bluuebunny/crossref_metadata_2025 huggingface dataset:
B7-H1 negatively regulates IL-12 production by dendritic cells (165.10)
Authors: Gibbons Rachel , Liu Xin , Dong Haidong
Published: January 2023

A promising strategy in tumor immunotherapy is the use of activated dendritic cells as vehicles for tumor vaccines. Clinical trials of dendritic cell-based immunotherapies have proven successful, resulting in recent FDA approval for their use in the treatment of late-stage prostate cancer. Current formulations for dendritic cell-based immunotherapies are capable of prolonging patient survival by only four months, providing motivation for further investigation of ways to enhance dendritic cell priming of anti-tumor T cell responses. Since the discovery of B7-H1 by our laboratory, we have been investigating the regulatory function of B7-H1 expressed by immune cells. In preliminary studies investigating the use of B7-H1 knockout dendritic cells as tumor vaccines, we found that B7-H1 knockout dendritic cells prime enhanced anti-tumor CD8 T cell responses. Further investigations revealed that the enhanced CD8 T cell response raised by B7-H1 knockout dendritic cells is not due to a lack of signaling through the receptors for B7-H1 on CD8 T cells. We observed that B7-H1 knockout dendritic cells produced increased levels of IL-12, a key cytokine that is centrally involved in both innate and adaptive immunity. Our results suggest that B7-H1 expressed by dendritic cells has a role in negatively regulating IL-12 production, and as a result B7-H1 deficient dendritic cells are able to prime enhanced anti-tumor CD8 T cell responses.
Adoptive transfer of Tc17 CD8 T cells as an approach to elicit a better immune response to vaccination (165.3)
Authors: Yen Hung-Rong , Huang Ching-Tai , Lin Tzou-Yien , Pardoll Drew , Drake Charles
Published: January 2023

CD8 T cells that produce IL-17 is an unknown field with lots to explore. In our previous studies, we found a novel subset of IL-17 producing CD8 T cells persisted longer in a self-antigen autoimmune model versus conventional IFN-g producing Tc1 cells; furthermore, Tc17 can convert to an IFN-g producing phenotype in vivo. In the present study, we adoptively transferred a relatively small numbers (100,000) of IL-17 secreting TCR-transgenic antigen-specific CD8 T cells one day before vaccination by recombinant vaccinia virus encoded with full-length hemagglutinin. Tc 17 cells activated, expanded better than Tc1 cells 9 days after vaccination (Mean = 7.425x10e6 versus 1.350x10e6, p<0.05). Interestingly, Tc17 can convert to an IFN-g producing phenotype after vaccination. Our preliminary data fit the niche that is required to improve vaccination and immunotherapy and we suggest that, to treat infectious disease, CD8 Tc17 could be a promising effector T cell subset to achieve the goal. Additional mechanisms are being explored.
A New Rip2 Kinase Inhibitor for the Treatment of Intestinal Inflammation (169.5)
Authors: Zhao Yun , Song Joo Hye , Ajami Alfred M , Reinecker Hans-Christian
Published: January 2023

AIM: RIP2 is a critical mediator of NLRs signaling for innate immune activation. GEF-H1 interacts with NLRs and is responsible for initiating tyrosine phosphorylation of RIP2 required for the activation of NF-κB. We identified DCAM-253, 2-dialkylamino-9-indazolyl-purine, as a potent small molecule tyrosine kinase inhibitor of Rip2. Results: Gene specific siRNA mediated knockdown and expression of signaling intermediates in macrophages, and HEK293 cells was used to determine the mechanisms by which DCAM-253 inhibits Rip2 mediated induction of cytokine expression. DCAM-253 prevented the recruitment of GEF-H1 to the Rip2/NLR signaling complexes and subsequent tyrosine phosphorylation of Rip2. DCAM-253 inhibited TNFα production induced by GEF-H1 dependent NLR activation. DCAM-253 was able to protect mice from intestinal inflammation induced by epithelial barrier disruption and microbial defense activation in the DSS-induced colitis model. Contol group Mice presented with transmural leukocyte infiltration and crypt damage with tissue destruction indicating severe persistent colitis. In contrast, low cellular infiltration and intact intestinal epithelium were observed in the colon sections of mice treated with 2 oral doses of DCAM-253. Conclusion: We have identified a new small molecule inhibitor, which protects mice from colitis and promotes mucosal recovery by preventing Rip2 tyrosine phosphorylation upon intestinal barrier disruption and subsequent innate immune activation.
Mechanism of sophorolipid (SL) suppression of nitric oxide production in models of septic or endotoxic shock. (169.9)
Authors: Watson Michelle , Wu Brian , Bluth Martin , Nowakowski Maja
Published: January 2023

