bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Toll-like receptor-stimulated dendritic cell cytokine production by exogenous osteopontin. (98.32)
Authors: Contractor Nikhat , Frantz David C
Published: January 2023

Osteopontin (OPN) is a pleiotropic cytokine produced by mast cells, macrophages, activated T cells, NK T cells and dendritic cells and is present at high levels in human milk. Several studies in OPN-deficient mice have suggested a role for OPN in the development of Th1 and Th17 mediated immune responses. Dendritic cells (DC) are central regulators of T helper cell responses, however, it is unknown whether exogenous OPN supplied by milk plays a role in the differentiation of DC upon activation by signals through the TLRs. To address this question, we assessed the role of OPN in the LPS and CpG induced cytokine responses in bone marrow derived dendritic cells (BMDC). In the presence of OPN, the production of LPS and CpG stimulated IL-10, IL-6 and TNFα by BMDC was reduced in a dose dependent manner. OPN had no effect on LPS or CpG stimulated IL-12p40 or IL-12p70 production. OPN by itself did not induce IL-12p40, IL-12p70, IL-10, IL-6 or TNFα production by BMDC. These data suggest that exogenous OPN can modulate dendritic cell responses to TLR stimulation, and suggest a mechanism by which orally ingested OPN might influence the function of the intestinal immune system. Further studies are required to determine whether T helper cell polarization is affected by OPN treatment of BMDC, as well as to determine if exogenous OPN has similar inhibitory effects on cytokine production by tissue derived DC.
Bcl2, PDGFRβ and M-CSF are upregulated in monocytes from SLE patients with defective HDL and atherosclerosis (99.42)
Authors: Skaggs Brian J , Sahakian Lori , Grossman Jennifer M , FitzGerald John , Hahn Bevra H , McMahon Maureen A
Published: January 2023

Purpose: Our previous studies suggest a defective form of HDL, pro-inflammatory HDL (piHDL), as an SLE-specific risk factor for developing atherosclerosis. We examined monocyte transcript differences in women with SLE that have piHDL and carotid artery plaque to SLE/no piHDL/no plaque and SLE/+piHDL/no plaque patients.

Methods: Monocytes were isolated from patient PBMC by Ficoll and magnetic bead separation. Transcript levels of atherosclerosis-specific genes were examined by real-time PCR-based arrays (SA Biosciences). Data reported are from 15 SLE/no piHDL/no plaque, 14 SLE/+piHDL/+plaque, and 14 SLE/+piHDL/no plaque patients. mRNA changes were also measured in the human monocyte cell line THP-1 after piHDL treatment.

Results: Bcl2, PDGFRβ and M-CSF are significantly upregulated in SLE/+piHDL/+plaque versus SLE/no piHDL/no plaque, suggesting piHDL plays a role in blocking monocyte apoptosis signals. MCP-1 and ICAM-1 were only upregulated in SLE/+piHDL/no plaque patients, suggesting a piHDL-driven inflammatory transition to atherosclerosis. Inflammatory genes were also upregulated in THP-1 cells treated with piHDL.

Conclusions: PDGFR, M-CSF, and Bcl2 are upregulated in SLE/piHDL/plaque patients and could be novel targets in the treatment of increased atherosclerosis observed in SLE patients.
CM1 Enhances Proinflammatory cytokine Production in Peripheral Blood Mononuclear Cells and Synovial Fluid Mononuclear Cells from Rheumatoid Arthritis Patients (99.53)
Authors: Bae Seyeon , Kang Jae Seung , Kim Hye Min , Maeng Hyung Gun , Kong Joo Myoung , Lee Na Eun , Hwang Young Il , Lee Wang Jae
Published: January 2023

CM1 (centrocyte/-blast marker 1) is expressed on the surface of activated peripheral blood mononuclear cells (PBMCs). Recently, we found that the production of prostaglandin E2 (PGE2) from activated PBMCs was increased by CM1 stimulation via the activation of nuclear factor kappa B (NF-kB). Therefore, this study was attempted to investigate the roles of CM1 in the regulation of rheumatoid arthritis (RA). PBMCs were purified from healthy donors, RA patients and degenerative osteoarthritis (OA) patients, and knee joint synovial fluid mononuclear cells (SFMCs) were purified from RA patients. The expression of CM1 on PBMCs and SFMCs were analyzed by flowcytometry. The production of inflammatory cytokines and PGE2 by CM1 stimulation was measured by ELISA and chromatin immunoprecipitation (ChIP) assay and immunoblotting were performed to examine signaling mediators for inflammatory cytokines by CM1 stimulation. CM1 is highly expressed on PBMCs and SFMCs from RA patients, but not from OA patients. TNF-alpha, IL-1 alpha/beta, IFN-gamma and PGE2 production were markedly increased in PBMCs and SFMCs of RA patients upon CM1 stimulation. The phosphorylation of p38 MAPK was increased in PBMCs and SFMCs from RA patients in a time dependent manner after CM1 stimulation. In addition, the binding of NF-kB to the promoters of TNF-alpha and IL-1 beta after CM1 stimulation was also confirmed by the ChIP assay. Our results strongly suggest that CM1 may play a critical role in the disease progression of RA via the induction of disease-related proinflammatory cytokines and PGE2 production.
Role of complement activation component C5a on tumor progression (100.9)
Authors: Gunn Lacey , Cai Yihua , Ding Chuanlin , Hu Xiaoling , Hansen Richard , Aggarwal Deep , Yan Jun
Published: January 2023

