bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene array analysis of PD-1H overexpressing monocytes reveals a pro-inflammatory profile (INC2P.417)
Authors: Shankar Premlata , bharaj preeti , Yi Guohua , Swamy Manjunath
Published: January 2023

We have previously reported that overexpression of Programmed Death -1 Homolog (PD-1H) in human monocytes leads to activation and spontaneous secretion of multiple pro inflammatory cytokines. Here we evaluated changes in monocytes gene expression after enforced PD-1H expression by gene array. Total RNA isolated from PD-1H-expressing and control monocytes were analyzed with human Illumina HT-12 v4 Expression BeadChip. Fold change of genes were confirmed by real time PCR. Data revealed alterations in 51 potential candidate genes that relate to immune response, cell adhesion and metabolism. Genes corresponding to pro-inflammatory cytokines showed the highest upregulation. Compared to vector control, PD-1H induced a 7, 3.2, 3.0, 5.8, 4.4 and 3.1 fold upregulation of TNF-α, IL-1beta, IFN-α, γ, λ and IL-27 respectively. Further, the difference was abrogated in presence of specific but not scrambled siRNA. The data are in agreement with our cytometric bead array analysis showing induction of proinflammatory cytokines, IL-6, IL-1β and TNF-α by PD-1H. Other genes related to inflammation, including transglutaminase 2, the transcription factor NF-kB (p65 and p50) and toll like receptors 3 and 4 were also upregulated 5, 4.5 and 2.5 fold respectively. The analysis also revealed a novel set of previously unreported genes that need to be further mined and validated. We conclude that PD-1H functions to enhance monocyte activation.
A Rho GDP dissociation inhibitor produced by apoptotic T cells inhibits growth of Mycobacterium tuberculosis (INC2P.415)
Authors: Venkatasubramanian Sambasivan , Dhiman Rohan , Paidipally Padmaja , Cheekatla Satyanarayana , Tripathi Deepak , Welch Elwyn , Tvinnereim Amy , Jones Brenda , Theodorescu Dan , Barnes Peter , Vankayalapati Ramakrishna
Published: January 2023

In the current study, we found that a subpopulation of apoptotic T-cells (CD4+CD25+ and 85% Foxp3+) from persons with latent tuberculosis (TB) infection, inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs) from 16.5 ± 1.2 x 106 vs 2.2 ± 1.1 x 106 CFU (p=0.001). Analysis of culture supernatants from T-cells by 2D gel electrophoresis and LC MS/MS indicated that a soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic T-cells is responsible for this inhibition of M.tb growth in MDMs and in mice. M. tb-expanded CD4+CD25+Foxp3+D4GDI+ cells do not produce IL-10, TGF-β and IFN-γ. D4GDI inhibited growth of M. tb from 10.4 ± 1.1 x 106 vs 2.8 ± 0.6 x 106 CFU (p<0.001) and D4GDI siRNA reversed T-cells-dependent M. tb growth inhibition in MDMs from 1.8 ± 0.3 x 106 to 5.5 ± 1 x 106 CFU (p=0.001). D4GDI enhanced production of IL-1β (43 ± 33 to 193 ± 104 pg/ml, p=0.02), TNF-α (88 ± 126 to 129 ± 111 pg/ml, p=0.03) and ROS production and enhanced apoptosis of M. tb-infected MDMs to inhibit M.tb growth. D4GDI was concentrated at the site of disease in TB patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from TB patients produced less D4GDI than PBMC from persons with LTBI (13.7 ± 9.3 vs. 40.6 ± 3.2, p=0.02). Our study provides the first evidence that a subpopulation of T-cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.
Macrophage production of innate immune protein C1q is associated with M2 polarization (INM1P.434)
Authors: Fraser Deborah , Melzer Emily , Camacho Alexis , Gomez Mariam
Published: January 2023

C1q is an important recognition molecule of the innate immune response. It plays a dual role in vivo, triggering inflammation via activation of the classical complement pathway, to clear pathogens. However, it can also directly opsonize targets including apoptotic cells and damaged-self molecules, leading to increased macrophage phagocytosis and M2-like anti-inflammatory cytokine production. C1q is primarily synthesized by macrophages. Therefore we investigated C1q production by macrophages under various conditions. Raw 264.7, a murine macrophage cell line, were incubated with different targets or under conditions associated with M1 or M2 polarization. C1q gene expression levels were measured by quantitative RT-PCR from isolated mRNA. C1q protein expression and secretion was measured in cell media by hemolytic titer. The results showed a decrease in C1q production at the gene and protein level when cells were treated with M1-associated inflammatory stimuli such as high levels of LPS, and/or IFNγ. Interestingly, treating the cells with M2-associated stimuli such as IL-4, or interaction of macrophages with “self” targets such as damaged self molecules oxidized or acetylated LDL yielded an increase in C1q production. These data support a non-complement associated role for C1q in resolving inflammation and prevention of autoimmune disease, and suggest it may act as a novel biomarker for M2-like inflammation resolving macrophage polarization.
Gata3 regulates the maturation program required for bone marrow exit and proliferation in Natural Killer cells (INM1P.435)
Authors: Lee Seung-Hwan , Ali Alaa
Published: January 2023

