bluuebunny/crossref_metadata_2025 huggingface dataset:
An NLRP7-containing inflammasome mediates recognition of microbial lipopeptides and restrict intracellular bacterial replication in human macrophages (114.6)
Authors: Stehlik Christian , Khare Sonal , Dorfleutner Andrea , Bryan Nicole , Yun Chawon , Radian Alexander , de Almeida Lucia , Rojanasakul Yon
Published: January 2023

Nod-like receptors (NLRs) emerged as central cytosolic pattern recognition receptors (PRRs). However, the function of the majority of NLRs is currently unknown. We identified that NLRP7 functions as a PRR in primary human macrophages. Activation of NLRP7 by cytosolic microbial acylated lipopeptides (acLP) causes ASC-dependent inflammasome formation in a cytosolic high-molecular weight complex, Caspase-1 activation and maturation and release of interleukin (IL)-1β and IL-18. Reconstitution of an NLRP7/ASC/pro-Caspase-1 inflammasome, but not of an NLRP3/ASC/pro-Caspase-1 inflammasome is sufficient to respond to intracellular delivery of acLP. However, in macrophages both NLRP7 and TLR2 are required for the acLP-mediated release of IL-1β. While TLR2 is necessary for the transcription of pro-IL-1β and pro-IL-18, NLRP7 mediates the Caspase-1-dependent maturation and release of both cytokines. Therefore, NLRP7 and TLR2 contribute to the host defense against intracellular Gram positive bacteria, since knock-down of either NLRP7 or TLR2 promotes intracellular replication of Listeria monocytogenes and knock-down of NLRP7 also increases the intracellular replication of Staphylococcus aures. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly and maturation of IL-1β and IL-18 in human macrophages.
Development of PLZF-expressing γδ NKT cells in Itk-/- mice (115.13)
Authors: Yin Catherine , Narayan Kavitha , Sylvia Katelyn , Immunological Genome Project The , Kang Joonsoo , Berg Leslie
Published: January 2023

In conventional αβ T cells, the Tec family tyrosine kinase, Itk is required for signaling downstream of the TCR. Itk also regulates αβ T cell development, lineage commitment, and effector function. A well-established feature of Itk-/- mice is their inability to generate T helper type 2 (Th2) responses that produce IL-4, IL-5, and IL-13; yet these mice have spontaneously elevated levels of serum IgE. We have previously shown the cause of this phenotype is due to the increase of the mature Vγ1.1+Vδ6.3+ (V6) subset of γδ T cells in Itk-/- mice. Wild type V6 γδ T cells display characteristics similar to αβ NKT cells including dual secretion of IL-4 and IFNγ along with expression of the transcription factor PLZF; therefore they are γδ NKT cells. Along with increased proportions of mature V6 cells in Itk-/- mice, V6 cells in Itk-/- mice have increased IL-4 secretion and elevated levels of PLZF expression. Based upon PLZF expression and the use of the IL-4 reporter (4Get) mice, our data indicate that γδ NKT cells have similar differentiation stages as αβ NKT cells, and that Itk-/- γδ NKT cells are blocked during development. Together, these data indicate that Itk signaling regulates γδ T cell lineage development and effector function, and is required to control IgE production in vivo.
Mechanisms of plasticity in the peripheral T cell receptor repertoire (115.15)
Authors: Thomas Paul , Wang George , Dash Pradyot
Published: January 2023

We recently reported a method for amplifying paired T cell receptor (TCR) αβ CDR3 regions from single cells in mice and have developed a similar protocol for human cells. Using this technique, we have discovered a surprising plasticity in peripheral TCR repertoire, particularly under conditions of inflammation. This plasticity includes two major mechanisms: TCR modulation and TCR revision. Modulation consists of the up or down regulation of secondary allele expression, almost exclusively from the TCRα locus. Revision results in changes to the amino acid sequence of the TCR by de novo rearrangement. Here we describe the upregulation of both processes in CD8+ T cells derived from influenza-infected lungs when compared to circulating naïve T cells. In particular, instances of modulation and dual chain expression are elevated over the course of a primary influenza T cell response, whereas in recall responses memory cells express only a single allele of each TCR chain. In repeated infections, revised TCRs are observed by using out of frame TCR sequences as an internal, lineage specific, barcode. The relative contribution of each mechanism to the activation of autoreactive T cells is assessed in genetic models.
S-Adenosylmethionine-dependent methylation modulates T cell immune response and lupus autoimmunity (116.13)
Authors: Yang Mei-Ling , Gee Alaric , Gee Renelle , Mamula Mark
Published: January 2023

