bluuebunny/crossref_metadata_2025 huggingface dataset:
Diet-induced obesity impairs induction of Experimental Autoimmune Encephalomyelitis and is restored by PD-1 blockade
Authors: Le Catherine , Khuat Lam T. , Dunai Cordelia , Soulika Athena , Murphy William J.
Published: January 2023

Obesity has marked effects on immune function and is associated with increased infection susceptibility and decreased adaptive immune response. We examined the effects of obesity on adaptive autoimmune responses using a diet-induced obesity mouse model. The pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model of autoimmune demyelinating disease of CNS, is mediated by autoreactive Th1 and Th17 CD4 T cell subsets. 8–9-month-old C57Bl/6 male mice were fed 60% fat or 10% fat control diet starting at 6–8 weeks old and were immunized with 300 μg of rodent myelin oligodendrocyte glycoprotein (MOG)35–55 peptide in complete Freund’s adjuvant (CFA) containing 5 mg/ml heat-killed M. tuberculosis. Control diet mice exhibited onset of severe clinical symptoms of hind limb paralysis similarly to young wild-type controls, while diet-induced obese mice experienced a significantly delayed onset. Clinical scores correlated with greater number of MOG-specific tetramer-positive CD4 T cells and number of IFN-gamma and IL-17a-expressing CD4 T cells in the CNS of control mice compared to obese mice. Decreased functional cytokine expression and proliferation called into question the exhaustive nature of T cells in obese mice through the upregulation of the PD-1/PD-L1 pathway, a known regulator of EAE. We treated EAE-induced mice on control and high-fat diet with anti-PD-1 blockade (29F.1A12) and restored EAE induction in both clinical score and number of pathologic CD4 T cells in the CNS of obese mice. Our findings may indicate an impairment of the adaptive immune system through the PD-1/PD-L1 pathway in obesity, possibly indicating an augmentation of the checkpoint pathway in settings of systemic inflammation.
Identifying novel regulators of T cell CNS infiltration in Experimental Autoimmune Encephalitis
Authors: Salzler Harmony R. , Carr Tiffany M. , Wheaton Joshua M. , Ciofani Maria
Published: January 2023

T helper 17 (Th17) lymphocytes promote immunity in many contexts, including infection clearance, mucosal barrier homeostasis, and autoimmunity. IL-17A producing Th17 cells differentiate from naïve precursors upon antigen encounter with specific cytokines. One important mediator of Th17 cell differentiation downstream of cytokine signaling, is the AP-1 family transcription factor, JunB. JunB is a master regulator of Th17 cell fate, both promoting expression of Th17 lineage-specific genes and suppressing those of alternative effector fates. Studies in mice with JunB deficient CD4 T cells reveal that JunB is a key arbiter of pathogenesis in the experimental autoimmune encephalitis (EAE) model of autoimmunity. In addition to displaying an increased proportion of alternative T helper cell fates, JunB CD4 T cell knockout (KO) mice are completely rescued from EAE symptoms. CD4 T cells fail to infiltrate the central nervous system (CNS) in these animals, despite retaining a significant population of IL-17A producing cells in other niches. Thus, JunB-deficient mice represent a compelling model for investigating the molecular determinants of encephalitogenic T cell infiltration into the CNS. Additionally, in vitro RNAseq experiments comparing JunB KO and WT cells differentiated under Th17 polarizing conditions, suggest several promising migration related JunB target genes which are currently under investigation. Discovery of one or more JunB targets mediating this migration defect is of great potential interest in developing targeted therapies for Th17 mediated autoimmunity.
Autoantibody-inducing CD4 T (aiCD4 T) cell that induces systemic lupus erythematosus (SLE) includes not only follicular helper T cell but also IL-21-producing CXCR5−ICOShiPD-1hi cell
Authors: Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective

We found that repeated immunization with antigen induces systemic lupus erythematosus (SLE) in mice, and we have then proposed a novel ‘self-organized criticality theory’ that autoimmunity takes place when host’s immune system is overstimulated by repeated exposure to antigen to levels that surpass the immune system’s stability limit. A novel T cell type which we termed autoantibody-inducing CD4 T (aiCD4 T) cell was newly generated via TCR revision at periphery. They not only stimulated B cells to generate varieties of autoantibodies but also helped differentiation of CD8 T cell into CTL to induce tissue injuries. We also showed that the aiCD4 T cell belongs to CD45RBlo122lo CD4 T cell subpopulation. Here we further dissected the phenotype of aiCD4 T cell.

Methods

BALB/c mice were repeatedly immunized with OVA and SLE was induced. Cell surface marker of CD45RBlo CD4 T cell was detected using flow cytomery. These CD4 T cells were stimulated with anti-CD3 and CD28 antibodies in vitro, and cytokine in culture supernatant was detected using ELISA.

Results

After repeated immunization with OVA, CD4+CXCR5+ICOShiPD-1hi follicular helper T (Tfh) cell was increased, and these Tfh cells were CD45RBlo. Further, CXCR5−ICOShiPD-1hi cell was also increased in CD45RBlo CD4 T cell of OVA-immunized mice. We found that CXCR5−ICOShiPD-1hi CD4 T cell highly produced IL-21 rather than Tfh cell after repeated immunization with OVA.

