-specific IgG in suppressing TI-2 antibody recall responses, but questions remain as to how TI-2 memory B cell formation and reactivation is regulated. In the current study, we investigated the role of the PD1-PDL regulatory axis in regulating B cell recall responses to TI-2 Ags. Using adoptive transfers of B cells from VHB1–8 transgenic mice in which a fraction of B cells express specificity for NP, we demonstrate B cell-intrinsic PD-1 expression suppresses memory B cell (Bmem) generation as well as Bmem reactivation in response to NP-Ficoll. B cell-extrinsic PDL1 and PDL2 both contribute to suppression of Bmem generation and reactivation. B cell-extrinsic PDL1 and Bmem-intrinsic PDL2 expression play key roles in PD-1-mediated suppression of Bmem reactivation and antibody production. In summary, this work highlights a key role for PD-1 and its ligands in regulating the formation and functional reactivation of TI-2 Ag-specific memory B cells and reveals new avenues for improving TI-2 Ag-based vaccine responses.
Neonatal B cell receptor induces a STAT-dependent negative regulator
Authors: Sakai Jiro , Akkoyunlu Mustafa
Published: February 2023

Neonates and infants have impaired immune responses to immunization compared to adults, resulting in high susceptibility to microbial infection. B lymphocytes have a central role in humoral immunity to immunization and infection, and signals via B cell receptors (BCRs) are essential for the development and maintenance of B lymphocytes. Neonatal B cells, however, do not respond to BCR activation as robustly as adult cells do.

We have identified that signal transducer and activator of transcription (STAT)-related signaling pathways were uniquely activated in neonatal B cells following BCR cross-linking. Among the STAT proteins, we found that STAT3 and STAT5 were highly phosphorylated in neonatal B cells compared to adult B cells in response to BCR activation. Using small molecule inhibitors and small interfering RNA, we identified that STAT5-induced IL-6 activates STAT3 in an autocrine or paracrine manner. Moreover, IL-6-induced STAT3 activation leads to the production of anti-inflammatory cytokine IL-10.

Neonatal spleen is known to contain higher numbers of IL-10 producing Breg cells than adult spleen and IL-10 is known to act as a negative regulator of T cell functions including survival and cytokine production. Our studies unveiled the pathways involved in the production of IL-10 in BCR-activated neonatal cells. We showed that BCR-activated neonatal cells secrete IL-6 in a STAT5-dependent manner. Subsequently, IL-6 signaling through STAT3 leads to the production of IL-10 from B cells.
Marginal zone B cells have higher basal BCR signaling that correlates with altered mobility and spatial organization of IgM-BCRs
Authors: Abraham Libin , Scurll Joshua M , Tafteh Reza , Lu Henry Y , Falcão Rebeca Cardim , Chou Keng C , Coombs Daniel C , Gold Michael R
Published: February 2023

Marginal zone (MZ) B cells exist in a partially-activated ‘primed’ state but the molecular basis for this priming is not fully understood. We found that MZ B cells exhibit greater antigen-independent ‘tonic’ BCR signaling than naïve follicular (FO) B cells. Because BCR signaling output is dependent on BCR spatial organization and BCR-BCR interactions, we hypothesized that the increased tonic BCR signaling in MZ B cells is due to altered lateral mobility and nanoscale organization of BCRs. Single-particle tracking showed that surface IgM-BCRs on MZ B cells have higher diffusion coefficients and decreased confinement compared to IgM-BCRs on FO B cells. In contrast, the mobility and confinement of IgD-BCRs was similar on MZ and FO B cells. To assess BCR nanoscale organization, we used dSTORM and a novel graph-based clustering algorithm. This revealed that IgM-BCRs were more dispersed and less clustered on MZ B cells than on FO B cells, whereas IgD-BCR spatial organization was similar on the two cell populations. Importantly, 3-color STED imaging revealed that phospho-CD79 nanoclusters overlapped to a much greater extent with IgM-BCRs than with IgD-BCRs on FO B cells, and that this IgM-pCD79 overlap was even greater in MZ B cells. MZ B cells also exhibited greater pCD79 signaling in response to membrane-bound antigens than FO B cells. Thus, MZ B cells have greater tonic and antigen-dependent BCR signaling, which correlates with altered IgM-BCR mobility and organization
The link between Complement System and Anemia in Gaucher disease
Authors: Pandey Manoj K , Magnusen Albert Frank , McKay Mary Ashley , Hatton Shelby Loraine , Magnusen Daniel Nii Aryee , Rapien Jillian , Nyamajenjere Tsitsi Carol , Dipasquale Betsy Ann , Woods Christopher
Published: February 2023