Natural killer (NK) cells are innate lymphocytes capable of killing virally infected cells or transformed cells. NK cells differentiate in the bone marrow before they migrate out to peripheral organs. The developmental program leading to functionally mature NK cells has been studied in the context of several transcription factors. However, the role of GATA-3 in NK cell development has not been completely understood. Using NK-cell specific Gata3 knockout mice (NK-Gata3-/-), we demonstrated that GATA-3 is required for the NK cell maturation beyond the CD27 single positive stage and for efficient IFN-g production. The frequencies of NK cells from NK-Gata3-/- mice were found higher in the bone marrow but lower in peripheral organs compared to control littermates, indicating that GATA-3 controls the maturation program required for bone marrow egress. Despite this, upon MCMV infection, NK cells from NK-Gata3-/- mice were able to expand vigorously, achieving NK cell frequencies surpassing those in controls, and therefore provided comparable protection. The enhanced proliferation of GATA-3-deficient NK cells was associated with enhanced upregulation of CD25 expression. Thus, the NK-Gata3-/- mice serve as a model to study the NK cell developmental process linked to cell migration, effector functions and proliferation.
p47phox, a subunit of NADPH oxidase, enhances the function of Nrf2 (INM1P.441)
Authors: Joo Myungsoo , Kim Kyun Ha
Published: January 2023

Although an essential component to clear invading bacteria in innate immunity, reactive oxygen species (ROS) can adversely affect tissue. However, ROS activate Nrf2 and induce the expression of anti-oxidant enzymes including NQO1, HO-1 and GCLC, which in turn reduce the adverse effects of ROS. Given a high reactivity of ROS, we postulate that NADPH oxidase, a major producer of ROS in macrophages, exists proximal to Nrf2-Keap1 complex to effectively activate Nrf2. Here, we show that p47phox, a cytosolic subunit of NADPH oxidase, physically interacted with Nrf2 and facilitated the protective function of Nrf2 in inflammation. Biochemical and functional analyses show that p47phox bound to Nrf2 and enhanced the expression of Nrf2-dependent genes such as NQO1, HO-1 and GCLC. Immunoprecipitation assays indicate that p47phox did not interfere with the binding between Nrf2 and Keap1 but suppressed the ubiquitination of Nrf2, suggesting that p47phox enhances Nrf2 transcriptional activity by disturbing the ubiquitination of Nrf2 by Keap1. Consistent with these results, bone marrow-derived macrophage (BMDM) from p47phox -/- mice showed reduced expression levels of Nrf2-dependent genes, compared with BMDM from wild type littermates. In addition, overexpression of p47phox in the mouse lung was sufficient to suppress LPS-induced neutrophilic lung inflammation. Together, our results suggest that p47phox binds to Nrf2, enhancing the protective function of Nrf2 in inflammation.
The heightened inflammatory response makes females are more susceptible to F. tularensis-mediated death (INM1P.444)
Authors: Spencer Charles , Martinez Danielle , Setzu Nicole , Ramos Mireya
Published: January 2023

The zoonotic infectious disease, Francisella tularensis, the causative agent for human tularemia, elicits high cytokine levels as a result of rampant inflammation, a condition referred to as “cytokine storm.” This cytokine storm is responsible for the severity of symptoms and ultimately for the death of the host. Though men have a tendency to be exposed to the pathogen more frequently than women, do to its presence in wild animals, women have a significantly greater inflammatory response than do men. Therefore, we asked whether their enhanced inflammatory response might make women more susceptible to tularemia-mediated death. Male and female mice were infected with F. tularensis and their survival and inflammatory response monitored. Subsequently, the role of sex hormones was considered by infecting gonadectomized mice. On average, 15% female mice survived infection compared with 90% of male mice. In addition, the female mice displayed an increased serum cytokine storm compared with male mice despite similar levels of bacterial growth. We seek to identify the factors controlling this increased susceptibility of female mice. This line of investigation may lead to the development of novel therapeutics and/or treatment strategies specific to women exposed to F. tularensis.
Regulating role of transmembrane protein 173 (Tmem173) on RANKL-induced osteoclastogenesis (INM1P.446)
Authors: Jang Yong-Suk , Choe Chung-Hyeon , Park Jisang , Yu Kang-Yeol , Kim Ju
Published: January 2023

Tmem173, also known as STING, MITA and MPYS, is a potential stimulator of IFN-β production which is essential for innate immune response in monocyte/macrophage cells. Based on the known function of IFN-β as a negative feedback controller for RANKL-induced osteoclast differentiation of monocytic macrophage cells, we investigated the effect of Tmem173 on RANKL-induced osteoclastogenesis via monitoring the osteoclast-associated gene expression and protein activation in RAW264.7 cells. TRAP activity and bone resorption activity were significantly suppressed in Tmem173-overexpressed RAW264.7 cells compared to control vector-transfected RAW264.7 cells. Tmem173 overexpression also down-regulated expression of osteoclast marker genes, such as TRAP, MMP-9 and cathepsin K, in RANKL-treated RAW264.7 cells. These marker genes were up-regulated efficiently in Tmem173 knock-downed RAW264.7 cells. Tmem173 overexpression down-regulated the expression of osteoclast-specific transcription factors, such as c-Fos and NFATc1, and RANKL-induced activation of ERK mitogen-activated protein kinase. In addition, we observed the down-regulation of RANKL-induced expression of COX-2 and iNOS. Finally, we found that Tmem173 expression was up-regulated by osteoprotegerin (OPG). These results suggest that regulatory role of Tmem173 in RANKL-mediated osteoclast differentiation.
A miRNA regulatory circuitry tightly tunes macrophage inflammatory response (INM1P.426)
Authors: Mann Mati
Published: January 2023