Conclusion

In addition to Tfh cell, the CXCR5−ICOShiPD-1hi CD4 T cell that produces IL-21 was increased in line with induction of SLE. This novel CXCR5−ICOShiPD-1hi CD4 T cell population appears to be responsible for helping B cell to induce varieties of autoantibodies and driving CD8 T cell to cause lupus tissue injury.
Proteomic Analysis Reveals Distinctive Protein Profiles Involved in CD8 T Cell-Mediated Murine Autoimmune Cholangitis
Authors: Zhang Weici , Zhang Ren , Zhang Jun , Sun Yin , Leung Patrick , Yang Guo-Xiang , Shuai Zongwen , Ridgway William , Gershwin M Eric
Published: January 2023

Autoimmune cholangitis arises from abnormal innate and adaptive immune responses in liver and T cells are critical drivers in this process, but little is known about the regulation of their functional behavior during disease development. We previously reported that mice with a T cell-restricted expression of a dominant negative form of transforming growth factor beta receptor type II (dnTGFbRII) spontaneously develop autoimmune cholangitis that resembles human primary biliary cholangitis (PBC). Adoptive transfer of CD8+ but not CD4+ T cells into Rag1−/− mice reproduced the disease, demonstrating a critical role for CD8 T cells in pathogenesis. Herein, we took advantage of SOMA scan technology to perform proteomic analysis of serum samples from dnTGFbRII and B6 control mice at different ages. In addition, we analyzed CD8 protein profiles after adoptive transfer of splenic CD8+ cells into Rag1−/− recipients. The use of the unique aptamer technology of SOMA scans reveals critical distinct profiles of CD8 cells, which are key to biliary mediation. 254 proteins were significantly increased, while 216 proteins significantly decreased in recipient hepatic CD8+ cells compared to donor splenic CD8+ cells. In contrast to donor splenic CD8+ cells, recipient hepatic CD8+ cells express distinct protein profiles involved in chemokine signaling, focal adhesion, T cell receptor and natural killer cell mediated cytotoxicity pathways.
Transcriptomics analysis reveals new insights into the roles of Notch1 signaling on macrophage polarization
Authors: Hans Chetan Parkash , Sen Sidharth , Zeng Shuai , Sharma Neekun , Dev Rishabh , Hans Ranjana , Joshi Trupti
Published: January 2023

Naïve macrophages (Mϕ) polarize in response to various environmental cues to a spectrum of M1 or M2 macrophages with distinct biological functions. Our earlier in vivo studies demonstrated that Notch1 deficiency attenuates the development of abdominal aortic aneurysm by promoting TGF-β2 dependent M2-polarization, however, the underlying mechanism is not completely understood. The present studies aimed to determine the specific roles for Notch1 signaling in macrophage polarization. Bone marrow derived macrophages isolated from WT or Notch1+/− mice (n=12) were differentiated to Mϕ, M1 or M2-phenotypes by 24h exposure to vehicle, LPS/IFN-γ or IL4/IL13, respectively. Total RNA isolated from these cells was subjected to RNA-Sequencing in triplicate. All samples had a minimum of 51.3 million 50 nucleotide paired-end reads and aligned at 87.5% ± 0.5% to the reference genome. EasyRNAseq and WebGestalt analysis with log2 fold change demonstrated that Notch1 haploinsufficiency downregulated the expression of 262 genes at basal level, 307 genes with LPS/IFN-γ and 254 genes with IL4/IL13 treatment. Among these, 50 unique genes including fibromodulin (Fmod), caspase-4, Has1, Col1a1, Alpl and Igf were downregulated by Notch1 haploinsufficiency under all the conditions. Pathway analysis demonstrated that extracellular matrix, macrophage polarization and osteogenesis to be the major pathways affected by Notch1 haploinsufficiency. Gain and loss-of-function and mechanistic studies established a strong correlation between Notch1 haploinsufficiency and Fmod in regulating TGF-β signaling. Overall, our studies provide strong evidence that Notch1 haploinsufficiency increases M2 polarization through these newly identified genes.
G protein Coupled Receptor Kinase 2 Levels Regulate Differential Internalization of C-C Chemokine Receptor 7 (CCR7) in Primary Mouse T Cells
Authors: Vines Charlotte M. , Winebrenner Caitlin T. , Bill Charles A. , Sanchez Anahi , Wagener Brant , Bill Colin A.
Published: January 2023

G protein-coupled receptor (GPCR) signaling can be regulated by phosphorylation via GPCR kinases (GRKs) and recruitment of b-arrestins. The C-C chemokine receptor 7 (CCR7) GPCR, has two ligands, CCL19 and CCL21 that promote chemotactic migration of CCR7 expressing immune cells, including naïve T cells. Distinct signaling pathways are activated in T cells depending upon which ligand is bound to the CCR7. It remains unclear to what extent GRKs contribute to differential internalization of ligand-bound CCR7 and subsequent signaling that controls T cell migration.

We used naïve primary T cells from wild type C57 BL/6 mice (WT) or syngeneic mice heterozygous for the GRK2 gene that express 50% GRK2 protein levels compared to WT mice. T cells were incubated with 40 nM of CCL19 or CCL21 for up to 8 minutes. Incubation with CCL19, but not CCL21, led to CCR7 internalization; however, both ligands facilitated rapid, sustained CCR7 internalization in GRK2+/− T cells. The reduced GRK2 levels in the heterozygote T cells had no significant effect on migration to CCL19 or CCL21 compared to WT T cells. Titration transfection experiments in HEK293T cells or CRISPR-Cas9 knockdown of GRK2 in CEM T cells suggested that GRK2 is required for ligand-mediated CCR7 internalization, while higher GRK2 levels inhibit the internalization process. CCR7 internalization was blocked following transfection of a kinase dead GRK2 mutant indicating that GRK2 phosphorylation is required for CCR7 internalization. Immunomicroscopy suggested arrestin-3 formed clusters, independent of the ligand used. Overall, these data suggest that cellular concentration of GRK2 plays an important role in the internalization of ligand/CCR7.
The role and regulation of cell-intrinsic glycogen metabolism in dendritic cell immune responses.
Authors: Thwe Phyu M. , Pelgrom Leonard , Cooper Rachel , Beauchamp Saritha , Reisz Julie A. , D’Alessandro Angelo , Everts Bart , Amiel Eyal
Published: January 2023