Introduction: Severe sepsis often leads to endotoxic shock and is reported to account for 9% of all deaths in the United States annually. Clinical manifestations of severe sepsis and shock are believed to result from the host’s overwhelming inflammatory reaction to infection and bacterial endotoxins. There is a clear need for new therapeutic approaches and agents based on a better understanding of the physiologic pathways underlying endotoxic shock. Objective: The aim of this project is to determine the mechanism of action of sophorolipids (SL) as novel therapeutic agents to block severe sepsis and shock. We established that SL were protective in vivo when injected following the initiation of endotoxic shock and in vitro when added to macrophage cultures following the addition of LPS. Methods: Cultured macrophages (RAW 264.7) were incubated for 1-5 days with LPS (0-200 ng/ml) and SL (100 ng/ml). Accumulated nitric oxide was measured using Griess reaction. Surface expression of CD14 and TLR4 was determined using specific antibodies (Santa Cruz Biotechnology, immunohistochemistry). Results: LPS induced nitric oxide production was inhibited by 70% in the presence of SL (SL alone had no effect on nitric oxide production). Surface expression of CD14 (macrophage LPS receptor) was also inhibited by 70% in the presence of SL; expression of TLR4 was inhibited by 15%. Conclusions: SL may interfere with binding and intracellular steps in the LPS-mediated gene activation cascade.
Myeloid-Derived suppressor cells in murine retrovirus-induced AIDS: inhibition of the T-cell and B-cell responsiveness that define the immunodeficiency (170.7)
Authors: Green Kathy , Cook W. James , Green William
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) have been characterized in several tumor settings however, they have been less well studied in response to infectious disease processes, in particular against retroviruses that induce immunodeficiency. Here we demonstrate for the first time the development of a highly immunosuppressive MDSC population that is dependent on LP-BM5 retrovirus infection, which causes murine acquired immunodeficiency. This MDSC population is CD11b+, Gr-1+, Ly6C+ , exhibits profound inhibition of immune responsiveness by a cell-dose and substantially iNOS-dependent mechanism that is independent of arginase activity, PD-1/PD-L1 expression, and IL-10; and the degree of immunosuppression parallels the extent of disease in differentially LP-BM5 susceptible w.t. vs. knock-out mouse strains. Not only T-cell, but also interestingly B-cell host responses, were suppressed by these MDSCs. The direct immunosuppression of B cell responses was confirmed by use of: purified B responder cells, multiple B-cell specific stimuli, and independent assays measuring B-cell expansion. This suppressive MDSC subset is positive for proviral LP-BM5 RNA, albeit at a significantly lower level than LP-BM5 infected non-fractionated spleen cells. These results, including the strong, direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression, and support a possible pathogenic effector role for these retrovirus-induced MDSCs.
Cannabinoid receptor 2 agonists inhibit migration of activated dendritic cells via modulation of MMP-9 (173.23)
Authors: Adhikary Sabina , Kocieda Virginia , Ganea Doina
Published: January 2023

In murine models of multiple sclerosis (experimental autoimmune encephalomyelitis or EAE), cannabinoid receptor 2 (CB2) agonists have been shown to be beneficial, reducing the number of CNS infiltrating leukocytes. Adhesion molecules, chemokines, and matrix metalloproteinases (MMPs), particularly MMP-9, play important roles in migration to the CNS. Antigen presenting cells such as dendritic cells (DC) and macrophages, as well as activated microglia are major producers of MMP-9. In this study, we investigated the effects of CB2 receptor agonists on DC migration and MMP-9 production utilizing both in vitro and in vivo migration assays. DC activated with cytokine cocktail (TNFα+IL-1β+IL-6+PGE2) in the presence or absence of CB2R agonist exhibited similar migratory capacity in transwell migration assays. However, the CB2R agonist treated cells migrated less in matrigel migration assays as compared to untreated cells. We found that this effect was due to reduced MMP-9 production in CB2R agonist treated cells. Also, in the in vivo migration assay using cytokine cocktail-activated DCs injected into mice footpads, we found significantly fewer CB2R agonist-treated DCs in the draining lymph nodes. Our data indicate that one of the mechanisms by which CB2R agonists attenuate inflammation is the inhibition of MMP-9 release which results in decreased migration of activated dendritic cells.
Trem-like 4-expressing dendritic cells and macrophages exhibit distinct inflammatory chemokine receptors (173.33)
Authors: Moyron-Quiroz Juan , Ampudia Jeanette , Yang Xifeng
Published: January 2023

Trem-like 4 (Treml4) is a recently identified type I transmembrane protein, member of the Trem (Triggering receptor expressed on myeloid cells) family. It is mainly expressed on splenic CD8α+ dendritic cells (DCs) and marginal metallophilic and red pulp macrophages. The natural ligand of Treml4 remains to be identified, but a fusion protein containing the extracellular domains of Treml4 is able to bind apoptotic and necrotic cells, suggesting it potentially plays a role in the clearance of dead cells, a process associated with anti-inflammatory and immunosuppressive effects. To further characterize the cell subsets expressing Treml4, we stained splenic DCs and macrophages with various flourochrome conjugated antibodies, including the anti-Treml4 antibody, 16E5. Our data demonstrated that both Treml4+ DCs and macrophages also express CCR6, but neither population expresses CXCR2 nor CCR5. In addition, Treml4+ DCs, but not Treml4+ macrophages, express CXCR3. Conversely, Treml4+ macrophages, but not Treml4+ DCs, express the receptor for the anaphylatoxin C5a. Taken together, these findings suggest that Treml4+ DCs and Treml4+ macrophages might be recruited to inflammation sites depending on the different chemoattractants produced.
Antibiotic induced alterations in the commensal microbiome reduce CD4+Foxp3+ Tregs in the colonic lamina propria and increase allergic responses to food (175.14)
Authors: Tripathi Prabhanshu , Stefka Andrew , Feehley Taylor , Patton Tiffany , Chang Eugene , Antonopoulos Dionysios , Pei Zhiheng , Nagler Cathryn
Published: January 2023