Regulation of the immune system is vital to preventing many pathogenic disorders, and mammals have developed a complex system of checks and balances for immune regulation in order to allow immune responses to foreign pathogens while avoiding chronic inflammatory responses. Recently, it has become evident that micro-RNAs (miRNAs) play an important role in regulating immune response. Among these, miR-155 and miR-146a were identified as inflammatory response miRNAs that are upregulated by NF-κB signaling. Here, we set to characterize the hierarchy and genetic interaction of miR-146a and miR-155 in regulating macrophage inflammatory response during acute and chronic conditions. We show a regulatory network where miR-155 expression amplify the inflammatory response by prolonged NF-κB activity through PI3K signanling, while miR-146a attenuates the inflammatory response by inhibiting NF-κB activity and miR-155 expression. This regulatory circuitry enables strong acute inflammatory response while avoiding the development of chronic inflammation.
Sulforaphane blocks HIV infection of macrophages (IRC4P.459)
Authors: Furuya Andrea , Sharifi John , Jellinger Robert , Cristofano Paul , de Noronha Carlos
Published: January 2023

Despite highly active anti-retroviral treatment (HAART), HIV infection still claims millions of lives annually. HAART therapy is accompanied by high financial costs, the emergence of resistant viruses and short- and long-term side-effects. For this adjunctive therapeutic based on natural products should be considered to ameliorate complications due to HIV infection. Here we show for the first time that sulforaphane (SFN), a natural product produced in cruciferous vegetables blocks HIV infection in primary macrophages but not in primary T cells or several other cell types tested by mobilizing the transcription factor nuclear erythroid 2-related factor 2 (Nrf2). SFN blocked HIV infection in macrophages and macrophage-like cell lines by triggering a block before proviral integration. Furthermore we found that other compounds known to mobilize Nrf2 in macrophages also establish an intracellular state that counters HIV infection. Additionally we found that depleting Nrf2 boosts HIV infection in primary macrophage cultures. This led us to hypothesize that SFN and other compounds that trigger Nrf2 action, in macrophages, act through this protein to establish an intracellular antiviral state that counters HIV. We propose that SFN and other modulators of Nrf2 can be used to block HIV infection and therefore SFN could serve as an auxiliary therapeutic in ART experienced patients.
Expression of cationic amino acid transporter 2 is required for myeloid derived suppressor cell-mediated control of T cell immunity (IRC4P.460)
Authors: Cimen Bozkus Cansu , Elzey Bennett , Crist Scott , Ellies Lesley , Ratliff Timothy
Published: January 2023

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immature cells that expand during benign and cancer-associated inflammation. MDSC regulate innate and adaptive immunity, notably by inhibiting T cell functions. Increased metabolism of L-Arginine (L-Arg), through arginase 1 (ARG1) and nitric oxide synthase 2 (NOS2), is well documented as a major MDSC suppressive mechanism. However, the mechanism by which extracellular L-Arg is transported into MDSC is not defined. Using murine models of prostate specific inflammation and cancer, we show that MDSC recruited to localized inflammation and tumor sites upregulate cationic amino acid transporter 2 (Cat2) coordinately with Arg1 and Nos2. CAT2 contributes to the transport of L-Arg in MDSC and acts as an important regulator of MDSC suppressive function. MDSC that lack CAT2 have significantly reduced suppressive ability ex vivo and display impaired capacity for regulating T cell responses in vivo as evidenced by increased T cell expansion and decreased tumor growth in Cat2-/- mice. The abrogation of suppressive function is due to lower L-Arg uptake, which leads to the impaired ability of NOS2 to catalyze L-Arg metabolic processes. These findings demonstrate that CAT2 is an important regulator of MDSC suppressive function. In the absence of CAT2 MDSC display diminished capacity for controlling T cell immunity in prostate inflammation and cancer models, where the loss of CAT2 results in enhanced antitumor activity.
Distinct features of newly found liver-draining lymph nodes in mice (IRC4P.612)
Authors: Yu Jiali , Sun Rui , Tian Zhigang
Published: January 2023

Draining lymph nodes (LNs) are specialized sites where adaptive immune responses take place. Recently, the anatomical location of two LNs draining the mouse liver, named as portal LN (PLN) and celiac LN (CLN) respectively, has been characterized. However, the immune responses in the liver-draining LNs and their contribution to the establishment and maintenance of liver tolerance are still not fully understood. In our further study, we find that these two liver-draining LNs (PLN and CLN) have different lymphatic vessels, excluding the possibility that one LN is secondary to the other one. Interestingly, although both PLN and CLN can drain liver lymph directly, they are different in many aspects. The two liver-draining LNs have different cellular composition, with PLN being highly similar to mesenteric LN but different from periphery LNs, and CLN being special with unique composition of innate immune cells. Importantly, the development of these two liver-draining LNs might be controlled by different transcription factors. Moreover, our preliminary data shows that the immune responses of these two liver-draining LNs exhibit different features during exogenous pathogen infection. Overall, PLN and CLN are two liver-draining LNs with distinct characteristics and their contribution to liver tolerance remains to be defined.
CD14hi equine monocytes are preferential initial targets for infection with equine arteritis virus (IRC4P.615)
Authors: Chelvarajan R Lakshman , Mondal Shankar P , Cook R Frank , Go Yun , Sarkar Sanjay , Henney Pamela , Marti Franscesc , Bailey Ernest , Balasuriya Udeni
Published: January 2023