Enhancement of anti-tumor mAb immunological mechanisms for tumor destruction may be obtained by promoting complement activation. However, complement activation components, such as C5a, trigger inflammation and chronic inflammation promotes tumor growth. Recently, it has been shown that C5a in the tumor could recruit and activate myeloid-derived suppressor cells to inhibit the anti-tumor CD8 T cell response. However, this effect was observed in only one tumor model. We chose to address the role of C5a on tumor growth by transfecting tumor cells with C5a. A human ovarian carcinoma, SKOV-3, and a murine lymphoma, RMA, were transfected which allows us to study C5a in an immunocompromised xenograft model and an immunocompetent syngeneic model. In vitro growth kinetics observed revealed no significant difference between C5a, control vector (CV), or wild-type cells. In both models, tumor-bearing mice with C5a-transfected tumor cells have significantly less tumor burden as compared to CV tumors. In the xenograft model, CD11b+DX5+ NK cells were significantly more and VEGF and arginase levels were significantly less in C5a expressing tumors. The syngeneic lymphoma model revealed C5a modulates tumor-infiltrating CD11b+ cells to produce more IL-12 and there is a greater influx of NK and CD4 T cells. Thus our studies demonstrate C5a chemoattracts innate NK cells and downregulates negative mediators in the xenograft model while in the syngeneic model C5a promotes shift to a Th1 response.
A distinct role of CD4+ Th17 and Th17-stimulated CD8+ CTL in induction of antitumor immunity and experimental autoimmune encephalomyelitis (101.5)
Authors: Ankathatti Munegowda Manjunatha , Xiang Jim
Published: January 2023

Th17 and CD8+ cytotoxic T lymphocytes(CTL) cells eradicate established tumors and induce experimental autoimmune encephalomyelitis(EAE). To assess their potential relationship, we generated ovalbumin(OVA) or myelin oligodendrocyte glycoprotein(MOG) specific Th17 cells by in vitro cultivation of OVA-pulsed dendritic cells(DCova) with CD4 T cells derived from T cell receptor transgenic OTII mice or MOG peptide-pulsed splenocytes with CD4 T cells purified from MOG immunization induced EAE C57BL/6 mice. We found these Th17 cells expressed transcriptional factor RORγt and secreted IL17. We also found that OVA-specific Th17 cells which acquired major histocompatibility complex/peptide(pMHC) I and costimulatory molecules by DCova activation stimulated OVA-specific CTL response and antitumor immunity against OVA-expressing BL6-10ova tumor. The stimulatory effect is mediated by acquired CD80 costimulation and targeted to CD8 T cells in vivo via acquired pMHC I complexes. Th17-stimulated CTL, but not the Th17 cells shown to have no in vitro killing activity play a major therapeutic role in eradication of early stage of BL6-10ova tumors(3mm dia). We also found that MOG-specific Th17 cells stimulated MOG-specific CTL responses, and both Th17 cells and Th17-stimulated CTL are involved in induction of EAE with a major pathogenic role played by the former. In total our data elucidate a distinct role of Th17 cells and Th17-stimulated CTL in induction of antitumor immunity and EAE.
The role of NFATc1 in tumor T cell responses to lung cancer (101.6)
Authors: Reppert Sarah , Boross Ildiko , Koslowski Michael , Türeci Özlem , Lehr Hans-Anton , Glimcher Laurie H. , Finotto Susetta
Published: January 2023

Nuclear factor of activated T cells (NFAT) proteins represent a Ca2+/calcineurin- regulated family of transcription factors that control T cell differentiation processes. Specifically NFATc1 and NFATc2 are highly expressed in peripheral T cells and activate the IL-2 and IL-4 promoters, suggesting that the Ca2+-regulated calcineurin/NFAT cascade controls alternative pathways of T cell activation and peripheral tolerance. NFATc1 is strongly activated upon antigen dependent T cell receptor engagement and induces genes that are involved in T cell effector function. In this study we describe that patients affected by lung Adenocarcinoma showed increased NFATc1 mRNA and protein levels in their lungs suggesting an involvement of NFATc1 on lung tumor development. To define the role of NFATc1 on lung cancer we start to analyze NFATc1 deficient mice in a murine model of lung Melanoma. Since NFATc1-deficient mice die at the embryonic stage, we bred conditional NFATc1 mice with CD4-Cre mice to gain insight into the role of NFATc1 in tumor T cell responses. Our preliminary results in this model showed a reduced tumor area in NFATc1-deficient mice compared to wild type mice. We also demonstrated here, that naïve T cells, isolated from NFATc1-deficient mice, produced less IL-17 under Th17- skewing conditions compared to those isolated from wild-type mice indicating a pathogenetic effect mediated by NFATc1 via IL-17 in lung tumor that must be further investigated.
The role of human Dectin-1 in eliciting antigen specific CD8+ T cell responses (130.35)
Authors: Ni Ling , Li Xiao-Hua , Ingrid Gayet , Duluc Dorothee , Klucar Peter , Clayton Sandra , Zurawski Sandy , Palucka Karolina , Zurawski Gerard , Banchereau Jacques , Oh SangKon
Published: January 2023