Stimulation of dendritic cells (DCs) through toll-like receptors (TLRs) is accompanied by an increased metabolic demand, which provides metabolites and energy required for DC activation. This metabolic requirement is fulfilled by TLR-driven rapid glycolytic burst, and inhibition of glycolysis impairs the survival and effector function of DCs. Although previous studies have shown an important role for extracellular glucose uptake via glucose transporter 1 (Glut1) in supporting TLR-driven glycolysis, the contributions of intracellular glucose stores to these processes have not been well-defined. While non-immune cells store glucose as glycogen, the role of glycogen in DCs has not been identified. Here, we demonstrate that cell-intrinsic glycogen metabolism in DCs supports the early glycolytic reprogramming essential for TLR activation, particularly before Glut1 upregulation. DCs express key enzymes for glycogen metabolism, possess intracellular glycogen stores, and inhibition of glycogen breakdown severely impairs the effector function of DCs, including the ability to stimulate T cells. Our preliminary data show a time-dependent increase in intracellular glycogen levels upon TLR stimulation, implying that intricate regulatory mechanisms of glycogen metabolism are at play for proper activation responses. Interestingly, our metabolomics data indicate that the glycogen metabolism in DCs generates both glycolytic and TCA cycle intermediates and that glycogen-derived carbons preferentially engage in metabolic pathways distinct from free glucose catabolism. Our work reveals a novel metabolic regulatory pathway by which DCs differentially utilize glycogen and glucose metabolism to support their activation.
Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils
Authors: White Mitchell R. , Hsieh I-Ni , De Luna Xavier , Hartshorn Kevan L.
Published: January 2023

Innate immunity is vital for the early control of influenza A virus (IAV) infection. Serum amyloid A (SAA1) is an acute phase reactant produced in the liver and lung that rises dramatically during IAV infection. SAA has been reported to directly activate neutrophils and to recruit them to the lung during infectious and inflammatory processes. In this study we characterized the interactions of several forms of SAA with IAV and with neutrophils. The complex of SAA with HDL had some neutralizing activity against IAV. Similarly purified SAA containing amino acids 19–94 prepared from human serum had some viral neutralizing activity. In contrast recombinant SAA containing amino acids 19–123 (rSAA) did not. SAA preparations, including rSAA, increased various responses of neutrophils to the virus. Neither serum nor rSAA directly stimulated neutrophil respiratory burst responses on their own; however, both preparations increased these responses to IAV. rSAA also increased neutrophil uptake of IAV. rSAA alone did trigger neutrophil IL-8 production but modestly increased the strong neutrophil IL-8 response triggered by IAV. rSAA alone did trigger a neutrophil intracellular calcium response and also increased the peak calcium response to IAV. Combining rSAA with surfactant protein D (a known lung inhibitor of IAV) resulted in potentiation of viral neutralizing and aggregating activity of IAV. We provide evidence that the effects of SAA on neutrophil responses to IAV were not mediated by formyl peptide receptor 2 but were in part mediated by TLR2. These results suggest that SAA produced in the lung may significantly modulate the early neutrophilic host response to IAV.
T-bet-expression in dendritic cells is essential for T. gondii clearance
Authors: Lopez-Yglesias Americo H. , Burger Elise , Araujo Alessandra , Martin Andrew T. , Yarovinsky Felix
Published: January 2023

The host’s type I immune response is essential for resistance to diverse groups of intracellular pathogens, including Mycobacterium, Leishmania, Salmonella, and Toxoplasma gondii. Type I immunity is traditionally characterized by the transcription factor T-bet, encoded by Tbx21, which has been considered the master regulator of determining the lineage commitment of CD4+ TH1 cells and their production of the effector molecule IFN-γ. However, recent studies by our and other groups have shown that T-bet expression in CD4+ T cells is dispensable for IFN-γ production, and that T-bet-independent T cell-derived IFN-γ is sufficient to cause immunopathology during infection. Despite comparable levels of IFN-γ generated by Tbx21−/− mice, they remain highly susceptible to infection. Furthermore, Tbx21−/− mice succumb significantly faster to T. gondii infection than animals lacking B and T cells, yet retain an intact myeloid innate compartment, suggesting that T-bet deficiency in innate immune cells results in loss of host resistance to intracellular parasite, and rapid host mortality during infection. Thus, we investigated T-bet expression levels in myeloid innate immune cells and observed that T-bet expression in monocyte-derived CD11c+ DCs occurs in an IFN-γ-dependent manner. We also established that T-bet expression in DCs, but not macrophages, is required for resistance to intracellular T. gondii infection. Overall, we revealed an essential role for T-bet expression in DCs for controlling host immunity to intracellular parasite infection.
Prolonged cell survival may contribute to the cytokine storm observed during the coinfection of influenza A virus (IAV) and Streptococcus pneumoniae.
Authors: Rodriguez Angeline E. , Lupfer Christopher R.
Published: January 2023

Coinfection with influenza A virus (IAV) and Streptococcus pneumoniae is known to cause severe pneumonia. Numerous reports suggests an inappropriate immune response plays an important role in this disease. Many reports have examined signaling pathways responsible for the excessive immune response to coinfection, but the role for cell death in mediating inflammation during coinfection is not well understood. The inflammasome is a caspase-1 containing protein complex that activates interleukin-1β (IL-1β) and induces a pro-inflammatory form of cell death termed pyroptosis. We have discovered enhanced IL-1β levels during coinfection of macrophages with IAV and S. pneumoniae. Furthermore, inflammatory macrophages/monocytes, but not alveolar macrophages, are the major cell type producing IL-1β in the lung. However, this is not due to increased inflammasome activation. Intriguingly, coinfected macrophages are less prone to cell death than those singly infected with S. pneumoniae. Thus, we have elucidated a mechanism whereby prolonged cell survival may contribute to the cytokine storm observed during coinfection.
A systematic approach integrating genetics, computational biology, chemical genomics and gene editing to identify novel targets in the Type I interferon signaling pathway for autoimmune and infectious diseases
Authors: Zeng Xin , MacNamara Aidan , Xie Qing , Lu Quinn , Hou Wangfang , Rao Yanhua , Maratuo Klio , Rea Ceara , Madsen Heather , Larminie Chris
Published: January 2023