Equine arteritis virus (EAV), the causative agent of equine viral arteritis (EVA) in horses, is distributed worldwide with clinical signs including an influenza-like illness in adult horses, abortion in pregnant mares, interstitial pneumonia in neonatal foals and persistent infection in genetically predisposed stallions. EAV is a small,enveloped virus with a positive-sense, single-stranded RNA genome and primarily infects epithelial and endothelial cells, plus unidentified subpopulations of monocytes and macrophages. The primary objective of this study was to identify the susceptible CD14+ monocyte subpopulation using a synthetic recombinant virus expressing mCherry derived from the prototypical virulent Bucyrus stain (VBS) of EAV. The recombinant virus had identical tropism to parental VBS supporting its use as a surrogate for EAV infection. Multi-color flow cytometric analysis showed that a minor population of CD14hi cells were preferentially infected by EAV at 12 h post infection (hpi), whereas by 36 hpi (three virus replication cycles) almost all infected monocytes were CD14lo. However, this CD14lo population was also characterized by expression of CXCL16, a molecule implicated in the establishment of persistent EAV infections in stallions. CXCL16 expression was not detected in CD14hi infected monocytes at 12 hpi. Work is in progress to determine the eventual differentiation status of CD14hi/CXCL16- and CD14lo/ CXCL16+monocyte populations in terms of M1/M2 polarization.
Molecular chaperoning facilitates the inflammasome activation and an innate immune response to microbial pathogens (IRC4P.455)
Authors: Wang Hongxia , Wang Xiang-Yang
Published: January 2023

While certain molecular chaperones are known to regulate an adaptive immune response by shutting antigenic polypeptides and facilitating cross-presentation, its potential impact on innate immune responses to pathogens is not well understood. Assembly and activation of NLRP3 inflammasome plays a central role in sensing microbial infections and the host innate immunity. We report that extracellular glucose-regulated protein 170 (Grp170), a large molecular chaperone, profoundly enhances microbial CpG oligodeoxynucleotides (CpG-ODN) induced NLRP3 inflammasome activation, which is independent of ATP. Mechanistic studies reveal that Grp170 potentiates the endocytosis of CpG-ODN, followed by lysosomal damage and the cathepsin B release. Further, the elevation of intracellular calcium and calcium-dependent activation of calpain are crucial for Grp170-potentiated lysosomal instability and the inflammasome activation. Lastly, chaperoning-enhanced inflammasome activation results in more efficient bacterial elimination in mice compared with CpG-ODN alone. These results indicate that molecular chaperoning of pathogen-associated molecules (e.g., microbial DNA) by Grp170 facilitates the immune recognition of invading pathogens via modulation of NLRP3 inflammasome activation. Our findings underscore an important role of extracellular chaperone molecules, possibly released from tissues injury following pathogen infection, in amplifying an innate immune response.
Localized, sustained presentation of VEGF-C in mouse dermis alters the local immune microenvironment (IRC5P.628)
Authors: Yu Shann , Fankhauser Manuel , Aigner Petra , Kocher Juliane , Swartz Melody
Published: January 2023

Lymphangiogenesis is induced in areas of chronic inflammation, including in both autoimmunity (associated with immunoreactivity) and in many solid tumors (associated with metastases and immunosuppression). We asked whether and how local VEGF-C-driven lymphangiogenesis alone could affect adaptive immunity to a model antigen. To this end, we designed a hydrogel system for sustained local delivery of the lymphangiogenic protein VEGF-C. These proteins were engineered to express a peptide tag which are covalently crosslinkable onto the hydrogel matrix, and are thus better retained by the hydrogels. Following implantation into healthy mice, local delivery of VEGF-C increased lymphangiogenesis at the implant site as well as at the draining lymph nodes compared to empty hydrogel controls. This promoted increased local expression of CCL21, similar to observations in lymphangiogenic tumors, and associated with increased recruitment of CCR7+ cells, most notably CD4 T cells expressing markers of naïve or memory phenotypes. To further examine the immunological relevance of these observations on antigen-specific T cell populations, hydrogels delivering both VEGF-C and the model antigen OVA were implanted into healthy mice which had previously received an adoptive transfer of OT-II T cells. These hydrogels also recruited CD4 T cells expressing markers of naïve and memory phenotypes. Our studies suggest a role for lymphangiogenesis in the recruitment of immune cells during inflammation.
CD27 costimulation promotes IL-7Rα re-expression in effector CD8+ T cells by transcriptional regulation and enhances memory precursor population during acute viral infection (IRC5P.463)
Authors: Dong Han , Bullock Timothy
Published: January 2023

IL-7 plays a critical role in the generation and maintenance of memory CD8+ T cells, and IL-7Rα has been regarded as a defining marker of long-lived memory precursor effector cells. While IL-7Rα is downreguated acutely upon TCR stimulation, little is known about the stimuli that promote the recovery of IL-7Rα in Ag-activated CD8+ T cells. Using vaccinia virus, we previously demonstrated that CD27 costimulation during primary response is critical for the generation of IL-7Rα-expressing effectors and promotes CD8+ T cell memory. By utilizing agonistic mAb and transgenic models, here we show 1) direct CD27 stimulation on CD8+ T cells is sufficient and necessary to enhance IL-7Rα-expressing effectors; 2) CD27 stimulation neither alleviates the downregulation of IL-7Rα upon TCR signaling nor promotes the expansion/survival of IL-7Rα-expressing effectors, but facilitates IL-7Rα re-expression; 3) CD27 stimulation regulates Il7ra transcription but not protein distribution. Importantly, CD27 stimulation promotes not only IL-7Rα, but also the common γ chain of the receptor and the downstream signaling mediated by pSTAT5. Intriguingly, stimulation of other TNFSF members OX-40 and 4-1BB showed limited ability to promote IL-7Rα. Our results provide insights into the mechanistic basis by which CD27 costimulation influences CD8+ T cell memory differentiation, and highlight the potential of targeting CD27-CD70 axis to enhance IL-7 signaling for antiviral/antitumor immunotherapy.
Splenic MAdCAM-1+ marginal reticular cells deliver antibody-inducing signals and confer gut-homing properties to human marginal zone B cells (IRC5P.622)
Authors: BASCONES GLEAVE SABRINA , Magri Giuliana , Cassis Linda , Comerma Laura , Gentile Maurizio , Barra Carolina , Puga Irene , Cerutti Andrea
Published: January 2023