Antibiotic (Abx) use in early life has been implicated in the increasing prevalence of food allergy, presumably due to its effects on the commensal bacterial microbiome. To gain insight into the mechanisms by which commensal bacteria regulate allergic responses to food, C57Bl/6 mice were treated with a cocktail of Abx, beginning by gavage at two weeks of age and continuing in the drinking water after weaning. 16S rDNA based pyrosequence analysis demonstrated that Abx treatment significantly altered the composition and diversity of the fecal microbiota; Lachnospiraceae (Clostridia) were significantly reduced and Bacteroidaceae were enriched in fecal samples obtained from Abx treated mice. Reduced Clostridia abundance correlated with significantly decreased proportions of CD4+Foxp3+ Tregs in the colonic lamina propria, but not the spleen or mesenteric lymph node, of Abx treated mice compared to untreated controls. Elevated allergen specific IgE and IgG1 responses were induced in Abx treated (but not untreated) mice sensitized with purified peanut extract plus cholera toxin. Transfer of CD4+Foxp3+Tregs from untreated mice, prior to sensitization, blocked the allergen specific IgE response induced by Abx treatment. Our data supports the hypothesis that bacteria (Clostridia) induce CD4+Foxp3+ Tregs in the colonic mucosa. These Tregs are depleted (and protection against an allergic response to food is reduced) in Abx treated mice.
Anti-Allergic Asthma Properties of Brazilin through Inhibition of TH2 Responses in T Cells and in a Murine Model of Asthma (175.8)
Authors: Wang Chien-Neng , Kang Jaw-Jou , Liao Jiunn-Wang , Chiang Bor-Luen , Chen Hui-Chen , Lin Chia-Der , Huang Shih-Hsuan , Lai Yu-Ting , Lee Chen-Chen
Published: January 2023

The aim of the study was to determine whether brazilin exhibits any anti-inflammatory effects that inhibit T helper cell type II (TH2) responses and whether it suppresses allergic inflammation reactions in a murine model of asthma. We found that brazilin inhibited phorbol myristate acetate (PMA) + cyclic adenosine monophosphate (cAMP)-induced IL-4 and IL-5 expression in EL-4 T cells with respect to both RNA and protein levels in a dose-dependent manner. After intratracheal instillation of brazilin in ovalbumin (OVA)-immunized mice, we found that brazilin-treated mice exhibited decreases in the release of IL-4, IL-5, IL-13, eotaxin, and TNF-α in bronchoalveolar lavage fluid (BALF), inhibited TH2 functioning by a decrease in IL-4 production, and the attenuation of the OVA-induced lung eosinophilia, and airway hyperresponsiveness and remodeling. These results suggest that brazilin exhibits an anti-TH2 reaction both in vitro and in vivo. Brazilin as demonstrates therapeutic potential for allergic diseases.
B cell antigen presentation plays a limited role in primary and secondary CD4 T cell responses (176.16)
Authors: Wu Gregory , Archambault Angela , McGee Gretchen , Dragatsis Ioannis , Allenspach Eric , Raabe Tobias , Russell John , Laufer Terri
Published: January 2023

The activation and differentiation of effector CD4 T cells depends on interactions with MHCII-expressing antigen presenting cells (APCs). While several APC populations coordinate CD4 T cell function, several lines of evidence indicate that antigen presentation by B cells is a critical element to the cellular immune response. We have sought to determine the contribution of B cell antigen presentation to CD4 T cell priming and secondary responses. To address the hypothesis that B cells are sufficient APCs for CD4 T cell responses, we have designed a new tool in which individual subsets of APCs conditionally express MHCII in vivo. We successfully targeted a loxP-flanked stop sequence to the murine IA-β chain locus to utilize the Cre/lox system for conditional expression of MHCII. Conditional manipulation was achieved using CD19-Cre mice, restricting expression of MHCII to B cells. After priming, sub-optimal proliferation and activation of CD4 T cells was observed in mice expressing MHCII only by B cells. Further, limiting MHCII expression to B cells constrained secondary CD4 T cell responses in vivo. In a CD4 T cell-dependent model of autoimmunity, EAE, B cell expression of MHCII was not sufficient to support secondary autoreactive CD4 T cell responses targeting the CNS. These results demonstrate a novel capacity to conditionally express MHCII in vivo and highlight the limitations of B cell antigen presentation during the initiation and propagation of CD4 T cell function
A Distinct Population of Lipid Sensing iNKT Cells are Enriched in Fat and Protect Against Diet-Induced Obesity and Insulin Resistance (176.26)
Authors: Lynch Lydia , Tatitori Raju , Patrick Brennan , Stutte Susanne , Varghese Bindu , Hogan Andrew , Balk Steven , O'Shea Donal , O'Farrelly Cliona , von Andrian Ulrich , Brenner Michael , Mark Exley
Published: January 2023

Immunometabolic interactions regulate adipose tissue inflammation in obesity and subsequent development of insulin resistance. We have shown that human and murine fat contains the largest population of iNKT cells in the body. Fat resident iNKT cells are a distinct subset with unique phenotype and anti-inflammatory cytokine profile. These iNKT cells are depleted in diet-induced obesity and removal of high fat diet results in iNKT restoration in mice and humans. Furthermore, mice lacking iNKT cells have enhanced weight gain, larger adipocyte size, fatty liver infiltration and insulin resistance. Adoptive transfer of iNKT from WT mice into obese mice or activation of remaining iNKT in obese fat by through injection of alpha- galactosylceramide (aGC) had a profound improvement in insulin sensitivity and glucose handling, a decrease in serum triglycerides, leptin, body fat, fatty liver infiltration and proinflammatory macrophage levels. It is therefore beneficial to identify what causes iNKT cells depletion in obesity. We have purified lipids from obese and lean adipose tissue, to identify which, if any, lipid fraction may activate iNKT cells. Early preliminary data suggest that a HFD abherrently activate iNKT cells in obesity, leading to their loss, and subsequent development of insulin resistance. In summary, we have identified a distinct population if iNKT cells in fat and describe a unique role for iNKT in protection against obesity and related metabolic disease. .
Identification of the v-SNARE required for lytic granule fusion in cytotoxic T lymphocytes (176.27)
Authors: Pattu Varsha , Matti Ulf , Halimani Mahantappa , Weins Lisa , Rettig Jens
Published: January 2023