The type I interferon (IFN a/b) signaling pathway plays a critical role in microbial host defense, including mediating the interaction between innate and adaptive immunity. A growing body of evidence suggests that IFN is aberrantly regulated in human autoimmune diseases and chronic infections, such as Systematic Lupus Erythematosus, HIV infection and the rare Mendelian diseases, Aicardi-Goutier Syndrome and the Singleton Merton Syndrome (also called “interferonopathies”). This has raised significant interest in exploring the cellular signaling associated with type I IFNs for potential targets for autoimmunity and infectious disease. In this study, we systematically interrogated the type I IFN signaling pathway to identify and validate potential therapeutic targets based on the following workflow: 1. Perform computational biology analysis using genetics, interaction networks and gene expression in human diseases to generate a list of targets that have an association with increased type I IFN. 2. Establish the THP-1 cell-based type I IFNbeta release assay to screen a focused set of target annotated tool compounds. 3. Integrate the inferred targets from the tool compound hits into the in-silico network analysis. 4. Generate a short list of targets (10–20) with high confidence to perform CRISPR analysis in THP-1 cells. 5. Generate a long list of targets (~200) to perform a pooled CRISPR screen using single cell technology and reporter-based flow sorting and enrichment. Here we present validation data on a few representative targets and a summary of the outcomes and learning from this study.
Commensal Bacteria Suppress Vitamin A Metabolism in the Intestinal Epithelium to Modulate IL-22 Activity in the Gut.
Authors: Grizotte-Lake Mayara , Vaishnava Shipra
Published: January 2023

Antimicrobials secreted by the intestinal epithelium are critical for regulating host-microbe interactions in the gut. However, certain pathogens can exploit excessive antimicrobial response during infection to outcompete commensal bacteria. We have uncovered a novel intestinal epithelial cell intrinsic mechanism by which commensal bacteria modulate Interleukin (IL)-22 induced antimicrobials to provide colonization resistance against pathogens. Vitamin A metabolite, retinoic acid (RA), is critical for coordinating multiple immune responses in the intestine, including IL-22 production, yet, we barely understand how conversion of vitamin A into RA is regulated in the intestinal tissue. Our work shows that commensal bacteria limited RA by suppressing expression of retinol dehydrogenase 7 (rdh7) in the intestinal epithelium. Deletion of rdh7 resulted in reduced RA signaling in immune cells, lower levels of IL-22, and an enhanced resistance to colonization by pathogen that exploits IL-22 induced antimicrobials to thrive in the gut. Supplementing mice deleted for rdh7 with RA reversed the colonization resistance, revealing the significance of downregulation of rdh7 by commensal bacteria. Our findings thus provide a novel insight into molecular mechanism by which commensal bacteria promote resistance to colonization by pathogenic bacteria. More importantly, we are demonstrating for the first time that bacterial regulation of RA provides a crucial feedback loop to curb excessive IL-22 activity in the gut.
Vasodilator Stimulated Phosphoprotein (VASP)-Mediated Actin Polymerization Drives Natural Killer Cell Granule Convergence
Authors: Wilton Katelynn Marie , Billadeau Daniel D.
Published: January 2023

Natural killer (NK) cells eliminate transformed and malignant cells through a highly orchestrated series of actin and microtubule cytoskeletal rearrangements, culminating in the secretion of preformed lytic granules. Despite the importance of actin and microtubules, the roles of many molecular cytoskeletal regulators in this process have not yet been ascertained. In this study, we investigated the role of Vasodilator Stimulated Phosphoprotein (VASP), an actin regulatory protein, in NK cell-mediated cytotoxicity. Interestingly, we found that depletion of VASP inhibits NK cell cytotoxicity and that VASP co-localizes with F-actin at the NK cell – target cell cytotoxic synapse. Surprisingly, despite their co-localization, VASP depletion did not affect either F-actin accumulation at the cytotoxic synapse or conjugate formation. Instead, we found that a minority of VASP localized and biochemically co-purified with cytolytic granules. Although VASP knockdown did not affect microtubule organizing center (MTOC) polarization, it did dramatically impact lytic granule convergence to the MTOC. Significantly, VASP depletion decreased F-actin accumulation on cytolytic granules and depolymerization of F-actin with Latrunculin A impaired lytic granule convergence. Taken together, these results demonstrate a novel requirement for VASP-mediated actin polymerization in NK cell granule convergence and cytotoxicity and highlight a role for F-actin in lytic granule convergence.
Foreign antigen-specific Foxp3+ regulatory T cells expand with immunization and limit Th1 polarization by Foxp3− conventional cells specific for the same antigen
Authors: Krueger Peter D. , Jenkins Marc K.
Published: January 2023