The splenic marginal zone (MZ) generates prompt antibody responses to blood-borne antigens through a unique subset of innate-like B cells that closely interact with poorly characterized stromal cells. We found that human MZ contained fibroblast-like marginal reticular cells (MRCs) that expressed MAdCAM-1 along with the stromal molecules Thy-1, thrombomodulin, ICAM-1 and VCAM-1, but lacked the endothelial molecules CD31, CD34 and von Willebrand factor. Similar to stromal lymphoid tissue organizers, MRCs up-regulated the adhesion molecules ICAM-1 and VCAM-1 in response to LT and TNF. Moreover, MRCs exposed to microbial TLR ligands, released the B cell-stimulating factors BAFF, APRIL and IL-6, which delivered survival, proliferation, class switching and plasma cell differentiation signals to MZ B cells. Plasma cells emerging from this pathway showed either T cell-dependent or T cell-independent ontogenetic features, and released IgM antibodies to conserved carbohydrate and lipid antigens shared by commensal and pathogenic bacteria. These antibodies decreased in splenectomized individuals. Finally, TLR-primed MRCs produced retinoic acid, which elicited MZ B cell expression of the gut-homing receptors a4b7 and CCR9. These findings indicate that MRCs have unique B cell-helper properties that could be harnessed to enhance vaccine-induced antibody responses systemically and perhaps at mucosal sites of antigen entry.
DNA sensing via STING regulates autoimmunity and tumor immunity. (IRM5P.648)
Authors: Mohamed Eslam , Lemos Henrique , Huang Lei , Ou Rong , Pacholczyk Gabriella , Hayakawa Yoshihrio , Munn David , Mellor Andrew
Published: January 2023

The Stimulator of Interferon Genes (STING) is activated by cytosolic DNA sensors such as cyclic GMP-AMP Synthase (cGAS) to induce interferon type I (IFN-I). Dendritic cells (DCs) that engulf dying tumor cells incite anti-tumor T cell immunity by sensing tumor cell DNA to activate STING/IFN-I signaling. Paradoxically, we find that DNA sensing to activate STING/IFN-I signaling induces DCs to express indoleamine 2, 3 dioxygenase (IDO), which activates regulatory T cells (Tregs). Thus treatment with DNA nanoparticles (DNPs) or cyclic dinucleotides (CDNs) to activate STING attenuated Experimental Autoimmune Encephalomyelitis (EAE), arthritis, and therapeutic responses were dependent on STING/IFN-I signaling to induce IDO. DNP and CDN treatments were also effective in slowing Type I Diabetes (T1D) progression in susceptible female Non-Obese Diabetic (NOD) mice. STING ablation also abolished IDO-dependent regulatory responses to apoptotic cells, suggesting that DNA from dying cells is sensed to activate the STING/IFN-I pathway and induce IDO to suppress autoimmunity. Moreover, cGAS and STING ablation abolished the ability of Lewis Lung carcinoma (LLC) tumor cells to induce local IDO, which suppresses anti-tumor immunity. These findings support the hypothesis that DNA from dying cells is sensed to activate STING/IFN-I in regulatory DCs that suppress T cell immunity and autoimmunity at sites of chronic inflammation associated with autoimmunity and tumor growth.
miR-23a supports survival of activated CD4+ T cells by maintaining reactive oxygen species equilibrium (IRM5P.638)
Authors: Liu Siqi , Zhang Baojun , Lin Regina , Li Chaoran , Li Qi-Jing
Published: January 2023

To identify key miRNAs that regulate CD4+ T cell responses, we profiled miRNA expression in CD4+ T cells at different phases post-antigen stimulation. As miR-23a is a miRNA that correlated strongly with effector CD4+ T cell expansion, we generated a T-cell specific miR-23a knockout mouse line (23T-KO). We found that miR-23a in T cells is essential for effective anti-bacteria protection, as the loss of effector T cells during the early phase of antigen activation rendered 23aT-KO mice susceptible to high-dose listeria infection. As early as a few hours post-activation in vitro, naïve 23T-KO CD4+ T cells exhibit an accelerated death, which is not rescued by caspase inhibition. Further analysis reveal that miR-23a deficiency in CD4+ T cells severely impairs their mitochondrial structural and functional integrity; Consequently, TCR activation of 23T-KO CD4+ T cells induce the rapid loss of mitochondrial potential and accumulation of reactive oxygen species. Forced expression of the antioxidant enzyme GPX1 protects activated naïve 23T-KO T cells from accelerated cell death. Mechanistically, we determine that miR-23a loss results in the accumulation of its direct target, PPIF, a structural component of the mitochondria permeability transition pore complex. Taken together, our results indicate that PPIF-controlled ROS equilibrium is essential for the survival of effector CD4+ T cells, and that miR-23a is indispensable for maintaining ROS homeostasis during early antigen activation.
Essential role of STAT3 in the upregulation of CD28 expression by IL-21 (IRM6P.658)
Authors: Wang Junmei , Frey Amanda , Li Haiyan , Park Jungsun , Lai Ivy , Watowich Stephanie , Lee Dean , Lee Kyung-Mi , Yee Cassian
Published: January 2023