Cytotoxic T lymphocytes (CTLs) kill target cells by secretion of cytotoxic components such as perforin and granzymes, which are contained in lytic granules. Fusion of lytic granules occurs at the contact zone between the target cell and the CTL, the immunological synapse. Soluble NSF attachment receptor (SNARE) proteins are required for all fusion events in cells, but the SNARE proteins involved in lytic granule fusion in CTLs remain unknown. Using a targeted gene knock in strategy we identified the v-SNARE that is required for lytic granule fusion in CTLs. We first used super resolution microscopy combined with careful quantification to identify the v-SNARE that is localized exclusively to lytic granules in primary mouse CTLs. By specifically cleaving the v-SNARE with a bacterial neurotoxin and by gene knock down we show a complete loss of lytic granule fusion by two independent methods - 1) LAMP1 based degranulation assay and 2) real time imaging of exocytosis using TIRF microscopy. We therefore conclude that the v-SNARE that is localizing to the lytic granules is mediating their final fusion at the IS, thereby delivering the lethal hit needed for target cell killing.
Determination of protein-protein interactions of human eleven-nineteen lysine-rich leukemia protein ELL2 (176.8)
Authors: Arumemi Fortuna , Milcarek Christine
Published: January 2023

Eleven-nineteen lysine-rich leukemia (ELL) genes produce protein factors that aid in transcription elongation. ELL1 was originally found as a fusion partner with MLL in some multiple lineage leukemias. ELL2 mRNA and protein are induced 6-fold in plasma cells. ELL1 and ELL2 are very similar except for 2 regions between amino acids 352 - 443 and 473 - 516, indicating that they may have different sets of interacting proteins. Yeast two-hybrid (Y2H) assays were performed to determine what proteins might interact with each ELL. ELL2, was subcloned into four main sections (A1, A2, B, and C) to determine specific locations of interaction. The C portion of ELL2, aa 516 to 640, autoactivated transcription in the Y2H and was not further analyzed. Meanwhile, the A1 region interacted with CEP192 (regulator of pericentriolar material recruitment, centrosome maturation, and centriole duplication in mammalian cells); the A2 region interacted with RP3 gene (XLRP gene 3), FIGNLI (new subfamily member of ATPases associated with diverse cellular activities); and the B region interacted with HIFIA (transcription factor found in mammalian cells under reduced oxygen tension that plays an essential role in hypoxia) and SEC63 (a translocation protein). Further mapping of the interactions of the ELLs may help us to clarify their functions in gene regulation and plasma cell development.
Intracellular adenosine inhibits IgE-dependent degranulation of human skin mast cells (177.19)
Authors: Gomez Gregorio , Schwartz Lawrence
Published: January 2023

Adenosine is a purine nucleoside that modulates the immediate release (degranulation) of allergic mediators from mast cells. We show that adenosine at relatively high concentrations (IC50=245 µM) inhibits IgE-dependent degranulation of human skin mast cells by a non-toxic intracellular mechanism. We demonstrate that pre-treatment with the specific inhibitor of equilibrative nucleoside transporter 1 (ENT1/SLC29A1), NBMPR, dose-dependently prevented the inhibitory effect of adenosine. In contrast, blocking adenosine A2a receptor (A2aAR) signaling with the specific antagonist ZM241385 was ineffective. Thus, blocking the influx of extracellular adenosine, but not A2aAR signals, was necessary and sufficient to prevent the adenosine-mediated inhibition of degranulation. The inhibitory effect of adenosine was not due to acute toxicity since human skin mast cells cultured up to 3 days with adenosine at concentrations as high as 0.5 mM remained completely viable and activation-competent. Importantly, the inhibitory action of intracellular adenosine was specific for FcϵRI-induced signaling pathways since degranulation induced by Complement Factor 5a, Substance P, or calcium ionophore A23187 was not affected. Moreover, activation (phosphorylation) of the FcϵRI-proximal and critical signaling molecule Syk was significantly inhibited in adenosine-treated mast cells. Overall, these findings provide the first evidence of an inhibitory anti-allergic effect of intracellular adenosine.
STAT5 activity downstream of IgE and IgG receptors in mast cells (177.8)
Authors: Pullen Nicholas , Barnstein Brian , Falanga Yves , Lama Tamang Tenchee , Ryan John
Published: January 2023

Removal of STAT5 impairs mast cell development, survival, and IgE-mediated cytokine production, justifying a need to closely investigate its activity downstream of Fc receptors. We found that STAT5 tyrosine phosphorylation occurs shortly after FcϵRI crosslinking in a Fyn-dependent manner, while the suppression of Lyn, and other negative regulators of IgE signaling, appears to enhance STAT5 activation. Tyrosine phosphorylation of STAT5 also occured during IgG-mediated activity, suggesting some overlap in the signaling proximal to different Fc receptors in mast cells. Additionally, IgE- and IgG-mediated signaling elicited serine phosphorylation of both STAT5A and STAT5B, possibly in a divergent manner. Inhibition of Syk ablated IgE-mediated pS-STAT5, but affected to a lesser extent, if at all, pS-STAT5B and IgG-mediated total pS-STAT5. Furthermore, inhibition of JNK reduced pS-STAT5 during FcγR crosslinking. Collectively, these data highlight similarities and differences from variable receptor inputs as well as the different STAT5 proteins, supporting their essential roles in mast cell biology.
SLP-2 regulates T cell activation by optimizing cardiolipin compartmentalization in mitochondrial membranes and enhancing cellular respiration (178.7)
Authors: Christie Darah , Elias Isaac , Chau Luan , Lian Dameng , Wang Hao , Ball Eric , Dunn Stanley , Hatch Grant , Madrenas Joaquín
Published: January 2023