Methylation modification is a key factor to maintain T and B cell immune responses and contributes to the development of autoimmune diseases. Failure to maintain the methylation status of CpG dinucleotide triggers T cell autoreactivity and results in the pathogenesis of systemic lupus erythematosus (SLE). However, methylated proteins have been considered as auto-antigens such as arginine-methylated myelin basic protein in multiple sclerosis (MS) and symmetrical dimethyl-arginines of SmD1 and SmD3 in SLE. Importantly, transmethylation reaction cannot achieve without the methyl donor supply. Herein, we demonstrate that the disruption of the metabolism of s-adenosylmethionine (SAM) cycle, the major methyl donor source for transmethylation, can modulate T cell functions. By giving excess methyl donor, SAM, or methylation inhibitor, methylthioadenosine (MTA), T cell immune responses were suppressed includes TCR signaling defect, impaired multiple Th1/Th2 cytokine productions, decreased T cell activation and proliferation. Moreover, intraperitoneal treatment of MTA markedly ameliorates splenomegaly, lymphadenopathy, peripheral autoantibodies production, renal IgG deposition and lymphocyte infiltration into the kidney in lupus-prone MRL/lpr mice. These studies demonstrate the central role of SAM-dependent methylation associated with T cell function and MRL lupus autoimmunity.
Cannabinoid receptor-2-selective agonists improve recovery in experimental autoimmune encephalomyelitis (116.7)
Authors: Kong Weimin , Li Hongbo , Adhikary Sabina , Tuma Ronald , Ganea Doina
Published: January 2023

Previous studies showed that cannabinoids have anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE), but the mechanism is elusive. In the present study, we investigated the effects of Gp1a (a new CB2 selective agonist) in EAE. EAE developed in a similar way in both vehicle and Gp1a treatment group, but mice treated with Gp1a recovered faster than the vehicle group. Cellular analysis was performed in spleen, brain and spinal cord. In spleen, there was no difference in numbers of CD3+ T cells, Th1 and Th17 cells, or macrophages. In contrast, CNS mononuclear cell analysis showed fewer CD3+ T cells, Th1 and Th17 cells, macrophages and activated microglia in spinal cord but not in brainin the Gp1a treatment group. Accordingly, for the Gp1a-treated group, histology studies also showed fewer CD3+ T cells, CD11b+ macrophages/microglia and less Iba-1 expression in spinal cord but not in brain. In vivo cell proliferation as measured through BrdU incorporation in CD3+CD45hi, CD11b+CD45hi and CD11b+CD45int mononuclear cells was not affected by Gp1a treatment. We hypothesize that Gp1a inhibits cell migration to the CNS. We also analyzed gene expression in brain and spinal cord following EAE development and found lower levels of VCAM-1, CXCL-10 and MMP9 in spinal cord. We also found that Gp1a inhibits MMP9 and TNFα production in BV.2 cells (a microglia cell line). We conclude that Gp1a improves EAE recovery through inhibiting immune cell migration into CNS.
IFN-γ induced antimicrobial response against M. leprae in human macrophages (117.12)
Authors: Realegeno Susan , Teles Rosane , Kelly-Scumpia Kindra , Modlin Robert
Published: January 2023

The role of IFN-γ in inducing an antimicrobial response in macrophages was investigated in leprosy, a disease caused by intracellular bacterium M. leprae. Tuberculoid leprosy (T-lep) is characterized by few skin lesions with low bacterial numbers, whereas lepromatous leprosy (L-lep) is characterized by disseminated skin lesions and increased bacilli numbers. The host immune response in T-lep is characterized by Th1 cytokines and expression of the vitamin D dependent antimicrobial pathway, in contrast to L-lep, which is predominately Th2. We have linked expression of the Th1 cytokine IFN-γ and antimicrobial response in leprosy by induction of vitamin D dependent antimicrobial response genes CAMP and DEFB4. IFN-γ is present in T-lep patients, where the disease is self-limited, however, the role of IFN-γ in antimicrobial activity is unclear. IFN-γ mediates antimicrobial activity against M. tuberculosis, a related pathogen, in the presence of vitamin D sufficient serum, but not in the presence of vitamin D deficient serum. Our preliminary data shows that M. leprae infected macrophages pretreated with IFN-γ prior to infection in combination with post-infection IFN-γ treatment in vitamin D sufficient serum reduced bacterial viability by approximately 80% compared to media control. Our studies suggest that IFN-γ can trigger a vitamin D dependent antimicrobial response in human macrophages as well as antimicrobial activity against M. leprae.
Efferocytosis induces macrophages to produce IL-4 and activate invariant NKT cells to suppress inflammation (117.17)
Authors: Zeng Melody , Pham Duy , Brutkiewicz Randy , Kaplan Mark , Dinauer Mary
Published: January 2023