The CD4+ T cell repertoire contains thousands of populations, each with antigen receptors specific for a different major histocompatibility complex class II (MHCII)-bound foreign peptide. Before exposure to the foreign peptide, an average population in a mouse consists of about 100 naïve Foxp3− conventional cells and 10 Foxp3+ regulatory T (Treg) cells. After infection containing the peptide, the population increases several hundred-fold and contains Foxp3− and Foxp3+ effector cells. Because conventional T cells have been reported to generate Treg cells and vice versa, it is not clear which precursors generate which effector cells. Using progeny-tracing systems, we found that Foxp3− conventional cells and Foxp3+ Treg cells specific for the same peptide:MHCII ligand proliferated at the same rate after immunization. Foxp3− naïve cells generated only Foxp3− effector cells, and Foxp3− naïve cells generated primarily Foxp3+ effectors cells. The Treg cells suppressed the Th1 differentiation of the Foxp3− cells but did not cause them to become Treg cells. These results demonstrate that Foxp3− and Foxp3+ naïve T cells are remarkably lineage-committed during the primary response to infection, and although their progeny interact, this interaction does not result in lineage instability.
Negative costimulation constrains T cell differentiation
Authors: Wei Spencer C. , Sharma Roshan , Anang Nana-Ama , Levine Jacob , Zhao Yang , Wang Jing , Pe’er Dana , Allison James P.
Published: January 2023

T cell costimulation is a principal mechanism by which T cell activation is regulated, but it remains unclear whether costimulatory pathways also control T cell differentiation. This unresolved question is germane to our understanding of how T cell function is controlled and has therapeutic implications in the context of cancer immunotherapy. Using a mass cytometry based systems approach and murine models we investigated whether negative costimulation regulates T cell differentiation as well as activation. Unsupervised population identification reveals 30 T cell subsets in Ctla-4−/− and littermate control mice including 6 CD8, 3 Treg, 14 CD4 effector T cell populations. Expectedly, loss of CTLA-4 led to dramatic changes in the relative frequency of CD8 and CD4 T cell subsets. Strikingly, however, loss of CTLA-4 led to the generation of multiple non-canonical T cell populations outside of the normal boundaries of T cell phenotypes, which interestingly, were all in the CD4 T compartment. To determine if the role of negative costimulation in differentiation may be a generalizable phenomenon, we performed similar analyses of Pdcd-1−/− mice. This profiling revealed that loss of PD-1 leads to a subtle expansion of CD8 T cell phenotypes in addition to changes in the relative frequencies of CD4 and CD8 T cell populations. These observations indicate that CTLA-4 restricts CD4 T cell phenotypes while PD-1 restricts CD8 T cell phenotypes. Consistent with this model, loss of CTLA-4 differentially affects the expression of individual markers in CD4 and CD8 T cells. Together these findings indicate that in addition to attenuating T cell activation, negative costimulation acts to constrain T cell differentiation.
Differential surface phenotype and context-dependent reactivity of functionally diverse NKT cells
Authors: Cameron Garth , Godfrey Dale I.
Published: January 2023

Natural Killer T (NKT) cells are a functionally diverse population that recognize lipid-based antigens in association with the antigen-presenting molecule CD1d. Here we define a technique to separate the functionally distinct NKT1, NKT2 and NKT17 cell subsets by their surface expression of CD279 (ICOS) and the activation-associated glycoform of CD43, enabling the investigation of subset-specific effector-functions. We report that all three subsets express the transcription factor GATA-3 and the potential to produce IL-4 and IL-10 following activation. This questions the notion that NKT2 cells are the predominant source of IL-4 within the NKT cell pool, and suggests that IL-10-production may be more indicative of NKT cell plasticity than the existence of a distinct regulatory lineage or subset. We also show that many NKT17 cells are CD4+ and are biased towards Vβ8.3 TCR gene usage. Lastly, we demonstrate that the toll-like receptor (TLR) ligand lipopolysaccharide (LPS) can induce a NKT17 cell-biased response, even in the absence of exogenous antigen, and that combining LPS with α-GalCer resulted in enhanced IL-17A-production, and reduced levels of the immunosuppressive cytokine IL-10. This study provides a novel means to examine the context-dependent reactivity of the functionally heterogeneous NKT cell population and provides important new insight into the functional biology of these subsets.
CD8+ T-cell effector molecules are differentially expressed in blood and gastrointestinal mucosa in chronic HIV-1 infection
Authors: Shacklett Barbara L. , Kiniry Brenna E. , Somsouk Ma , Hunt Peter W. , Deeks Steven G.
Published: January 2023

CD8+ T-cells in human gastrointestinal mucosa exhibit reduced perforin expression and weak cytotoxicity compared to their counterparts in blood. We previously reported that these cells are predominantly T-bet-low and Eomesodermin-negative. Nevertheless, in response to antigenic stimulation these cells degranulate and express cytokines and chemokines. The goal of this study was to elucidate expression patterns of granzymes (Gzm) A, B and K in conjunction with perforin in rectosigmoid CD8+ T-cells during chronic HIV-1 infection and determine whether these patterns vary with disease status. We used flow cytometry to compare intracellular Gzm A, B, K and perforin in resting CD8+ T-cells from blood and rectosigmoid biopsies of HIV-1+ controllers; viremic individuals; virologically suppressed individuals on antiretroviral therapy; and seronegatives. Expression of perforin and GzmB, but not GzmA or GzmK, was reduced in mucosa compared to blood. A large fraction of rectosigmoid CD8+ T-cells did not express granzymes or were single-positive for GzmA. Rectosigmoid CD8+ T-cells appeared skewed towards cytokine production rather than cytotoxicity, with cells expressing multiple cytokines/chemokines lacking perforin and granzyme expression. Percentages of mucosal CD8+ T-cells expressing cytotoxic effectors were higher in HIV-1+ groups, compared to seronegatives. These data support the interpretation that perforin and granzymes are differentially regulated, and display distinct expression patterns in blood and rectosigmoid T-cells. These findings also reinforce our previous work, suggesting that rectosigmoid CD8+ T-cells are programmed for cytokine polyfunctionality rather than cytotoxicity.
TRIM34 attenuates colon inflammation and tumorigenesis by promoting Muc2 exocytosis from goblet cells
Authors: Sun Bing
Published: January 2023