Dectin-1 recognizing fungal pathogens and mycobacteria, delivers intracellular signals to activate DCs. Here we report that Dectin-1 expressed on human DCs possesses unique immunological function that potentiates antigen-specific CD8+ T cell responses. We first generated an agonistic anti-Dectin-1 monoclonal antibody (mAb), which recognized Leu192-Asn202 in Dectin-1 and efficiently bound to both in vitro monocyte-derived DCs and in vivo CD1c+ skin dermal DCs, but not Langerin cells. Syk-dependent activation by anti-Dectin-1 mAb induced DCs to upregulate T cell costimulatory molecules, particularly CD70 and 4-1BBL, as well as cytokines and chemokines. This signaling via Dectin-1 allowed DCs to potentiate both memory and naïve CD8+ T cell responses by promoting their expansions and functional activities. Furthermore, both neo- and non-self-antigens delivered to DCs via Dectin-1 resulted in potent antigen-specific CD8+ T cell responses. This was largely dependent on CD70 and 4-1BBL upregulated by the signaling via Dectin-1. We also demonstrated that Dectin-1-mediated activation of DCs did not promote Foxp3+ and IL-10-producing regulatory CD4+ T cell responses, but potentiated CD4+ helper T cell function for inducing effector CD8+ T cells. We conclude that Dectin-1 on human DCs has unique function for eliciting antigen-specific CD8+ T cell responses by taking up antigens and delivering intracellular signals favoring effector CD8+ T cell responses.
Flagellin does not promote a CD8+ T cell response to the SIINFEKL epitope (130.42)
Authors: Bates John , Graff Aaron , Mizel Steven
Published: January 2023

Flagellin, the ligand for TLR5, is a potent adjuvant for CD4+ T cell-dependent humoral immune responses and has been reported by several groups to promote CD8+ T cell responses in vivo. However, other groups have failed to detect an effect of flagellin on the CD8+ T cell response. In an attempt to resolve this controversy, we created a protein composed of the SIINFEKL epitope and flanking amino acids fused to the N-terminus of full length Salmonella flagellin for use with TCR transgenic OT-I mice. We found that the CD8+ T cell stimulatory activity of this protein was, as expected, dependent on proteasome processing and was markedly more potent than ovalbumin by itself. However, the adjuvant effect of the fusion protein was TLR5 and IPAF independent. A fusion peptide containing the hypervariable region Salmonella or Pseudomonas flagellin and the SIINFEKL epitope also stimulated antigen-specific CD8+ T cell activation. These results are consistent with the conclusion that flagellin does not promote antigen-specific CD8+ T cell activation. Since the flagellin fusion protein containing the SIINFEKL epitope was far superior to ovalbumin as an inducer of CD8+ T cell activation, it is likely that uptake and processing of the SIINFEKL epitope by antigen-presenting cells is enhanced outside the context of ovalbumin.
Targeting CD73 to improves tumor immunotherapy (131.23)
Authors: Zhang Bin , Jin Dachuan , Fan Jie , Wang Long , Thompson Linda , Liu Aijie , Daniel Ben , Shin Tahiro , Curiel Tyler
Published: January 2023

During tumor progression, tumor cells foster a tolerant microenvironment and the activation of many immunosuppressive mechanisms that act in concert to counteract effective immune responses. Therefore, to accomplish cancer immune eradication, tumor-induced immunosuppression must be overcome. We demonstrate that CD73 is widely expressed on many tumor cell lines and upregulated in cancerous tissues with active enzymatic activity. Notably, CD73 expression in malignant cancer patients is closely associated with poor immune status of tumor infiltrating effector T cells. Because CD73 catalyzes AMP breakdown to immunosuppressive adenosine, we propose that CD73-generated adenosine prevents tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that inhibition of CD73 by selective inhibitor APCP slowed tumor growth and increased mice survival, and more importantly, augmented CTL therapy of cancer. Using genetic ablation of CD73 on tumor cells by siRNA and CD73 deficient mice, we found that both tumor and host CD73 synergistically controlled tumor progression by impairing antitumor T cell immunity. Thus, counteracting adenosinergic effects of CD73 in the tumor microenvironment represents a novel and feasible strategy of cancer treatment and is complementary to other approaches directed at improving the immune function of antitumor T cells such as adoptive T cell therapy or dendritic cell vaccines.
Lentiviral-siRNA blockade of immunosuppressive genes increases DC vaccine efficacy. (131.42)
Authors: Lebson Lori , Rosenzweig Jason , Manicini Justin , Whartenby Katharine
Published: January 2023

Dendritic cells (DCs) play an essential role in the activation of T cells. Because of their potent activation capabilities, DCs have been used to stimulate anti-tumor responses via vaccines. While these vaccines have elicited immune responses, they have not been curative. In a tumor environment, suppressive factors can downregulate their ability to generate effective responses through modifying gene expression in DCs. We have been identifying such genes and proposed that blocking them in DCs would enhance their efficacy. To test our hypothesis, we silenced one immunosuppressive gene using a lenti viral vector siRNA construct. Our results demonstrate that blocking gene expression enhances activation and anti-tumor responses, leading to prolonged survival of tumor bearing mice. This approach may serve as a novel means to increase the efficacy of ex vivo developed DC vaccines.
Immunization with lentivector expressing HBV Ag markedly improves the tumor microenvironment and result in regression of established tumors (131.44)
Authors: He Yukai , Hong Yuan , Xiao Haiyan , Peng Yibing , Wang Guiqiang
Published: January 2023

One of the main reasons for the limited antitumor efficacy of current tumor vaccines is the failure of immune effector cells inside the tumor environment due to the infiltration of immune suppressor cells and immune dysfunction of effector cells. In this study, we investigated if the immune suppressive tumor environment can be improved by lentivector immunization. We found that lentivector immunization could not only stimulate potent T cell responses against HBV surface Ag and cause tumor influx of effector T cells, but also markedly changed the tumor microenvironment. Immunization with lentivector induces strong proliferation of CD4 and CD8 T effector cells but does not increase the proliferation of Treg in the vaccine draining LN. More importantly, lentivector immunization profoundly increase proliferation of the CD4 Teff cells in the tumor lesions, resulting in more CD4 Teff cells accumulation in the tumor lesions and decrease of Treg ratio in the tumor. Analysis of the tumor environment showed that lentivector immunization can skew immune suppressive tumor environment to Th1 and Tc1 like environment, with marked increase of T-bet, IFNγ, Perforin, and granzyme in the tumor lesions. Lentivector HBV-S-lv immunized mice were completely protected from HBV positive B16 tumor cells (B16-S) challenge. Furthermore, established B16-S tumor can be eradicated by lentivector immunization.
TSC2 regulates CD8+ T cell effector function and the generation of memory (132.14)
Authors: Pollizzi Kristen , Waickman Adam , Delgoffe Greg , Powell Jonathan
Published: January 2023