Efferocytosis of apoptotic neutrophils by macrophages is fundamental to the resolution of inflammation. In this study we demonstrate using a mouse sterile peritonitis model, a circuit requiring the activation of NKT cells by efferocytosing macrophages to resolve inflammation. Following ingestion of apoptotic neutrophils, macrophages produced IL-4 and IL-13, which in an autocrine manner, resulted in cells with an M2 phenotype. Efferocytosing macrophages displayed increased cell surface expression of CD1d and activated invariant NKT (iNKT) cells that produced IL-4 during the later phase of inflammation. Peritoneal inflammation was exaggerated in mice lacking IL-4 or NKT cells, suggesting both are required to suppress inflammation. Furthermore, sterile peritoneal inflammation was also enhanced and prolonged in NADPH oxidase-deficient mice with X-linked chronic granulomatous disease (X-CGD). We found that X-CGD exudate macrophages expressed reduced surface CD1d and were impaired in activating iNKT cells, which contributed to the persistent inflammation in these mice. Therefore, efferocytosis-induced IL-4 production and activation of iNKT cells by macrophages are immunomodulatory events in an innate immune circuit required to resolve sterile inflammation.
TLR agonist treatment elicits an increase in DNA repair machinery (117.19)
Authors: Harberts Erin , Fishelevich Rita , Gaspari Anthony
Published: January 2023

The toll-like receptor (TLR) pathway is stimulated by pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Activation of this pathway leads to an inflammatory and immunomodulating response appropriate for the stimulus by which it was activated. Recently a body of literature has emerged indicating that TLR stimulation may not only result in an inflammatory response, but may also lead to an up-regulation of DNA repair genes and an increase in functional DNA repair after injury. These conclusions are drawn from studies conducted in the mouse animal model, however the human DNA repair response to TLR activation has yet to be investigated. Using an in vitro cell culture system, we find that human DNA repair genes are similarly up-regulated. Previously, only TLR 7 and TLR 9 agonists have been shown in mice to stimulate increased DNA repair; in this study we expand the panel of TLR agonists investigated and show that the induction of this phenomenon may not be unique to the previously studied TLR agonists. This functional increase in repair after injury may lead to a wide range of outcomes for the cells involved and warrants further study into the ever broadening role of TLR signaling in controlling cell fate.
Intradermal route of infection with Francisella tularensis subspp novicida elicits differential induction of a wide array of cytokines and chemokines (117.7)
Authors: Biffa Demelash , Buntzman Adam , Barrigan Lydia , Frelinger Jeffrey
Published: January 2023

Francisella tularensis is highly virulent and infectious bacterial pathogen and a category A select agent. Many aspects of immunity to the infection are not well understood. This study is part of ongoing work to understand the immune response following cutaneous exposure. The aim of the study was to determine cytokines/chemokines induced in response to intradermal infection with F. tularensis subspp novicida. Further aim was to determine the correlation between induction of cytokines/chemokines with bacterial load. C57BL/6J mice were intradermally infected with F. novicida U112 WT and DotU. DotU mutants do not grow inside macrophages and are greatly attenuated. The results investigated wide array of induced anti-and pro-inflammatory cytokines/chemokines in spleen. Higher levels of INF-γ and P-10 were expressed at 24hrs post-infection in spleen of mice infected with WT compared to DotU or mock control. The recovered number of viable bacteria from the spleen peaked at 24hrs and correlated with increased induction of INF-γ. Studies are underway to elucidate the role of cytokines/chemokines in shaping adaptive immunity.
Neutrophil mediated IL-1β processing in Pseudomonas aeruginosa infection is IPAF and Caspase-1 independent and mediated by serine proteases (118.1)
Authors: Karmakar Mausita , Sun Yan , Hise Amy , Rietsch Arne , Pearlman Eric
Published: January 2023

Inflammasome and caspase-1 dependent cleavage of IL-1β to the mature, active form is the predominant, but not sole mechanism of IL-1β processing. To investigate the role of IL-1β, IPAF/NLRC4 inflammasome and caspase-1 in bacterial keratitis, which is an important worldwide cause of blindness, mouse corneas were infected with Pseudomonas aeruginosa invasive strain PAO1 (expressing ExoS, T and Y but not ExoU), and corneal inflammation and bacterial survival were examined. We found that IL-1β-/- mice had significantly fewer infiltrating neutrophils, and significantly higher CFU than C57BL/6 mice. In contrast, there was no difference in either corneal disease or bacterial survival between caspase-1-/-, IPAF1-/- and C57BL/6 mice. In all mouse strains, we found that neutrophils are the major producers of IL-1β during corneal infection, and that although IL-1β processing by macrophages was completely dependent on caspase-1, neutrophil mediated IL-1β processing in vivo was not impaired in caspase-1-/- or in IPAF1-/- mice. In vitro studies showed that inhibition of neutrophil elastase significantly inhibited IL-1β processing by human and mouse neutrophils. Taken together, these results demonstrate a novel caspase-1 independent mechanism of IL-1β processing by neutrophils during bacterial infection.
Improved production of human beta-defensins (HBDs) in grafted site skin tissues of human IMC/mouse chimeras treated with glycyrrhizin (GL) (118.14)
Authors: Okita Yamato , Lee Jong , Nakamura Kiwamu , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