Loss of colonic inner mucus layer leads to spontaneously severe colitis and colorectal cancer. However, it is rarely reported whether there are some key host factors to control the generation of inner mucus layer. Here, we identify a novel function of TRIM34 in goblet cells (GCs) in controlling the inner mucus layer generation. Upon DSS treatment, TRIM34 deficiency led to a reduction in Muc2 secretion by GCs and subsequent defects in the inner mucus layer and massive commensal bacterial penetration. This rendered TRIM34-deficient mice more susceptible to DSS-induced colitis and colitis-associated colorectal cancer. Mechanistic experiments demonstrated that TRIM34 controlled TLR signalling-induced, Nox/Duox-dependent ROS synthesis, thereby promoting the compound exocytosis of Muc2 by colonic GCs that were exposed to bacterial TLR ligands. Clinical analysis revealed that TRIM34 levels in patient samples were highly correlated with the outcome of ulcerative colitis (UC) and the prognosis of rectal adenocarcinoma. This study indicates that TRIM34 expression in GCs plays an essential role in generating inner mucus layer and preventing excessive colon inflammation and tumorigenesis.
Selective knockdown of A2AR in CD8+ T cells using CD8-targeting nanoliposomes
Authors: Newton Hannah , Arnold Michael J. , Chimote Ameet A. , Conforti Laura
Published: January 2023

Adenosine accumulates in the tumor microenvironment and contributes to the inhibition of T cell function via the A2A receptor (A2AR). Blockade of the A2AR has been shown to rescue T cell function and decrease tumor burden. However, A2AR is present in a variety of cells and thus a limitation of non-selective A2AR pharmacological blockers is the development of side effects. Our goal is to selectively knockdown the A2AR in cytotoxic CD8+ T cells through lipid nanoparticles (NPs) decorated with anti-CD8 antibody and containing A2AR siRNAs. The A2AR knockdown by A2AR siRNAs was verified in human peripheral blood T cells (PBT). Nucleofection of PBTs with A2AR or scramble siRNAs downregulated mRNA and protein by 61% and 14–42%, respectively (as determined by RT-qPCR and flow cytometry). A functional assay for IL-2 production (a cytokine produced by activated PBTs and suppressed by the A2AR pathway) in PBTs treated with siRNAs and activated in the presence of CGS21680 (an A2AR agonist) was used to verify the A2AR knockdown. Treatment of PBTs with A2AR siRNAs prevents the CGS21680-induced inhibition of IL-2 production by 27%. Additionally, NPs were fabricated using biotinylated lipids as previously described (Hajdu et al., Biomaterials 2013) and labeled with anti-CD8 antibody. Their selectivity and internalization were determined. Flow cytometry revealed that CD8-labeled NPs are specific to CD4− PBTs and suggested that the NPs are subsequently internalized. Thus, these data indicate the feasibility of fabrication and potential efficacy of CD8-specific A2AR siRNAs-loaded NPs that should restore the functionality selectively in CD8+ tumor infiltrating lymphocytes—a potential targeted cancer therapy with limited side effects.
Obesity-induced changes in baseline immune responses to pre-clinical breast cancer
Authors: Gibson Justin Tyler , Nagareddy Prabhakara , Norian Lyse
Published: January 2023

Obesity is known to increase morbidity and mortality in breast cancer patients; however, the immunological contributions to such changes remain incompletely understood. Our long-term goal is to investigate the broad impact of obesity on anti-tumor immunity, with a particular focus on understanding the mechanisms underlying obesity-induced immune alterations and how these alterations may influence the use of immunotherapeutics in breast cancer patients. For our studies, we utilized a diet-induced model of obesity (DIO) in which mice were fed either a high-fat or low-fat diet to generate DIO or age-matched lean controls, respectively, then challenged with the syngeneic E0771 mammary carcinoma cell line. Tumor outgrowth was quantified by caliper measurements, bioluminescent imaging (BLI) via firefly luciferase-expressing E0771 (E0771-fLUC) cells, and endpoint tumor weights. Immune populations were evaluated via multi-parameter flow cytometry. BLI revealed that obesity increases primary tumor outgrowth and significantly alters intra-tumoral immune populations. Changes include decreases in the frequency of CD4+ and CD8+ T cells, increases in granulocytic myeloid-derived suppressor cells (G-MDSCs) and dendritic cells (DCs), and a trending decrease in total CD45+ tumor infiltrating leukocytes. These alterations were specific to the tumor environment, as they were not present in the spleens of tumor-bearing mice. Collectively these data implicate obesity as a causal agent in impairing anti-tumor immune responses to murine breast cancer. Furthermore, they suggest several cellular candidates for mechanistic investigations into obesity-induced dysfunction in breast cancer immunity.
Changes in Chronic Inflammatory Cells by Bariatric Weight Loss Surgery
Authors: Sanchez-Pino Maria D. , Kim Yonghyan , Liu Jiao , Wyczechowska Dorota Danuta , Richardson William , Wooldridge James , Mynatt Randall , Berthoud Hans-Rudolf , Ochoa Augusto C.
Published: January 2023