Gaucher disease (GD) is the classical example of lysosomal storage disease, which happens due to mutations in GBA1 as well as the resultant deficiency of glucocerebrosidase (GCase) and the excess tissue accumulation of glucosylceramide (GC). GD patients have shown high risk for developing anemia, which is characterized by Red blood cells (RBCs) and the hemoglobin (Hb) abnormalities. The exact mechanism by which such blood aberrations progress in GD is not clear. We have described immune complexes of GC-specific IgG autoantibodies in GD, which induced massive complement activation and the resulting complement 5a (C5a) and its cognate receptor 1 (C5aR1)-mediated immune inflammation and tissue disruption in GD. Complement activation had been linked to RBCs and/or Hb abnormalities in several diseases, (e.g., atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus, malaria, and β-thalassemia). Hence, we hypothesized that the C5a-C5aR1 activation is critical for anemia development in GD. To indorse this theory, blood from C5aR1 deficient (C5aR1−/−) and sufficient (C5aR1+/+) experimental mouse model of GD as well as the background matched control WT and C5aR1−/− mice were measured for RBCs and Hb with uses of automated system and the FACS staining methods. Our data illustrate that C5aR1+/+ mouse model of GD causes increased shortage of RBCs and Hb when compared to control WT or C5aR1−/− strains. Strikingly, C5aR1−/− mouse model of GD were protected from the loss of RBCs and Hb. These findings suggest that C5a-C5aR1 axis is a critical driver of disease processing of anemia in GD. Targeting C5a-C5aR1 axis may stop or slow down anemia development in GD.
Production of antigen-specific IgE requires the properly timed help from TFH cells in a food allergy model
Authors: Chen Qiang , Dent Alexander
Published: February 2023

Food allergy is characterized by food antigen (Ag)-specific IgE-mediated hypersensitivity. Follicular helper T cells (TFH) have been shown to control IgE production. However, the mechanisms for how TFH cells regulate IgE is not well understood. Utilizing a food allergy model in which mice are sensitized using peanut extract and cholera toxin through oral gavage, we found that peanut-specific IgE but not non-specific IgE requires two intragastric gavage (ig) Ag primings given 7 days apart. QPCR for germline expression of IgE showed that class-switching to IgE mainly occurs after second priming, which is different from IgG1. When NP-OVA and NP-KLH are used as Ags, we found that NP-specific IgE is still produced and thus maintaining the Ag specificity of TFH cells at both primings is not necessary. The expression of several IgE-promoting cytokines including IL-4 and IL-13 were upregulated in TFH cells upon the second priming. The requirement of IL-4 in antigen-specific IgE is validated using antibody blocking and Il4-heterzygote mice. Furthermore, we found that Ag-specific IgE is not produced when the timing between first and second timing is too short (2 days) or too long (2 weeks). TFH cells from these different timing showed different gene profile, indicating a possible role for the regulatory factors Fgl2 and CD39. These suppressive genes have not been associated with TFH function in food allergic IgE before. In conclusion, the production of Ag-specific IgE in gut priming requires a secondary priming and TFH cells play a crucial role through cytokines and other factors.
Effect of non-tuberculous mycobacteria on the protective efficacy of BCG vaccine via modulation of mucosal immunity
Authors: Dutt Taru S , Fox Amy , Karger Burton Richard , Rampacci Elisa , Dhadhwal Rhythm , Youssef Nathan , Obregon-Henao Andres , Henao-Tamayo Marcela
Published: February 2023

Tuberculosis (TB) continues to increase worldwide despite vigorous attempts to control it. Bacillus Calmette-Guerin (BCG) is the only licensed vaccine currently available for protection against TB, however, its efficacy is highly variable between countries. It has been hypothesized that BCG’s variable protection is due, amongst others, to immunological interference by environmental, non-tuberculous mycobacteria (NTM). However, a definitive mechanism has not been identified so far. Considering the foregoing, we developed a murine model closely resembling the natural history of human exposure to different mycobacterial species, including: 1) BCG vaccination at an early age; 2) exposure to viable NTMs (Mycobacterium avium subsp. avium) via the oral route and 3) maintaining continuous NTM exposure even after TB infection, as occurs in endemic places. Surprisingly, we found that a low dose of NTM via the oral route enhanced BCG-mediated protection 30 days post infection, as determined by decreased TB burden in lungs and spleens of infected mice. We hypothesized that upon oral intake, NTMs would interact with intestinal Peyer’s Patches (PPs) and activate mucosal immune responses. Therefore, we evaluated immune cell populations present in PPs and lungs using flow cytometry. Fifteen days after initiating NTM feeding, higher influx of B220hiMHC-IIhi B cells was observed in PPs of NTM exposed mice, regardless of BCG vaccination status. Similarly, NTM exposure augmented pulmonary CD62LintMHC-IIhiCD19hiB and CD4+ T cells. These results suggest that NTM exposure via the oral route elicit humoral mucosal immunity against TB, and this will be further dissected to leverage mucosal NTMs as a strategy to boost BCG.
A single center, double blind, randomized, placebo controlled trial of anakinra in adult patients with features of Cytokine Storm Syndrome in COVID-19
Authors: Cron Randy Q , Jackson Lesley E , Khullar Nitasha , Chapleau Christopher A , Chatham W Winn
Published: February 2023

Methods:

(ClinicalTrials.gov, NCT04362111) Adult inpatients were recruited with PCR confirmed SARS-CoV-2 infection, radiographic confirmation of COVID-19 pneumonia, new or increasing oxygen requirement, ferritin ≥700 ng/mL, and any 3 of the following: D-dimer ≥500 ng/mL, platelet count <130,000/mm3, white blood cell count <3500/mm3 or lymphocyte count <1000/mm3, AST or ALT >2X the upper limit of normal (ULN), LDH >2X the ULN, C-reactive protein >100 mg/L. Eligible patients were randomly assigned (1:1) to standard of care (SoC) plus anakinra (100 mg subcutaneous every 6 hours for 10 days) or SoC plus placebo. Primary outcome was survival and discharge from the hospital without need for intubation/mechanical ventilation at day 10. Analyses were done on a modified intention-to-treat basis.