Recent studies implicate mTOR activity in the generation of memory CD8+ T cells. Tuberous Sclerosis Complex 2 gene (TSC2) is an upstream inhibitor of mTOR function. In this report, mice in which TSC2 is deleted in T cells were utilized to determine the role of TSC2 in controlling T cell function. TSC2-/- T cells demonstrate enhanced and constitutive mTOR Complex 1 (TORC1) activity. Using a recombinant vaccinia virus expressing OVA peptide (vac-OVA) as the primary stimulation we have found that loss of TSC2 promotes an enhanced effector response in CD8+ T cells in terms of killing ability and cytokine production 7 days after injection. However, when assessing function at 30 days post-infection the absolute number of OVA specific CD8+ T cells are reduced compared to wildtype controls. This correlated with diminished killing ability as shown by in-vivo CTL activity. Interestingly, when the TSC2-/- OVA specific CD8+ T cells were analyzed at day 30 for cytokine production, these cells secreted increased IFNγ and TNFα over their Wt counterparts. Together these data indicate that TSC2 deficiency impairs the effector to memory transition. However, the absence of TSC2 leads to enhanced CD8+ effector function during the primary immune response and in the remaining clonotypic CD8+ T cells that are present 30 days post infection. Overall, our findings implicate a novel and complex role for TSC2 signaling in the regulation of both CD8+ T cell effector function and memory generation.
Population level effects on Vγ2Vδ2 T cell frequencies in healthy adults: a difference in baseline values and resting phenotypes among Caucasian and African American donors (132.16)
Authors: Armstrong Cheryl , Cairo Cristiana , Cummings Jean-Saville , Deetz Carl , Davis Charles , Pauza C.David
Published: January 2023

Adult human γδ T cells are dominated by the Vγ2Vδ2 T cell receptor (TCR) which modulates MHC-unrestricted responses to non-peptidic antigens. This subset is so numerous that between 1 in 40 and 1 in 400 circulating CD3+ T cells will respond to stimulation with isoprenoid pyrophosphates. The normal frequency of Vγ2Vδ2+ cells reflects TCR selection and amplification by chronic antigen stimulation, but the regulatory mechanisms controlling population size have not been described. During clinical studies related to Vγ2Vδ2 T cells in cancer and AIDS, we identified a difference in the circulating populations between Caucasian and African American healthy volunteers. When expressed as the percentage of total lymphocytes, Caucasian donors had 5-times higher levels of Vγ2Vδ2 cells compared to African American donors and phenotypes characteristic of mature functional cells were also different among the groups, including CD56 expression that is linked to cytotoxicity. Analysis of the Vg2 repertoire showed a strikingly lower proportion of cells expressing the Vγ2-Jγ1.2 rearrangement. Our data suggest that distinct regulatory mechanisms, in addition to positive selection and chronic amplification, control Vγ2Vδ2 cell levels in blood and that further study of population level effects may uncover unique regulatory mechanisms that control this interesting population.
Multichromatic phenotypic analysis of peripheral lymphocytes in non-human primates (NHP) following total body irradiation (TBI) with 5% bone marrow shielding (BM5) (132.19)
Authors: Higgins Adam , Maciel Milton , Sztein Marcelo , MacVittie Thomas
Published: January 2023

High dose TBI of NHP leads to severe myelosuppression, neutropenia, thrombocytopenia, and immunosuppression, concurrent with an acute gastrointestinal syndrome. BM shielding mitigates these lethal effects by preserving a reservoir of unexposed hematopoietic progenitors. Rhesus macaques were exposed to either 10Gy (sub-lethal) or 11Gy (LD15 8-10 days) in the TBI/BM5 protocol and administered supportive care (fluids, antibiotics, transfusions). Peripheral blood was drawn over 150 days post irradiation (DPI) and lymphocyte subsets were assessed by flow cytometry. 10/11Gy TBI/BM5 caused a significant, early decrease in CD3+ T cells, sustained through 42 DPI, which reached baseline at 56 DPI. CD8+ T cells dominated T cell repopulation while CD4+ T cells showed a slower recovery. Most of the CD8+ T cells were CD62L+CD45RA+. CD20+ B cells reached 168% of baseline at 56 DPI and remained above baseline through 150 DPI. CD20+CD27+ memory B cell recovery kinetics were similar to B cells up to 77 DPI, with a sharp decrease at 90 DPI. CD3+ integrin α4β7+ (mucosal marker) cells recovered to 249% of baseline levels by 35 DPI and peaked at 400% of baseline at 120 DPI. Conversely, α4β7+ B cell recovery was slower, requiring 120 DPI to reach pre-irradiation levels. Functional assays will help understand the recovery pattern of lymphocyte subsets.
Various Sources, Types and Levels of Reactive Oxygen Species Make Distinct Contributions to Cell Cycle Progression And Survival (132.25)
Authors: Carey Gregory , Phatak Pornima , Koontz Jason
Published: January 2023