CD28 plays an essential costimulatory role in T cell activation and survival by promoting IL-2 expression. However, the regulation of CD28 expression on CD8 T cells has not been well defined. Our previous studies demonstrated that tumor antigen-specific CD8 T cells generated in the presence of IL-21 stably express high levels of CD28 on the cell surface and exhibit enhanced replicative potential and in vivo persistence following adoptive transfer. The mechanism by which IL-21, a gamma-chain receptor cytokine, known to activate STAT3, and to a lesser degree STAT1 and STAT5, regulates CD28 expression is currently not known. Here we report that IL-21 regulates expression of CD28 at the transcriptional level through pSTAT3 binding to the CD28 promoter region. Using a reported small molecule inhibitor of STAT3, we demonstrate that STAT3 is critical for IL-21-induced expansion of tumor antigen-specific T cells and upregulation of CD28 expression. In addition, using CD8 T cells from Job’s syndrome patients with a sole dominant negative stat3 mutation and T cells transfected with stat3 knockdown shRNAs, we further confirmed that STAT3 is essential for the IL-21 mediated CD28 effect on human CD8 T cells. Moreover, we discovered that STAT3 directly regulates CD28 expression by binding to consensus STAT sites in human CD28 promoter. Taken together, our study demonstrates for the first time a specific role for STAT3 in IL-21-mediated CD28 expression in activated human CD8 T cells.
Evidence for a pivot upon TCR engagement (IRM7P.700)
Authors: Kuhns Michael , Lee Mark , Glassman Caleb , Deshpande Neha , Parrish Heather
Published: January 2023

The T cell receptor (TCR)-CD3 complex is the chief determinant for T cell development, activation, differentiation and the execution of effector functions. The TCR module (TCRα+TCRβ) relays highly specific information about antigenic peptides embedded within major histocompatibility complex molecules (pMHC) to the intracellular signaling domains of the associated CD3γε, CD3δε, and CD3ζζ signaling modules to initiate intracellular signaling. It is unclear how mechanical information is transferred from the TCR-pMHC interface, across the plasma membrane, and on to the intracellular signaling domains of the CD3 subunits. The complexity of this molecular machinery, and the lack of obvious parallels with other receptor systems, has hindered progress in resolving these issues. We tested the hypothesis that the juxtamembrane regions of the CD3 signaling subunits are held in a spatial relationship that keeps the complex ‘off’ until TCR engagement triggers a change in this spatial relationship that converts the complex to an ‘on’ conformation. This would be akin to cytokine receptors, receptor tyrosine kinases, and even integrins. Our data indicate that unengaged TCRs enforce a divaricated conformation of the cytosolic juxtamembrane regions of key TCR-CD3 complex subunits at steady state. We interpret this to represent an off conformation. TCR engagement then triggers the intra-complex apposition of these regions into what we interpret to represent an on conformation.
Cereblon, a molecular target of lenalidomide (IMiDs), negatively regulates T cell activation (IRM7P.715)
Authors: Beatty Matthew , Han Ying , McDaniel Jessica , Mailloux Adam , Painter Jeffrey , Bronk Chrystina , Yoder Sean , Beg Amer , Yu Xue-Zhong , Fu Jianing , Engelman Robert , Liu Chen , Rajadhyaksha Anjali , Epling-Burnette Pearlie
Published: January 2023

Thalidomide analogs classified as immunomodulatory drugs (IMiDs) are rapidly emerging T-cell stimulants for the treatment of cancer. Cereblon, an E3-Ub ligase receptor with no known immune regulatory function, is the first identified target of IMiDs. The drug is hypothesized to have antagonistic properties by interfering with substrate recruitment to the thalidomide-binding domain in cereblon, and agonistic activity through an exposed ring that may recruit new proteins to the Ub complex. To explore this further at the molecular level, we studied immune regulation in cereblon deficient mice (crbn-/-), which exhibited an expanded white blood cell and neutrophil compartment, and higher numbers of peripheral and splenic lymphocytes. Mature crbn-/- T cells were capable of proliferation and IL-2 production following CD3 ligation in the absence of CD28 co-ligation. This positively associates with proximal phosphorylation events including pZap70, pAKT, pro-survival Bcl-XL and Bcl-2 protein expression, and corresponds with decreased pro-apoptotic proteins Bim, Bad, and Bax. Consistent with this data in a lethal graft-versus-host (GVHD) model, MHC-class I mismatched T cells from crbn-/- mice showed shortened post-transplant survival, greater weight-loss, and higher IFN-γ compared to transplanted wild-type T-cells. These data suggest that native targets of cereblon restrain the activation threshold of T-cells and that IMiD-associated blockade of cereblon leads to T-cell potentiation.
Direct interaction between PKCθ and ZAP70 kinases is required for T cell activation (IRM7P.716)
Authors: Xie Jiji , Han Xuemei , Silletti Steven , Lillemeier Bjorn
Published: January 2023