Results:

Between August 2020 and January 2021, 32 patients (235 screened) were recruited: 15 assigned to anakinra group and 17 to placebo group. 2 patients in the placebo group withdrew in the initial 48 hours and were excluded from analysis. Median age was 63 years (IQR 56 – 70.3), 20 (66.7%) were men, and 20 (66.7%) Caucasian. At day 10, 1 (6.7%) patient in the anakinra group had died and 2 (13.3%) in the placebo group had died (p=0.54). At hospital discharge, 4 (26.7%) patients in the anakinra group and 4 (26.7%) in the SoC group had died. Confirmed infections occurred in 4 (anakinra) and 2 (placebo), respectively (p=0.36).

Conclusion:

Anakinra added to SoC with dexamethasone did not significantly impact outcomes in study patients with clinical lab features of early cytokine storm syndrome and mild-to-moderate COVID-19 pneumonia. Additional studies are needed to assess efficacy and optimal dosing duration of anakinra in patients with more severe COVID-19.
Bromodomain protein BRD4 directs CD8 T cell differentiation during infection and cancer
Authors: Milner Justin , Toma Clara , Quon Sara , Omilusik Kyla , Scharping Nicole E , Dey Anup , Reina-Campus Miguel , Nguyen Hongtuyet , Getzler Adam J , Diao Huitian , Yu Bingfei , Delpoux Arnaud , Yoshida Tomomi M , Li Deyao , Qi Jun , Vincek Adam , Hedrick Stephen M , Egawa Takeshi , Zhou Ming-Ming , Crotty Shane , Ozato Keiko , Pipkin Matthew E , Goldrath Ananda W
Published: February 2023

In response to infection and malignancy, antigen-specific CD8 T cells differentiate into functionally diverse populations that are critical for resolving disease and providing durable immunity. Here, we utilized an in vivo RNAi screening strategy to identify transcriptional and epigenetic regulators controlling CD8 T cell fate. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we revealed an essential role for the chromatin modifier and BET family member BRD4 in supporting T cell differentiation during infection and cancer. BRD4 was found to bind diverse regulatory regions critical to effector T cell differentiation and was essential for transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion or chemical inhibition of BRD4 resulted in impaired maintenance of a terminal effector phenotype. In preclinical models of cancer, BRD4 was also required for formation of a terminally differentiated CD8 T cell state in the tumor microenvironment, which we show holds implications for immunotherapies. Tailored inhibition of BRD4 enhanced immune checkpoint blockade efficacy; however, complete ablation of BRD4 activity blunted T cell effector function and adoptive cell therapy efficacy. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis-regulatory elements to control CD8 T cell fate and stability of lineage identity, and provide insight for understanding the efficacy and full therapeutic potential of BET inhibition and T cell immunotherapies.
Histone deacetylase inhibition can revert induced resistance to immune checkpoint blockade in a mouse model of prostate cancer.
Authors: Rahmy Sharif , Lu Xin
Published: February 2023

Castration resistant prostate cancer (CRPC) is one of the leading causes of death in men in the United States, with a 5-year survival rate of 28%. Immune checkpoint blockade (PD-1 & CTLA4 inhibition, ICB) is a novel therapeutic strategy based on restoring the tumoricidal activity of cytotoxic T-lymphocytes. ICB therapy has produced spectacular results in the treatment of melanoma but shows almost no effect in CRPC.

We have developed a model system of advanced prostate cancer (Pten−/−/Tp53−/−/Smad4−/−) consisting of a parental (ICB sensitive) cell line and an ICB resistant subline (PPS-ICBR). We analyzed the transcriptome and found significant changes associated with ICB resistance. Subsequently we reversed some of those changes with Vorinostat, a histone deacetylase inhibitor. Mice injected subcutaneously with the PPS-ICBR cell line showed no response to Vorinostat or ICB therapy, but when combined 40% of the mice were completely cured, and another 40% showed significant reductions in tumor growth. We dissociated the tumors and used mass cytometry to determine the changes in the tumor immune microenvironment. ICB + Vorinostat therapy causes a significant increase in CD4+ & CD8+ T-cells, and a significant decrease in M2 macrophages. Concurrent with this decrease in M2 macrophages we observed a significant increase of TNFα+/iNOS+ monocyte derived dendritic cells.

Our goal is to study the underlying mechanisms that can transdifferentiate an immuno-suppressive tumor immune microenvironment towards a tumor-suppressive immune microenvironment and improve clinical outcomes.