We examined several sources, types and roles of ROS in B cell lymphoma (BCL) survival. Initially following B cell receptor (BCR) crosslinking, hydrogen peroxide (H2O2), superoxide (O2•-) and cellular respiration were stimulated. Conversely, chronic BCR activation led to the loss of H2O2 and to massive O2•- induction, which were associated with tumor death. ROS scavengers reduced cellular ROS, but enhanced BCR-stimulated apoptosis, suggesting that ROS regulate BCL homeostasis positively. Next, we examined the RedOx- and activation-status of the survival protein, Akt after BCR-crosslinking. Interestingly, increased H2O2 production paralleled increased, reversible oxidation of Akt whereas the loss of H2O2 tone and massive O2•- induction paralleled irreversible Akt oxidation and its deactivation. Fractions of ROS were attributable to either NADPH oxidases, xanthine oxidase, the electron transport chain or glycolysis. Interestingly, however, it appeared that either particular ROS or distinct metabolic pathways differentially impacted the cell cycle. Thus, blockade of glycolytic respiration reduced ROS and arrested cells at Go/G1. In contrast, blocking oxidative respiration at complex I reduced a fraction of basal ROS and allowed the cells to exit Go/G1 and progress through S-phase, but to become arrested in G2/M. Together, these data suggest that the temporality, spatiality and magnitude of ROS production ultimately influence cell signaling and consequently, determine cell fate.
CXCL13 inhibition prevents bone metastasis in hormone-refractory prostate cancer (133.8)
Authors: Lillard James , Singh Rajesh , Sharma Praveen , Singh Shailesh
Published: January 2023

We have recently reported, chemokine receptor CXCR5 and its ligand CXCL13 expression positively correlates with prostate tumor staging. We have also noted differential expression of CXCR5 by prostate cancer (PCa) cell lines. By virtue of the presence of elevated CXCL13 levels in patient serum, CXCL13 secretion by bone marrow endothelium, and expression of CXCR5 on the surface of PCa cells, we hypothesized that gradient-dependent CXCR5-CXCL13 interactions are responsible in part for PCa cell bone metastases and inhibition of this axis may prove beneficial for prevention of bone metastasis and tumor progression. Luciferase-expressing PC3 cells were injected into the right carotid artery or directly into the tibia of nude mice. Before and after the development of bone metastases, groups were treated with control or anti-CXCL13 antibodies every third day and monitored for tumor progression or regression by non-invasive bioluminescent and microCT in vivo imaging. We demonstrate CXCL13 is present in bone marrow of xenograft tumor-bearing mice following intra-cardiac /intra-tibial injection of PC3 cells. This hormone-refractory PCa cell line established skeletal (osteolytic) metastases and tumor growth, but CXCL13 blockade significantly delayed prostate tumor formation, osteolysis, and spread to bones as well as growth, than compared to mice treated with control antibody. In conclusion, our study shows that the CXCL13-CXCR5 axis supports PCa bone metastasis and growth.
Differential effects of Human Interferon-alpha subtypes on CD4 T cell function. (134.10)
Authors: Gold Doria , Hillyer Philippa , Raviv Nataly , Heuer Melissa , Chi Bo , Rabin Ronald
Published: January 2023

We compared the effects of each of the 12 IFN-α subtypes and IFN-β on CD4 T cell proliferation and cytokine expression in response to stimulation with SEB. Purified CD4 T cells or PBMC from healthy donors were treated with 4 doses of each IFN for 16 hours, and stimulated with SEB for either 4 hours or 4 days, to measure cytokine expression with flow cytometry or proliferation by 3H-thymidine uptake, respectively. While type I IFN did not affect the percentage of SEB-activated (CD40L+) CD4 T cells, they did enhance the percentage of CD40L+ cells that expressed IFN-γ, IL-2 and IL-4 in a dose-dependent manner. The greatest difference among the subtypes was at 25 U/mL (for example, the percent of IL-2+ cells in response to SEB alone, IFN-α1 and -α8 was 19.9, 23.4, and 26.2, respectively). Surprisingly, type I IFN decreased the level of expression of CD40L less than or equal to 50% of SEB alone, and this decrease highly correlated with the enhancement of cytokines. Proliferation was decreased by each IFN subtype with maximal differences among subtypes in roughly the same hierarchy as the effects on cytokine and CD40L expression. These data suggest that type I IFN enhance both types 1 and 2 CD4 T cell responses with differences among the subtypes at “physiological” levels. The decrease in CD40L expression may affect the level of B cell activation and DC-CD4 T cell cross-talk.
Genetic ablation of steroid receptor coactivator-3 promotes PPAR-β-mediated alternative activation of microglia in experimental autoimmune encephalomyelitis (135.1)
Authors: Zhang Yanyun , Xiao Yichuan
Published: January 2023

The importance of links between lipid metabolic regulation and immune functions is increasingly acknowledged, yet not enough. SRC-3 has been demonstrated to play important roles in the regulation of lipid metabolism. In the present study, the potential role of SRC-3 in EAE was examined by analyzing disease progression in SRC-3-/- mice. SRC-3-/- mice are lean and exhibit lower lipid deposition through modulating the expression of lipid metabolism genes by PPAR-β. Moreover, we found that SRC-3 deficiency significantly attenuated the disease severity of EAE along with decreased inflammatory infiltration and demyelination. However, these effects are not caused by inhibition of peripheral T cell response, but by up-regulated expression of PPAR-β in CNS, which induced an alternative activation state of microglia in SRC-3-/- mice. These alternatively activated microglia inhibited CNS inflammation through inhibition of the expression of pro-inflammatory cytokines and chemokines well as up-regulation of anti-inflammatory cytokines, and promoted myelin regeneration through increased accumulation of oligodendrocyte precursors (OPCs) in white matter and elevated expression of myelin genes in the spinal cords of SRC-3-/- mice. Our data uncover the mechanism by which PPAR-β promotes tissue repair in response to inflammatory injury in SRC-3-/- mice, which builds up important links between lipid metabolic regulation and immune functions.
IL-17 and IL-22 affected keratinocytes via IL-1β inducing NALP3-caspase-1 pathway (135.18)
Authors: Cho Kyung-Ah , Woo So-Youn , Lee Kyung Ho
Published: January 2023