Stomatin-like protein 2 (SLP-2) is a mitochondrial protein of the highly conserved Stomatin, Prohibitin, Flotillin, HflC/K superfamily. The function of SLP-2 has remained unclear but members of this superfamily have been linked to membrane organization. We have previously reported that SLP-2 partitions within glycolipid-enriched, detergent-insoluble microdomains, and its expression is up-regulated upon T cell activation. This correlates with increased mitochondrial biogenesis and function. Using human inducible over-expression/down-regulation systems and newly generated T cell-specific SLP-2 knockout mice, we now report that SLP-2 binds directly and selectively to cardiolipin (CL) and interacts with prohibitins. Overexpression of SLP-2 leads to increased recruitment of prohibitins to mitochondrial membranes. This translates into optimal assembly and function of complexes I and II+III of the respiratory chain. SLP-2 deficiency in T cells is associated with abnormal CL compartmentalization and decreased respiration, which correlates with a selective post-transcriptional defect in the production of IL-2 and defective T cell (mostly CD4+) responses. Therefore, we propose that SLP-2 functions as an organizer of cardiolipin-enriched domains in mitochondrial membranes. These domains are required for optimal respiration and metabolic capacity during T cell activation. Our work also reveals a novel post-transcriptional checkpoint for IL-2 production under metabolic control.
Aging is associated with an increase in regulatory T cells, MCP-1 and RANTES (179.13)
Authors: Mansfield Aaron , Nevala Wendy , Dronca Roxana , Leontovich Alexey , Markovic Svetomir
Published: January 2023

We have observed Th2 polarization in patients with metastatic malignant melanoma. We hypothesized that changes in the immune system with age may parallel changes we have seen in patients with cancer. We analyzed the peripheral blood of 389 healthy blood donors. We profiled T cell subsets in 389 of the subjects, and antigen-specific tetramers for CMV, influenza and MART-1 in 58 of the subjects. We used a cytokine multiplex to analyze 42 cytokines in 95 of the subjects. We observed significant increases in CD4+ Th2 cells, and decreases in CD3+ T cells, cytotoxic T cells and helper T cells with age. There were fewer CMV-, influenza- and MART-1- specific naïve and CD8+ T cells with age. There were also significant increases in MCP-1 and RANTES with age. We observed differences between males in females in their cytokine profiles in their 20-40’s but not in their 50-60’s. In summary, we have found that aging is associated with an increase in CD4+ Th2 cells and concomitant decreases in total T cell count, cytotoxic T cells and helper T cells. Furthermore, aging is associated with increases in MCP-1 and RANTES. Females had different cytokine profiles than males before menopause, but not after.
Sequential expression of CD39 regulates late developmental Th17 plasticity imparting a regulatory cell phenotype (179.20)
Authors: Longhi Maria Serena , Bai Aiping , Wu Yan , Strom Terry , Robson Simon
Published: January 2023

Background: CD39 is an immune cell phenotype marker that exhibits ectonucleotidase activity, converting extracellular nucleotides to nucleosides. Although CD39 has been associated with Treg, the ectoenzyme is also expressed by a subpopulation of memory T-cells (CD4mem) with effector functions. We postulate that CD39 imparts plasticity to T helper type 17 (Th17) as well as co-ordinating Treg cellular programs of differentiation. Methods and Results: CD4mem cells were exposed to 1) IL6+IL1β+rTGFβ or 2) IL6+IL1β+IL23 or 3) IL6+IL1β+rTGFβ+IL23 to induce Th17 polarization. Cells were then treated with high dose IL2+anti-CD3/anti-CD28 to favour Treg differentiation and then re-exposed to Th17 differentiating conditions to induce putative reverse or suppressive Th17 (revTh17) cell. Impacts of purinergic mediators on the effector phenotype and functions were assessed. CD4mem could be differentiated sequentially to Th17, Treg and revTh17. In contrast to the inflammatory properties associated with prototypic Th17 cells, revTh17 exhibited a suppressive phenotype. Cells responded in a context-dependent manner to adenosine that boosted revTh17 suppression in the absence of TGFβ. Conclusion: Differential levels of expression of CD39 designate early Th17 cells from later Treg/revTh17 cell plasticity. The potential for Treg to revert to the inflammatory Th17 phenotype is mitigated by expression of CD39, as indicated by enhancements of suppressive function in vitro.
Modulation of T cell receptor and interleukin-2 receptor signaling in human primary CD4+ T cells (179.7)
Authors: Perry Daniel , Thompson James , Han Zhao , Brusko Todd
Published: January 2023

Type 1 Diabetes (T1D) results from the aberrant autoimmune targeting and destruction of insulin-producing β-cells, and dysregulation of T cell activation is central to this break in tolerance. Regulatory T cells (Tregs) are fundamental to maintaining immune homeostasis and functional defects of this T cell subset can potentiate autoimmunity. Therefore, Tregs represent an attractive target for clinical intervention of T1D. Genetic variants of PTPN2 and PTPN22 have been linked to several autoimmune diseases, including T1D, by genome wide association studies. These genes encode for phosphates that dampen IL-2R and TCR signaling, respectively. In this study, we have utilized lentiviral vectors for dual overexpression and knockdown of these genes in primary human CD4+ T cells. We hypothesize that the suppressive function and phenotypic stability of human Tregs is enhanced by increased IL-2R and TCR signaling and deficient following downregulation of IL-2R and TCR signaling. Pathway-modified human effector T cells (Teff) will display altered lineage skewing and may be resistant to AICD and suppression when IL-2R and TCR signaling are increased. Further, simultaneous modulation of these pathways will synergistically affect T cell function. These data will provide insights into how direct modulation of these autoimmune signature pathways controls adaptive Treg and Teff activity, and further, have important implications for adoptive Treg therapy for the treatment of autoimmunity.
TLR2 activation of TRIF controls both early inflammatory and late tolerance responses to Borrelia burgdorferi (180.11)
Authors: Petnicki-Ocwieja Tanja , Chung Erin , Acosta David , Ramos Laurie , Shin Ok , Ghosh Sanjukta , Kobzik Lester , Li Xin , Hu Linden
Published: January 2023