PKCθ plays a key role in T cell activation, but the mechanisms that control its activation are not fully understood. We previously reported that the PKCθ N-terminal C2 region is a novel regulatory phosphotyrosine (pTyr)-binding domain. However, the physiological pTyr-containing C2-binding ligand has remained unknown. Using a GST-PKCθ-C2 recombinant fusion protein in pull-down assays, we detected a ~75-kDa pTyr-containing protein that associated with wild-type PKCθ-C2, but not with a pTyr non-binding PKCθ-C2 mutant after TCR/CD28 costimulation. Mass spectrometry analysis identified this protein as ZAP70 kinase. The stimulation-induced association of ZAP70 with full-length PKCθ or PKCθ-C2 in T cells was confirmed by co-immunoprecipitation assays, and was mediated by residues pY315 and pY319 in interdomain B of ZAP70. This transient association upon T cell activation required Lck kinase, followed by rapid complex dissociation that led to ZAP70 phosphorylation on Y493, resulting in full activation of ZAP70 and downstream signals. Mutation of two PKCθ-C2 residues essential for pTyr binding abolished PKCθ’s ability to rescue TCR- and CD28-induced IL-2 production, proliferation and Th2 differentiation in PKCθ-deficient T cells, without affecting T cell development or Th1 differentiation. These data suggest anchoring of PKCθ-C2 domain to phosphorylated interdomain B of ZAP70 promotes the activation of both kinases, thereby provided a novel pharmaceutical target for immunosuppression.
T cell development is regulated by the coordinated function of two Lck promoters (IRM7P.701)
Authors: Chiang Jeffrey , Hodes Richard
Published: January 2023

Lck is a T cell lineage-specific tyrosine kinase that is critical for T cell development and activation. Expression of an identical Lck protein can be mediated by two promoters, the proximal and distal lck promoters, but the function of these promoters during T cell development has not been directly assessed. We generated mutant mice by inactivating either one of the two lck promoters. Lck-PROX mice, in which only the proximal promoter is functional, had normal levels of Lck protein and thymic development through the CD4-CD8- double negative (DN) stage of T cell development, but reduced levels of Lck later in development and a dramatic reduction of mature single positive thymic T cells. In contrast, Lck-DIST mice, in which only the distal promoter was functional, were deficient in Lck and severely defective in early T cell development, with a block at the DN3 stage equivalent to the defect seen in complete Lck knockouts. Interestingly, the small number of peripheral T cells that developed in Lck-DIST mice expressed Lck protein and proliferated normally in response to TCR/CD3 cross-linking, while those of Lck-PROX mice were deficient in Lck and defective in response to TCR stimulation. These results define the coordinated requirements for proximal and distal lck promoters during T cell development and represent a unique direct demonstration of the requirement for alternative promoters in allowing developmental stage-specific regulation of a single protein product.
MicroRNA-183/96/182 cluster regulates iNKT cell development and function (IRM7P.706)
Authors: Zhou Li , Li Guihua , Zhang Xiling , Xu Shunbin , Mi Qing-Sheng
Published: January 2023

MicroRNAs (miRNAs), a class of non-coding small RNAs. Invariant Natural Killer T (iNKT) cells are potent regulators of diverse immune responses. Our previous study indicated that lack of miRNAs following the deletion of Dicer, a miRNAs-processing enzyme, affect iNKT cell development and function (PNAS, 2009). However, the roles of specific miRNA in iNKT cell development and function remain unclear. The miR-183/96/182 cluster, composed of 3 miRNA genes, is significantly upregulated upon T cell activation. In this study, we investigate the role of miR-183/96/182 cluster on development and function of iNKT cells, using miR-183/96/182 knockout (KO) mice. We found that the frequency of immature iNKT cells significantly increased, while mature iNKT cells significantly decreased in thymus of KO mice compared to littermate control (P<0.05), indicating the blockage of iNKT cell development in thymus. Furthermore, lack of miR-183/96/182 resulted in a substantial reduction of iNKT cell number (p<0.05) in the thymus and in spleen. The mutant iNKT cells displayed defective cytokine production, including IL-4 and IFNγ, compared to iNKT cells from littermate control (P<0.05). Finally, bone marrow chimera transferring experiments further suggested the miR-183/96/182 cluster controlled iNKT cell development and function by cell-intrinsic characteristics. Thus, our data define a specific role of miR-183/96/182 cluster in the development and function of iNKT cells.
Intrahepatic development of liver-resident natural killer cells (LYM2P.739)
Authors: Tian Zhigang , Peng Hui , Tang Ling , Bai Lu , Sun Rui
Published: January 2023

Natural killer (NK) cells are an important component of the innate immune system and play critical roles in host defense against infections and tumors. Accumulating studies have shown that the NK cell pool is highly heterogeneous, consisting of different cell subsets with distinct phenotypic and functional features. Recently, in collaboration with Professor Wayne Yokoyama, we showed that a novel NK cell subset expressing CD49a and lacking DX5 rarely recirculates and selectively resides in the liver, that is, liver-resident NK cells. While bone marrow (BM) cells are efficient in generating conventional (cNK) cells, the frequency of liver-resident NK cells is significantly reduced after BM transplantation. Conversely, lethally irradiated mice adoptively transferred with fetal liver cells exhibit normal frequency of liver-resident NK cells. Further analyses reveal that the adult liver retains hematopoietic potential and contains a few hematopoietic progenitor cells (HPCs), which are phenotypically distinct from BM lineage (Lin)-negative progenitor cells but resemble fetal liver counterparts. Moreover, adoptive transfer of these adult liver HPCs have the capacity to generate liver-resident NK cells, but not cNK cells. Finally, Liver HPCs express higher levels of the transcription factors that are essential for liver-resident NK cell development than their BM counterparts. Thus, our studies suggest an alternative development pathway for liver-resident NK cells.
Exacerbation of human astrocyte inflammatory responses to bacterial pathogens by the neuropeptide substance P (MPF2P.742)
Authors: Young Ada , Marriott Ian
Published: January 2023