Pathogenesis of inflammatory skin disease is involved in the interplay between immune cells and keratinocytes. Psoriasis, for example, can be triggered by dysregulation of T cell-mediated immune response and characterized by hyperplasia of the epidermis, infiltration of leukocytes into both the dermis and epidermis, and dilation and growth of blood vessels. In addition, chronic immune response of epidermal layer is involved in cytokine release from keratinocytes and IL-1β has been implicated in inflammatory skin disease. Th17 cells possess IL-1βreceptor and excess production of IL-1β in NALP3 mutation leads to augment Th17-dominant pathology. Th17 cells are induced by exposure of TGF-β, IL-6, IL-15 and IL-23 and Th17 cells are related to autoimmunity and inflammation by producing IL-17 and IL-22. To determine whether IL-17 or IL-22 may have an effect on IL-1β secretion from keratinocytes, we used human keratinocyte cell line, HaCaT, and capsase-1-deficient mice with the IL-17 or IL-22 stimulation. We found that under IL-17 and IL-22 treatment IL-1β was increased via NALP3 activation. These results suggest that cytokine from Th17 cells may potentiate IL-1β mediated skin inflammation and lead to phenotypic alteration of keratinocytes.
Profibrotic alternatively activated M2 macrophages are associated with increased severity of dilated cardiomyopathy in male BALB/c mice with cosackievirus B3-induced autoimmune myocarditis (135.20)
Authors: Coronado Michael , Frisancho-Kiss Sylvia , Bedja Djahida , Frisancho J. Augusto , Shin Jaewook , Gabrielson Kathleen , Fairweather DeLisa
Published: January 2023

Current research has shown heart disease as the leading killer in the US. Autoimmune myocarditis (AM) progresses to dilated cardiomyopathy (DCM) and occurs at a higher incidence in males. In order to study sex differences in AM we inoculated BALB/c mice with 103 PFU of coxsackievirus B3 with cardiac myosin ip to induce autoimmunity. At d10 post infection males develop severe myocarditis that progresses to DCM at d35. We investigated functional endpoints of DCM by echocardiography at d90 and found that males developed significantly more severe DCM. To investigate sex differences further we conducted a microarray comparing infected males and females. Approximately 500 genes were significantly up regulated in males. Genes increased with a > 1.5 fold change fell into categories associated with Th2 responses, M2 macrophages (Macs), and extracellular matrix (ECM) remodeling. We showed previously that male cardiac CD11b+ GR1+ Macs express the mannose receptor (MR), TLR4, caspase-1 and IL-1β. This microarray confirmed that males have increased M2 markers including the MR, CD14 and IL-33. We also found upregulation of ECM remodeling genes including proteoglycan-4, timp-1 and tenascin-C indicating cardiac remodeling. Th2 responses are known to increase ECM remodeling and can drive differentiation of Macs to an M2 phenotype. Our data suggests that Th2 associated profibrotic M2 Macs may increase cardiac remodeling in males during AM resulting in DCM. Funded by NIH-HL-087033.
Maladaptive effector CD8+ T cells distinguish clinical subtypes of Juvenile Idiopathic Arthritis (135.36)
Authors: Dvergsten Jeffrey , Mueller Robert , Abedin Sameem , Michel Joshua , Rosenkranz Margalit , Vallejo Abbe
Published: January 2023

Pervasive T cell clonality is a feature of Juvenile Idiopathic Arthritis (JIA), but the role of oligoclonal T cells in this chronic disease of childhood is poorly understood. We propose that T cell clonality in JIA is indicative of replicative pressure leading to accumulation of terminally differentiated cells. Hence, we examined the T cell repertoire of children with confirmed diagnosis of JIA and their age-matched healthy counterparts. Results showed that naive T cells of children with JIA had significantly lower telomere-FISH fluorescence indicating a long replicative history. Multicolor cytometry analyses also showed that patients had over abundance of CD8+CD31+CD28null T cells in blood and synovial fluid irrespective of disease duration and medication. Frequency of these unusual T cells was significantly higher among patients with the more severe, polyarticular form of JIA disease than those with oligoarticular disease. Furthermore, these T cells expressed high levels of γH2AX and pRB suggesting senescence. In proliferation assays using CFSE, these T cells remained CFSEhi but were CD69hiCD25lo/neg, results consistent with senescence and not quiescence. In receptor crosslinking assays, ligation of CD31 alone sufficiently induced tyrosine phosphorylation, cytolysis, and production of IFNγ, TNFα, and IL-10. These data indicate premature immune senescence in JIA, with CD8+CD31+CD28nullH2AX+ T cells as maladaptive effectors, and are potential targets for therapy.
Toll-like receptor 9 is alternatively cleaved to generate a soluble form of the receptor that downregulates response to CpG-DNA (136.25)
Authors: Chockalingam Annapoorani , Brooks James , Cameron Jody , Leifer Cynthia
Published: January 2023