TRIF is an adaptor molecule important in transducing signals from intracellularly signaling toll-like receptors (TLRs) 3 and 4, but not other TLR family members. Recently, TLR2 was found to signal from intracellular compartments. Using a synthetic ligand for TLR2/1 heterodimers as well as the bacterium Borrelia burgdorferi, which causes Lyme disease and is a strong activator of TLR2/1, we found that, contrary to current dogma, TLR2 signaling can utilize TRIF. Unlike TRIF signaling by other TLRs, TLR2-mediated TRIF signaling is dependent on the presence of another adaptor molecule, MyD88. However, unlike MyD88, TRIF deficiency does not result in diminished control of infection with B. burgdorferi. This appears to be due to the effects of MyD88 on phagocytosis via scavenger receptors such as MARCO which are not affected by the loss of TRIF. Although TRIF deficiency is associated with a decrease in pro-inflammatory cytokine induction in vitro, in an animal model of B. burgdorferi infection, loss of TRIF resulted in an increase in IL-6. In vitro studies mimicking longer exposures to B. burgdorferi show that TRIF mediates tolerance to the organism by suppressing pro-inflammatory cytokines disproportionately to anti-inflammatory cytokines in response to long term stimulation. Our studies suggest a dual role for TRIF in early and late TLR2 responses to infectious organisms.
Targeted degradation of DOK3 regulates LPS responses and endotoxin tolerance (180.13)
Authors: O'Loughlin Jason , Peng Qisheng , Humphrey Mary Beth
Published: January 2023

Innate immune activation via Toll-like receptors (TLRs), although critical for host defense against infection, must be regulated to prevent sustained cell activation that can lead to cell death. Cells previously stimulated with lipopolysaccharide (LPS) develop endotoxin tolerance making the cells hypo-responsive to additional TLR stimulation. We investigated the role of downstream of tyrosine kinase 3 (DOK3) in regulating TLR responses and endotoxin tolerance of macrophages and find that DOK3 is a negative regulator of TLR4 and TLR9. Mechanistically, LPS induces ubiquitin-mediated degradation of DOK3 and the Ras activator SOS1, in a DOK3-dependent manner, with subsequent inhibition of ERK activation and repression of IL-1β and IL-10 production. In LPS-induced tolerant macrophages, DOK3 expression remains stable allowing it to sequester SOS1 and Grb2 preventing ERK activation. DOK3-deficient cells demonstrate enhanced sensitivity to LPS and to LPS-induced tolerance becoming tolerant at lower levels of LPS than wild type cells. Additionally, DOK3 negatively regulates several mediators of tolerance including SHIP1, IRAK-M, and SOCS1. Taken together, the absence of DOK3 increases LPS signaling leading to more rapid induction of LPS-induced tolerance. Thus, DOK3 mediates LPS responses and the induction of endotoxin tolerance by a novel mechanism of SOS1 regulation.
A novel TRIF-independent role for the TLR adaptor molecule TRAM during infection with Francisella tularensis. (180.15)
Authors: Morris Ian , Linehan Leslie , Shen Haiqian , Medina Edward , Bergman Molly , Berton Michael
Published: January 2023

The only established function for the TLR adaptor molecule TRAM is as an adaptor linking TLR4 and TRIF to the induction of Type I interferon responses. However, in studies of pulmonary infection with the bacterial pathogen Francisella tularensis (LVS strain), we found that TRAM-/- mice, but not TRIF-/- mice, are more susceptible to F. tularensis infection than WT mice. TRAM-deficient mice exhibited significantly reduced median-time-to-death, increased mortality rate, and a significantly increased bacterial organ burden by days 1-2 after infection compared to WT and TRIF-/- mice. These results are surprising because numerous studies have shown that F. tularensis, despite being Gram-negative, signals the host via TLR2 rather than TLR4. We have also observed a similar increased susceptibility to infection in TRAM-/- mice systemically infected with Listeria monocytogenes. These novel findings suggested that TRAM may function not only in a TLR4 signaling pathway but also as part of a TLR2 pathway. To begin to characterize such a pathway, we expressed a TRAM-GFP fusion protein in macrophages and demonstrated via confocal microscopy that the TRAM-GFP colocalized with TLR2 and F. tularensis upon infection. Moreover, we found that TRAM-TLR2 complexes could be coimmunoprecipitated from TLR2-stimulated macrophages. Collectively, these findings suggest a heretofore unknown role for the TLR adaptor molecule TRAM in TLR2-dependent immune responses.
The omentum is a site of IgM production during T cell-independent bacterial infection in secondary lymphoid organ-deficient mice (43.6)
Authors: Jones Derek , Racine Rachael , Jones Maura , Wittmer Susan , Randall Troy , Winslow Gary
Published: January 2023