Obesity is characterized by chronic low grade systemic inflammation, where myeloid cells play a critical role. Myeloid-derived suppressor cells (MDSC; Monocytic [M-MDSC] and Granulocytic [PMN-MDSC] subsets) have been identified in cancer and other chronic inflammatory diseases. A few studies have shown a significant accumulation of MDSC in blood and different tissues in diet-induced obese mice and we also observed a significant accumulation of MDSC in mice made obese by a high fat-high sucrose diet compared with lean animals. The mechanisms linking obesity and MDSC activation or the effects of weight loss are unknown. We have compared the frequency of MDSC in blood and omental adipose tissue in obese patients (BMI≥40) with non-obese controls (BMI≤25), and evaluated the effect of bariatric surgery over time (3, 6 and 12 months). Our preliminary data shows that PMN-MDSC are significantly increased in patients prior to surgery, but drastically decrease 3 months after bariatric surgery, associated with improvement of obesity-related metabolic dysfunction. These data support the hypothesis that increased caloric intake and obesity stimulate the induction of MDSC, which promote a chronic inflammatory environment, while weight loss intervention reduces MDSC, eliminating the inflammation and controlling the metabolic dysfunction condition. Furthermore, our in vitro studies demonstrated that the uptake of lipoproteins transforms normal myeloid cells into highly immunosuppressive MDSC by inducing the expression of Arginase I. Thus understanding the cellular and molecular mechanisms by which obesity leads to the induction of MDSC is essential to understand the role of those MDSC in the obesity-related pathologies.
The Force Awakens: Illuminating the role of kynurenine in cancer progression and treatment.
Authors: Ashoura Norah E. , Dekker Joseph D. , Triplett Todd , Garrison Kendra , Blazeck John , Karamitros Christos , Lamb Candice , Tanno Yuri , Ehrlich Lauren I. , Zhang Michelle , Manfredi Mark G. , Stone Everett , Georgiou George
Published: January 2023

Cancer is the second leading cause of death in the US and, despite progress in treatment options, there is a critical need for novel treatments that specifically target cancerous cells. Our immune system routinely identifies potential cancer cells and eliminates them without the need for clinical intervention. However, to evade immune clearance, many cancers elevate tryptophan catabolism in the tumor microenvironment (TME) by upregulating the enzymes indoleamine 2, 3-dioxygenase (IDO) or, alternatively, tryptophan 2, 3- dioxygenase (TDO). This results in greater tryptophan turnover, accumulation of IDO/TDO product, kynurenine (L-kyn), and immune suppression in the TME. Whether the resulting immunosuppression arises from tryptophan depletion or L-kyn accumulation remains highly controversial. This work aims to (1) clarify L-kyn’s effect on T-cells and (2) whether its depletion can relieve tumor burden. Exposing T cells to L-kyn in vitro results in gene expression changes consistent with regulatory T-cell generation and the suppression of naïve T-cell proliferation; establishing L-kyn as a key therapeutic target for depletion to relieve TME immune suppression. Using a pharmacologically optimized kynureninase (KynU) enzyme, we tested L-kyn depletion therapy in murine cancers. KynU administration potently inhibits tumor growth, reduces L-kyn concentration, and results in a significant increase in the infiltration and proliferation of polyfunctional T-lymphocytes. Our ongoing study of KynU’s efficacy and L-kyn’s in vitro effects will illuminate details of L-kyn’s elusive mechanism of action, resolving critical mechanisms of tumor tolerance while creating a more innovative and effective cancer treatment strategy.
Nicotinamide adenine dinucleotide (NAD) oxidation preserves T cell function under lactic acidosis characteristic of the tumor microenvironment (TME)
Authors: Beier Ulf H. , Quinn William J. , Jiao Jing , TeSlaa Tara , Stadanlick Jason , Hancock Wayne W. , Eruslanov Evgeniy B. , Rabinowitz Joshua D. , Baur Joseph A.
Published: January 2023

Cancers are adept at employing metabolic events to evade host immunity. The TME is often glucose depleted and enriched with lactic acid, weakening effector T cell (Teff) function and interferon (IFN)-γ production. By contrast, immunosuppressive T-regulatory (Treg) cells are well adapted to low glucose and elevated lactate and enriched in the TME. Overcoming immunosuppressive metabolic conditions in the TME may be an important step towards improving anti-tumor immunotherapy. Here we show that NAD oxidation can protect Teffs from the immunosuppressive effects of lactic acid. We found that Teff proliferation and IFN-γ production can be controlled through NAD redox metabolism and NAD-dependent enzymes, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Metabolomic and bioenergetics studies showed that reducing NAD led to accumulation of pre-GAPDH glycolytic metabolites and impaired glycolytic flux similar to GAPDH inhibition, which was reversed by NAD oxidation. Human and murine Teffs exposed to lactate, oligomycin, rotenone, or antimycin lost GAPDH function despite sufficient glucose, limiting glycolysis and IFN-γ mRNA translation, while the Treg transcription factor Foxp3 was stabilized. Oxidizing NAD (pyruvate, mitochondrial uncoupling, β-lapachone, α-ketobutyrate) rescued Teff glycolysis and restored their function despite ambient lactate, while Foxp3 turnover increased in a poly-(ADP-ribose) polymerase dependent manner, using NAD as substrate. Our results identify a novel therapeutic approach to overcoming metabolic barriers to anti-tumor immunity, and provide new mechanistic insights into the role of NAD redox metabolism in controlling T cell function in the TME.
Cellular and molecular mechanisms regulating the development of tertiary lymphoid structures in tumor
Authors: Rodriguez Anthony B. , Peske J David , Woods Amber N. , Melssen Marit Martina , Cyranowski Salwador , Parriott Geoffrey Raymond , Engelhard Victor H.
Published: January 2023