Proper regulation of nucleic acid sensing Toll-like receptors (TLRs), including TLR9, is critical to prevent autoimmune disease and promote host defense. The objective of our research is to elucidate the molecular mechanisms that control TLR9 signaling. Localization of TLR9 in endolysosomes is one regulatory mechanism that facilitates recognition of CpG-DNA and prevents response to self DNA. Restricted localization of TLR9 controls access to proteases, including cathepsins B and L and asparagine endopeptidase, which generate an active 80 kDa (p80) form of the receptor. Here we use mass spectrometry analysis to identify a 100 kDa soluble form of TLR9 (sTLR9, amino acids 1-740). The proteolytic cleavage of sTLR9 is distinct from that of p80 since it is not blocked by Z-FA-fmk or E-64 protease inhibitors, or by bafilomycin A1 or chloroquine. These data suggest that generation of sTLR9 does not occur in acidic endosomes. We show that the generation of sTLR9 requires cathepsin-S, a protease inhibitor active at neutral pH. Most importantly we show that the sTLR9 binds to CpG-DNA and down regulates CpG-DNA induced TLR9 signaling. Balance between proteolytic cleavage occurring at two distinct sites on TLR9 ectodomain is likely a major factor that modulates host innate immune responses to microbial infection and prevents development of autoimmune disease.
Inflammasome-dependent IL-1β production is critical for complete Freund’s adjuvant-induced helper T cell polarization (136.44)
Authors: Shenderov Kevin , Barber Daniel , Mayer Katrin , Jankovic Dragana , White Sandy , Caspar Pat , Hieny Sara , Trinchieri Giorgio , Besra Gurdyal , Cerundolo Vincenzo , Sher Alan
Published: January 2023

The innate immune system recognizes pathogens/antigens via several families of pattern recognition receptors (PRRs). Often, several PRRs will be triggered, resulting in an innate immune response via a complex integration of signals. However, the importance of different PRR signaling pathways in determining adaptive responses such as helper T cell polarization is not fully understood. We studied the role of two pathways—TLRs and the inflammasome—in promoting helper T cell polarization in response to complete Freund’s adjuvant (CFA). Mice immunized with ovalbumin (OVA) in CFA developed an OVA-specific Th1/Th17 response that was dependent on the signaling adaptor MyD88. TLRs were only partially required for this response, whereas interleukin 1 (IL-1) receptor signaling and the inflammasome were critical. We found that heat-killed M. tuberculosis H37Ra, a component of CFA, induces mature IL-1β production in vitro in a process that requires potassium efflux and reactive oxygen species and is mediated by the NLRP3 receptor. We further showed that the major inflammasome-triggering component of H37Ra is the peptidoglycan. However, the peptidoglycan-sensing receptors NOD1 and NOD2 were not needed for this response. Our data suggest that NOD1/2-independent recognition of mycobacterial peptidoglycan by the NLRP3 inflammasome induces IL-1β production and may play an important role in CFA-induced helper T cell responses. This work was supported by the NIAID Intramural Research Program.
CD4+ T cell regulation of CD25 expression controls the development of effector CD8+ T cells in acute and chronic virus infections. (137.14)
Authors: Molloy Michael , Zhang Weijun , Usherwood Edward
Published: January 2023

Both CD4+ T cell help and IL-2 have been postulated to ‘program’ activated CD8+ T cells for memory cell development. However, the link between these two signals has not been well elucidated. We have studied effector and memory CD8+ T cell differentiation following infection with vaccinia virus and murine gammaherpes virus in the absence of both CD4+ T cells and CD25. Expression of CD25 on antigen-specific CD8+ T cells peaked 3-4 days after infection and was dependent on CD4+ T cell help. CD4+ T cell or CD25-deficiency led to normal early effector CD8+ T cell differentiation, but a subsequent lack of accumulation of CD8+ T cells resulting in overall decreased memory cell generation. Interestingly, KLRG1high CD127low short-lived effector cells were diminished in the absence of IL-2 signaling, while memory precursors remained intact. Interestingly, the mononucleosis-like expansion of Vβ4+ T cells seen in mice infected with MHV-68 was impaired in a similar fashion to antigen specific responses. Upon secondary antigen encounter, CD25-deficient CD8+ T cells were capable of undergoing robust expansion, but short-lived effector development was again impaired. These results demonstrate that CD4+ help and IL-2 signaling are linked via CD25 upregulation, which controls the expansion and differentiation of antigen-specific effector CD8+ T cells.
Cytokine production patterns identify novel CD8 effector subsets (137.18)
Authors: Rudenga Benjamin , Khanna Kamal , Lefrançois Leo
Published: January 2023

Underlying the continuing rational development of vaccines to infectious agents is a deeper understanding of the immunological determinants affecting the outcomes to various strategies of vaccination. For example, different forms of Listeria monocytogenes (LM) based vaccines elicit distinct immune responses. Thus, heat killed LM is less effective than irradiated LM which is in turn less effective than live LM in inducing protective CD8 T cell immunity. To characterize the underlying reasons for these differences we analyzed the genetic signature of CD8 T cells responding to the different immunogens. Interestingly, cytokine and chemokine genes were induced very early after activation and their levels correlated with the “strength” of the immunogen. We have begun to examine the expression of some of these cytokines at the protein level using multiplex bead assays and intracellular staining. Several cytokines and chemokines have been identified as potential targets; however, IL-3 was found to be expressed early in the immune response to live LM and was also induced after vesicular stomatitis virus (VSV) infection. Only a subset of CD8 T cells produced IL-3 and we are now determining the relationship between this population, other effector subsets, and memory development. Ongoing studies are focused on deciphering the complexity of cytokine production by CD8 T cells responding to infection in the hope of designating effector and memory lineages based on the identified patterns.
TULA-family proteins are key to the regulation of T-cell-driven inflammatory responses. (138.13)
Authors: Newman Tiffanny , Carpino Nick , Ganea Doina , Safadi Fayez , Yen Jimmy , Tsygankov Alexander
Published: January 2023