The neuropeptide substance P (SP) has increasingly been recognized to augment inflammatory immune responses. We have recently demonstrated that SP can significantly exacerbate the inflammatory responses of isolated murine glial cells to clinically relevant bacterial pathogens. Furthermore, we have shown that SP interactions with its receptor are an essential component in the initiation and/or progression of central nervous system (CNS) inflammation in in vivo mouse models of meningitis. In the present study, we demonstrate that SP similarly alters the inflammatory responses of human astrocytes to clinically relevant bacterial pathogens and their components. Specifically, we show that SP augments the production of inflammatory cytokines and the generation of nitric oxide by human astrocytes in response to Neisseria meningitidis. In addition, we showed that this effect is seen in astrocyte responses to the TLR4 ligand, LPS. However, such SP-mediated increases in inflammatory mediator production are not limited to TLR4 stimuli as human astrocyte responses to the disparate Gram-negative bacterial pathogen Borrelia burgdorferi, that does not express LPS, are also modulated in a dose and time dependent manner. Together, these results suggest that SP could play a significant role in the initiation and/or progression of bacterially induced CNS inflammation in humans in a similar manner to that previously seen in rodent models following infection.
Group 1 CD1-restricted T cells can recognize Staphylococcus aureus lipid antigen (MPF2P.751)
Authors: Visvabharathy Lavanya
Published: January 2023

CD1-restricted T cells are a unique subset of T cells that respond to lipid self- and foreign antigens presented by group 1 CD1 (CD1a, -b, -c) and group 2 CD1 (CD1-d) antigen presenting molecules. Previous studies have shown that group 1 CD1-restricted T cells can respond to lipid antigens derived from Mycobacterium tuberculosis. As group 1 CD1 molecules can accommodate a variety of lipid species, we sought to address whether group 1 CD1-restricted T cells can be activated by Staphylococcus aureus (SA) lipids. Using a transgenic mouse strain that expresses human group 1 CD1 molecules (hCD1Tg), it was found that group 1 CD1-restricted T cell responses against SA lipids can be observed in immunization and infection settings. When DCs coated with total lipid from MRSA strain USA300 were injected into hCD1Tg mice, lymphocytes produced significantly higher amounts of IFN-g in response to SA lipid in a group 1 CD1-dependent manner. Lymphocytes from SA-infected animals also produced more IL-17 in a group 1 CD1- and SA lipid antigen-dependent manner. Finally, long-term cultured CTL clones derived from immunized and infected animals displayed specificity toward SA lipid antigens. These clones produced IL-10 and TNF-α in response to SA lipid, demonstrating that group 1 CD1-restricted T cells isolated from immunized or infected animals can produce multiple cytokines in response to SA lipid.
The Mycobacterium tuberculosis-specific CD4 T cell immune repertoire in rhesus macaques overlaps with human responses (MPF2P.752)
Authors: Lindestam Arlehamn Cecilia , Mothe Bianca , Dow Courtney , Dillon Myles , Wiseman Roger , Bohn Patrick , Karl Julie , Golden Nadia , Foreman Taylor , Mehra Smriti , Scriba Thomas , Kaushal Deepak , O'Connor David , Sette Alessandro
Published: January 2023

Animal models to study the process of TB pathogenesis and vaccine development are of importance. NHP models, especially Rhesus macaques, are attractive because of available resources to evaluate their immunogenetic backgrounds and the aerosol model closely mimics the human infection route. In this study, we characterized the MHC immunogenetic background of a cohort of animals that have been vaccinated and/or infected with Mtb, defining 54 new immunodominant CD4 T cell epitopes using IFNγ ELISPOTs with antigens utilized in human clinical trials. Antigens which are immunodominant in humans are also immunodominant in rhesus macaques, including Rv3875 (ESAT-6) and Rv3874 (CFP10). Interestingly, the shared responses were not due to common ancestry or inbreeding upon identifying MHC alleles. Instead, the results indicate promiscuous binding, which is the first account of this phenomenon in the rhesus macaque TB model of infection. These results suggest that while the CD4 response of NHPs to TB is broad and heterogeneous, epitope sets can be defined to broadly follow and characterize these responses in genetically heterogeneous NHP animal cohorts. To our knowledge, this is the first comprehensive epitope identification effort for non-human primates. We reveal a profound repertoire overlap between epitopes recognized in NHPs and in humans. These findings have important implications for the evaluation of new vaccines and diagnostics in the setting of non-human primate model of TB.
Differential function of Ag-specific CD4 T cells from sepsis-induced lymphopenia is influenced by gut microbiota (MPF2P.744)
Authors: Griffith Thomas , Cabrera Javier , Babcock Jeffrey , Badovinac Vladimir
Published: January 2023

Sepsis changes the composition and functional potential of the naïve CD4 T cell repertoire, but the mechanism(s) that control such changes are unknown. We posited that the constituents of the gut flora play an important role in defining the responsiveness of CD4 T cell populations specific for Ag present in the commensal bacterial species that establish polymicrobial sepsis after cecal-ligation and puncture (CLP) versus those CD4 T cells undergoing sepsis-induced Ag-independent homeostatic proliferation. To test this hypothesis, we tracked the loss/recovery and function of CD4 T cells specific for an Ag present in segmented filamentous bacteria (SFB) in mice containing or devoid of this bacteria in their gut. SFB-specific CD4 T cells underwent Ag-driven proliferation in CLP-treated SFB+ mice, but not in SFB- mice. This correlated with their functional ability after secondary infection with SFB Ag-expressing Listeria. Interestingly, we found that a second CD4 T cell population specific for the influenza NP311 epitope underwent differential recovery and function depending on the mouse vendor, suggesting antigenic stimulation as a result of cross-reactivity with some yet-to-be defined epitope expressed by the gut commensal bacteria. Our data suggest the extent of recovery and function of a particular Ag-specific T cell population during sepsis can determined by the intestinal “health” of the individual, regardless of possible genetic similarities.