Infection of mice with the bacterium Ehrlichia muris results in a T cell-independent (TI) splenic IgM response, and this antibody is secreted largely by a population of CD11c-expressing B cell plasmablasts in the spleen. However, we found that the TI IgM is maintained in the absence of a spleen; infection of splenectomized mice resulted in a modest decrease in antigen-specific serum IgM, indicating that splenic B cells are not required for the generation of the protective antibody response. To identify the source of the IgM-secreting cells, we infected secondary lymphoid organ (SLO)-deficient mice, which lack both spleen and lymph nodes. SLO-deficient mice were similarly able to produce ehrlichia-specific IgM, and both splenectomized and SLO-deficient mice were protected against challenge infection with a lethal ehrlichial species. These findings led us to investigate the omentum as a source of IgM-secreting cells during infection. We identified the omentum as a large reservoir of antigen-specific IgM-secreting cells; these B cells expressed CD11c, but lacked expression of CD138. CD11c-expressing B cells were elicited by intraperitoneal, but not intravenous infection, revealing a route-dependent response to peritoneal antigen. Our data reveal the omentum as an important site of TI IgM production against peritoneal antigens, in the absence of conventional secondary lymphoid organs.
Fli-1 transcription factor directly regulates expression of inflammatory chemokine MCP-1 (44.20)
Authors: Zhang Xian , Suzuki Eiji , Karam Eva , Williams Sarah , Gikeson Gary
Published: January 2023

Fli-1, a member of the Ets family of transcription factors, is highly expressed in immune cells and endothelial cells and is implicated in the development of nephritis in murine models of systemic lupus erythematosus (SLE). Expression of monocyte chemotactic protein-1 (MCP-1) play an important role in the inflammation responses. In this study, we examined the role of Fli-1 on production of MCP-1 in endothelial cells. We found expression of MCP-1 was significantly reduced in endothelial cells isolated from Fli-1 heterozygous knockout NZM2410 mice (Fli1+/-; Fli-1 homozygous knockout is embryonic lethal) after stimulation with Lipopolysaccharides (LPS) compared with endothelial cells isolated from wild-type controls. The expression of Fli-1 protein was significantly reduced and the production of MCP-1 was significantly decreased in endothelial cells transfected with specific Fli-1 siRNA compared to cells transfected with negative control siRNA. We demonstrated that Fli-1 directly binds to the promotor of MCP-1 by Chromatin Immunoprecipitation (ChIP) assay. Our data indicate that transcription factor Fli-1 can binds to the promoter of MCP-1 and directly regulates the expression of inflammatory chemokine MCP-1.
Viral Infection Triggers Rapid Differentiation of Human Blood Monocytes into Dendritic Cells (45.12)
Authors: Hou Wanqiu , Gibbs James , Lu Xiuju , Brooke Christopher , Roy Devika , Modlin Robert , Bennink Jack , Yewdell Jonathan
Published: January 2023

Viruses post a constant threat of human existence. Although human immune system plays a central role in recognizing and eradicating pathogens, how it responds to virus infection remains unclear. Here we show that monocytes make up the majority of a wide variety of virus susceptible population in human peripheral blood mononuclear cells. Notably, infection with influenza virus, vesicular stomatitis virus and vaccinia virus promotes monocytes to differentiate within 18 hours into CD16-negative CD83-positive mature dendritic cells (DCs) with enhanced capacity to activate T cells. Virus-induced monocyte differentiation into DCs does not require cell division, and happens despite the synthesis of virus proteins, suggesting that monocytes thwart the capacity of these highly lytic viruses to hijack host cell biosynthesis pathway. Indeed, virus-induced DC differentiation from monocytes requires infectious virus and viral protein synthesis. Thus, our results demonstrate that monocytes are uniquely susceptible to virus infection among human peripheral blood mononuclear cells, with the likely purpose of differentiating into DCs with enhanced capacity to activate innate and acquired anti-virus immunity.
Involvement of notch in activation of human γδ T cells (45.22)
Authors: Gogoi Dimpu , Chiplunkar Shubhada
Published: January 2023

γδ T cells recognize phosphorylated antigens and play an important role in tumor immunity. The aim of the present study was to investigate the role of Notch signaling pathway in antigen specific responses of γδ T cells. Expression of mRNA for Notch1 and Notch2 genes was predominantly observed in peripheral γδ T cells. The presence of active Notch intracellular domain in γδ T cells was confirmed by confocal microscopy and western blotting. Proliferation of γδ T cells and IFN-γ release in response to phosphoantigens was inhibited in the presence of γ-secretase inhibitor confirming involvement of Notch signal. Delta like ligands 1 and 4 activated γδ T cells while Jagged1 inhibited the γδ T cell response. Oral and breast cancer cells showed dominant expression of Jagged1 ligand which may exert inhibitory signal as a way to evade immune recognition by γδ T cells. Decreased expression of CD107a and diminished lysis of tumor targets in the presence of γ-secretase inhibitor confirmed that Notch was involved in cytotoxic effector functions of γδ T cells. Thus, our data for the first time demonstrates that Notch signal plays an indispensable role in antigen specific responses of γδ T cells. Future immunotherapeutic strategies can exploit Notch signaling in γδ T cells to achieve therapeutic benefits in cancer patients.
Distinct functions of CXCL9/Mig and CXCL10/IP-10 in the priming of tumor-specific IFN-g producing effector T cells (46.2)
Authors: Gorbachev Anton , Kish Danielle , Petro Marianne , Fairchild Robert
Published: January 2023