The novel TULA family consists of two proteins recently implicated in cellular regulation. TULA-1 is expressed only in lymphoid cells and has been shown to exert a pro-apoptotic effect in T-cells, possibly through its interaction with AIF. TULA-2 is expressed ubiquitously and is an active phosphatase capable of dephosphorylating proteins involved in receptor-mediated signaling. CD4+ T-cells from mice lacking both family members (dKO) are hypersensitive to TCR stimulation. Initial characterization T-cell subpopulations in TULA dKO mice revealed an atypical CD45RB distribution among dKO CD4+ T-cells. Further analysis of memory and regulatory markers indicate that both memory and regulatory T-cell populations are expanded in dKO mice. To determine in vivo consequences of these changes, we utilized two mouse models of inflammation in which T-cells play a key role: trinitrobenzene sulfonic acid (TNBS)-induced colitis and colitis induced by the adoptive transfer of CD45RB-high CD4+ T-cells. Studies utilizing TNBS indicated that TULA dKO mice are more sensitive to colitis as compared to WT mice and that the increased sensitivity is dependent on T-cells. Consistent with these findings, dKO CD45RB-high CD4+ T-cells were significantly more colitogenic than the in WT mice in the transfer colitis model. Taken together, our data suggests that TULA-family proteins are key to the physiological regulation of T-cell reactivity that drives inflammatory responses in the intestine.
Licensing of naïve T cells for rapid TNF production (138.14)
Authors: Priyadharshini Bhavana , Welsh Raymond , Greiner Dale , Gerstein Rachel , Brehm Michael
Published: January 2023

TNF is a potent inflammatory cytokine with numerous immunological functions, including the ability to limit the magnitude of virus-specific T cell responses during infection. Naïve T cells in secondary lymphoid organs (SLOs) exhibit a unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. Peripheral T cells are a heterogeneous population of cells at various stages of post-thymic development. To determine how maturation influences the licensing of T cells to produce TNF, we compared cytokine profiles of single positive thymic T cells, recent thymic emigrants (RTEs) and matured-naïve T cells during TCR activation. Thymic T cells exhibited a poor ability to produce TNF when compared to splenic T cells, despite possessing a similar potential for activation. Moreover, thymic T cells had significantly reduced levels of TNF message and phosphorylated MAPK (Erk1/2) when compared to their splenic counterparts. Stimulation of thymic T cells in the presence of splenic B cells and APCs partially licensed the T cells to produce TNF but not to the same extent as peripheral T cells. RTEs had a significantly enhanced ability to produce TNF as compared to thymic T cells, but fully matured naïve T cells were the most efficient TNF producers. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.
Mechanism of exaggerated inflammation in A2A adenosine receptor-deficient mice. (138.28)
Authors: Subramanian Meenakshi , Kini Radhika , Madasu Manasa , Ohta Akiko , Sitkovsky Michail , Ohta Akio
Published: January 2023

Extracellular adenosine regulates inflammatory responses via A2A adenosine receptor (A2AR). Indeed, A2AR-deficiency results in much exaggerated inflammatory tissue damage, indicating non-redundancy of adenosine-A2AR pathway in the inhibitory mechanism of immune activation. In this study, we found that intravenous injection of concanavalin A into A2AR-deficient mice induced not only stronger hepatic damage than in wild-type mice but also higher levels of cytokines, i.e. TNF-α, IL-4 and IFN-γ. Since NKT cells are responsible for the early production of these cytokines after concanavalin A injection, NKT cells were suspected to be under physiological control of adenosine-A2AR pathway. To directly examine the role of A2AR in NKT cell activation, we specifically stimulated NKT cells with α-galactosylceramide in the presence of dendritic cells. As a result, cytokine production from NKT cells decreased in the presence of A2AR agonists, suggesting that A2AR signaling can down-regulate NKT cell activation. We further injected α-galactosylceramide into mice and observed higher cytokines production in A2AR-deficient mice comparing to wild-type mice. Correspondingly, α-galactosylceramide induced stronger liver damage in A2AR-deficient mice. These results strongly suggest that NKT cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation.
High-throughput multi-parameter analysis of human immune responses to the seasonal Influenza vaccine in the elderly and young subjects: defining metrics of immune health and immune senescence (138.4)
Authors: Furman David , Shen-Orr Shai , Kidd Brian , Dekker Corry , Maecker Holden , Davis Mark
Published: January 2023

Aging is a complex process in which the immune function is severely affected. Increased prevalence of infectious diseases and poor vaccine responses are characteristic in the elderly. Here we seek to define the immune response to the seasonal inactivated Influenza vaccine in young subjects and in an elderly population with the aim of finding signatures of immune health and immune senescence. Ninety-one subjects, age-ranges 20-30, 60-79 and 80-96, were drawn prior to the vaccine, 7 and 21 days after vaccination. PBMC were isolated to phenotype 18 immune cell subsets and for high-throughput analysis of signaling phosphoproteins in multiple cell-subsets, after various stimulations. Serum samples were used to determine cytokine levels and antibody titers to the vaccine strains and to non-circulating Influenza strains. Dramatic age-related changes in STAT signaling were observed in CD8 and CD4 T cells when stimulated with IL-6, IFN-α or IFN-γ. The level of PLCγ phosphorylation in B cells from older subjects was significantly lower than the observed in the younger group, independently of the reduced B cell frequency. Weak antibody response against the vaccine strains while high level of seroprotection against older non-circulating vaccines was seen in the elderly and strongly associated to the IL-6 or IFN-α pathways. A better comprehension of human immune aging is necessary for designing age-specific vaccines with higher efficacy.