Tertiary lymphoid structures (TLS) have been described in association with several tumor types, and they are usually associated with enhanced patient survival. However, mechanisms regulating their formation remain unknown. We found that intra- and peri-tumoral TLS develop in intraperitoneal (IP) but not subcutaneously (SC) implanted tumors. These TLS are characterized by a robust accumulation of B cells, and an associated reticular network of podoplanin+ fibroblasts expressing high levels of CXCL12, CXCL13, and the B-cell survival factors BAFF and APRIL. These results suggest that IP tumor-associated fibroblasts are polarized into organizer cells that orchestrate TLS formation. We’ve previously demonstrated that CD8 T effectors induce the development of lymph node-like vasculature in IP tumors. To determine whether these cells also regulate TLS development, we evaluated IP tumors grown in Rag1−/− mice and Rag1−/− mice repleted with bulk CD8 T cells. CD8 T cells upregulated the number of podoplanin+ fibroblasts and CXCL12 expression. However, CXCL13, BAFF and APRIL expression was not dependent on CD8 T cells. Thus, the representation of podoplanin+ fibroblasts and their expression of CXCL12 is regulated by adaptive immunity, while their expression of CXCL13, BAFF and APRIL is mediated by an innate element within the IP tumor microenvironment. We also found that increasing the load of the model tumor antigen ovalbumin led to an increased formation of peri-tumoral TLS with a classical organization, on top of the non-classical intratumoral structures evident at a low antigen load. These results identify novel mechanisms regulating different elements involved in organizing tumor-associated TLS.
Atrophied thymus can serve as a tumor reservoir for harboring melanoma cells
Authors: Sizova Olga , Kuriatnikov Denis , Su Dong-Ming
Published: January 2023

Tumor metastatic relapse is responsible for main cancer associated mortality and potentially arises from undetectable minimal number of tumor cells, which are able to resist radio-chemotherapy at a dormant state hiding in certain organs (termed: tumor reservoirs). A central T cell immune organ, the thymus, has been suggested as this kind of pre-metastatic tumor reservoir for B-lymphoma cells, which are a risk for eventual relapse. It remains unknown whether the thymus is able to harbor non-lymphoid solid tumor cells, why chemotherapy cannot thoroughly eliminate cancer cells in the thymus, and what the state of thymic occult cancer cells is during chemotherapy. With melanoma inoculated and genotoxic doxorubicin treated mouse model, we determined that the thymus, particularly the atrophied thymus, was able to harbor blood stream-circulating melanoma cells. We found that chemotherapy-resulted DNA-damage response triggered p53 activation in non-malignant thymic cells, which in turn resulted in thymocyte death and thymic epithelial cell senescence to develop an inflammatory thymic microenvironment. This inflammatory condition induced thymic occult minimal tumor cells to acquire heterogenic chemo-resistant dormancy. Therefore, the thymus, which becomes a pre-metastatic reservoir for non-lymphoid solid tumor cells under chemotherapy, should be a novel target in antitumor therapy for considering and preventing from tumor metastatic relapse.
IgD Ligation Promotes B Cell Proliferation in a Model of the Tumor Microenvironment.
Authors: Riggs James Edward , Londregan Jennifer E. , Goldman Naomi , Somerville John
Published: January 2023

Potent immune suppression within tumor microenvironments (TMEs) is a hallmark of cancer. We model the TME by culture of C57BL/6J peritoneal cavity (PerC) cells. PerC cells include a significant fraction of immune-suppressive macrophages that, depending on the form of activation, temper T and B cell proliferation. PerC B cell responses to both BCR (F[ab′]2 anti-IgM) and TLR4 (LPS) ligation are suppressed. Here, we show that IgD receptor ligation (anti-IgD) induces modest B cell proliferation in this suppressive environment. Unlike other forms of B cell stimulation, the anti-IgD response is optimal at high PerC cell concentrations. Both polyclonal anti-IgD and monoclonal anti-IgDa(AMS 9.1) reagents trigger B cell proliferation. Strain comparisons revealed better responses with PerC cells from BALB/c (“Th2-like”) mice. Interestingly, while IL-4 enhanced spleen B cell proliferation, this cytokine suppressed the PerC cell response to IgD ligation. We are testing other cytokines to optimize this response and are assessing the impact of IgD ligation on T cell survival in the PerC cell/TME model. These studies may inform strategies to enhance lymphocyte activation within TMEs.
Surface and cytoplasmic PD-L1 regulate distinct cell-intrinsic signaling and functional outcomes
Authors: Gupta Harshita B. , Lao Shunhua , Padron Alvaro , Zhou Mei , Liu Jinyou , Clark Curtis A. , Drerup Justin Michael , Garcia Myrna , Sareddy Gangadhara , Hurez Vincent , Li Rong , Vadlamudi Ratna , Curiel Tyler J.
Published: January 2023

We reported that PD-L1 regulates tumor cell-intrinsic mTOR, autophagy and tumor initiating cell (TIC) generation. We found significant cytoplasmic PD-L1 in various mouse and human tumor cell lines by Western, flow cytometry and confocal. To assess subcellular localized PD-L1 effects, we CRISPR knocked out PD-L1 (KO) from mouse B16 melanoma cells and re-expressed PD-L1 only on surface (surfKI; Display vector) or cytoplasm (cytoKI; PcDNA vector). KO cells proliferated slowly vs ctrl in vitro, partially restored by surfKI and cytoKI. KO suppressed mTORC1 (p70S6K), which was reversed by cytoKI but not surfKI. Rapamycin (mTORC1 inhibitor) reduced surfKI and KO proliferation equally, but both > cytoKI (57% vs 88%, p<0.0001). AZD8055 (mTORC1/2 inhibitor) effects were similar. In WT mice surfKI grew faster (p <0.0001) than KO and cytoKI. SurfKI grew similar to total KI when injected into immunocompromised NSG mice, whereas cytoKIgrew slower than either (p<0.01). CytoKI and surfKI partially restored TIC numbers in vitro. We similarly generated PD-L1KO, cytoKI and surfKI cells in human ES2 ovarian cancer cells and found similar growth, rapamycin and AZD8055 effects thus far. Thus, surface and cytoplasmic PD-L1 contribute distinctly to tumor-intrinsic signals and pathogenesis. Current works tests PD-1/PD-L1 cooperation and PD-L1 cytoplasmic tail effects. Understanding specific cell-intrinsic PD-L1 signals affords novel biology insights and suggests new treatments and drug discovery.