Major factors limiting immune-mediated elimination of tumors are poor activation and/or insufficient effector functions of tumor-reactive T cells. Chemokines CXCL9/Mig and CXCL10/IP-10 share many anti-tumor functions as potent T cell chemoattractants and angiostatics. However, the individual contribution of each chemokine to the activation of anti-tumor T cell responses remains unclear. Here we tested the role of Mig and IP-10 in the activation of tumor-reactive T cells to the immunogenic murine fibrosarcoma MCA205. Tumor antigen-presenting dendritic cells (DC) deficient in Mig were less potent in activating IFN-g producing MCA205-reactive T cells than were Mig-sufficient DC. In contrast, IP-10-deficient DC were significantly more stimulatory for IFN-g producing tumor-specific T cells than were wild type DC. Activation of tumor-reactive IFN-g producing T cells in tumor hosts was markedly increased in IP-10-/- mice and was decreased in Mig-/- mice when compared to wild type mice. These differences in T cell activation correlated with either accelerated or decreased tumor growth in Mig-/- or in IP-10-/- mice, respectively. In contrast, host DC deficiency either in Mig or in IP-10 did not affect IL-17 producing tumor-specific T cells. These results demonstrate antagonistic functions of Mig and IP-10 during priming of tumor-reactive T cells producing IFN-g and suggest that manipulating the expression of these chemokines by DC can generate more potent anti-tumor immune responses.
Identification and temporal monitoring of breast cancer-associated T cell receptors with high throughput sequencing (46.23)
Authors: Han Jian , Wang Chunlin , Sanders Catherine , Yang Qunying , Byrne-Steele Miranda , Ng Enders , Wong Chris , Kwong Ava , Lu Chiung-Mei
Published: January 2023

Tumor-specific antigens may trigger an immune response that leads to T lymphocytes infiltrating the tumor tissue. We have developed a method to study the immune repertoire of a sample by utilizing a patented multiplex PCR amplification strategy, arm-PCR (Patent No. 7,999,092), coupled with high throughput sequencing (Wang et al., PNAS 2010). Using this method, we studied a patient’s surgically removed breast cancer tissue, the nearby normal tissue, and sorted peripheral blood. The patient’s sorted peripheral blood was also examined three months, six months, and one year post-surgery. Dominant T cell clones with specific CDR3 sequences were identified from the breast cancer tissue. Some of these same clones were found expressed at high levels in the nearby normal tissue and peripheral CD8+ cells. After treatment, dynamic changes in these cancer-associated clones were apparent, demonstrating the capability of the current technology to identify specific T cells associated with a patient’s cancer tissue. These specific T cells can serve as personalized biomarkers for prognosis, treatment evaluations, and early detection of recurrence. They can also be used to develop personalized treatment strategies. Currently, this study has been extended to examine 10 additional breast cancer patients.
Investigating the Role of ERK2 in Peripheral T cells Function Using a Novel Transgenic Mouse Model (47.13)
Authors: Janardhanan Preethi , Quintana Gwendolyn , Byerly Susan , Ji Niannian , Forsthuber Thomas
Published: January 2023

Extracellular signal Regulated Kinase 2 (ERK2) is an important Ser-Thr Kinase involved in MAPK pathway. It is indispensable for early T cell development and is also believed to play a role in T-cell specific antigen recognition signaling, cell proliferation, differentiation, migration and survival. However, it remains unclear how ERK2 regulates peripheral T cell function. Our studies focused on the impact and mechanism of ERK2 deficiency in peripheral T cell activation/function utilizing a mouse model with a conditional ERK2 knockout identified and confirmed by expression of a YFP reporter. Our gene knockout strategy relied on Cre recombinase driven by either distal Lck or inducible promoters. Using flow cytometry analysis/cell sorting and cytokine ELISPOT assay, we have found that T cell activation, survival and cytokine production were impaired in the absence of ERK2. Our results also indicate a role for ERK2 in cell survival in serum starvation and ionomycin/Ca2+ mediated apoptosis. The presented approach will allow us to further investigate the role of ERK2 in other peripheral T cell functions and potentially identify cellular signaling pathways that could be explored for the treatment of CD4+ T cell mediated autoimmune diseases such as MS.
Effect of filgrastim (recombinant human granulocyte colony stimulating factor) on IgE responses in human asthma (48.1)
Authors: Smith-Norowitz Tamar , Kohlhoff Stephan , Ginsburg David , Norowitz Kevin , Joks Rauno , Chice Seto , Durkin Helen , Bluth Martin
Published: January 2023

Rationale: The role of peripheral blood progenitor cell mobilization on IgE responses has not been studied. Methods: Distributions of blood lymphocytes (CD4+, CD8+, CD8+CD60+, CD19+, CD23+, CD16/56+, CD25, CD45RA+, CD45RO+, CD34+), and levels of serum immunoglobulins (IgM, IgG, IgA, IgE) were studied in an allergic asthmatic serum IgE+ (181 IU/mL) adult (m/45 y/o) donor undergoing routine stem cell mobilization protocol (American Society of Hematology) before (day -30), during (day 4), and after (1 wk post last dose) filgrastim (subcutaneous, 480 mcg, 2qd) treatment (flow cytometry, nephelometry, UniCAP Total IgE Fluoroenzymeimmunoassay). Results: On day 4 of filgrastim treatment, numbers of CD8+CD60+ T cells and CD23+ blood cells dramatically increased (>2 fold); CD34+ cell numbers also increased. One week after treatment, numbers of these cells decreased to pre-treatment levels. On day 4 of treatment, serum IgE levels decreased (>50%). However, one week after treatment, IgE levels approached pre-treatment levels. Conclusions. Filgrastim treatment transiently increases numbers of CD8+CD60+ T cells known to regulate human IgE responses (Smith-Norowitz, et al., JI, 2008), while also transiently suppressing ongoing IgE responses. These results suggest that filgrastim might be useful in modulating allergic responses.