Skin HBDs are very important on the elimination of pathogens at the burn wound infections. Immature myeloid cells (IMC) appearing in tissues surrounding the burn area are shown to be inhibitory on skin HBD production. In this study, the effect of GL on the impaired production of skin HBDs was studied in X-irradiated NOD-SCID IL-2rγ−/− mice (NSG mice) grafted with human IMC and their syngeneic skin tissues. NSG mice were grafted with patient skin (8 × 8 mm unburned skin removed for auto-graft surgery; skin-chimeras) and inoculated i.d. with 1 × 106 cells/mouse of CD34+CD31+ IMC isolated from the same burn patients (skin/IMC-chimeras). These chimeras were treated with 0.2 to 40 mg/kg of GL (Minophagen Pharmaceutical CO., LTD., Tokyo, Japan) 6 and 24 hrs after IMC inoculation. Two days after grafting, tissue specimens excised from grafted sites were homogenized, and the amounts of HBDs in the homogenates were measured. In the results, HBD1 and HBD2 were detected in the skin homogenates of skin-chimeras, while the peptides were not detected in the skin homogenates of skin/IMC-chimeras. However, HBDs were detected in the skin homogenates of skin/IMC-chimeras treated i.p. or i.d. with 3 to 10 mg/kg of GL. The greatest efficacy of GL on recovering HBD production was seen when skin/IMC-chimeras were treated i.d. with 10 mg/kg of GL. These results indicate that the impaired HBD production is improved in skin/IMC-chimeras treated with GL.
Extracellular superoxide dismutase impairs neutrophil function and clearance of Listeria monocytogenes (118.4)
Authors: Break Timothy , Jun Sujung , Indramohan Mohanalaxmi , Dory Ladislav , Berg Rance
Published: January 2023

It is well established that reactive oxygen species (ROS) play a critical role during immune responses against pathogens. Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of ROS and protects tissues during non-infectious inflammatory insults. However, how ecSOD activity impacts immune responses directed against pathogenic microorganisms has not been thoroughly investigated. We utilized mice with varying levels of ecSOD activity to investigate the involvement of this enzyme in innate immune responses against Listeria monocytogenes. Our data demonstrate that ecSOD activity negatively impacted host survival and bacterial clearance from the spleen and liver. Paradoxically, ecSOD activity was associated with increased percentages of neutrophils in the livers of mice prior to, and during infection. Neutrophils from livers of mice with high ecSOD activity were altered in their ability to produce various molecules important for bacterial clearance. Additionally, ecSOD activity was associated with a higher percentage of neutrophils that phenotypically resembled myeloid-derived suppressor cells. Neutrophil depletion studies revealed that high ecSOD activity resulted in neutrophils with no protective capacity, whereas neutrophils from mice lacking ecSOD provided superior protection compared to neutrophils from wild-type mice. Collectively, our data demonstrate that ecSOD activity inhibits innate immune responses, thus impairing bacterial clearance.
Regulation of the inflammatory response: enhancing neutrophil infiltration under chronic inflammatory conditions (118.5)
Authors: Bian Zhen , Guo Yanan , Ha Binh , Zen Ke , Liu Yuan
Published: January 2023

Neutrophil (PMN) infiltration plays a central role in inflammation and is a major cause of tissue damage. Thus, PMN infiltration must be tightly controlled. Using zymosan-induced peritonitis as an in vivo PMN infiltration model, we show in this study that PMN response and infiltration were significantly enhanced in mice experiencing various systemic inflammation including colitis and diabetes. Adoptive transfer of leukocytes from mice with inflammation into healthy recipients or from healthy into inflammatory recipients followed by inducing peritonitis demonstrated that both circulating PMN and tissue macrophages were altered under inflammation and that they collectively contributed to enhanced PMN infiltration. Detailed analyses of dextran sulfate sodium (DSS)-elicited colitis revealed that enhancement of PMN infiltration and macrophage function occurred only at the post-acute/chronic phase of inflammation and was associated with markedly increased IL-17A in serum. In vitro and ex vivo treatment of isolated PMN and macrophages confirmed that IL-17A directly modulates these cells and significantly enhances their inflammatory responses. Neutralization of IL-17A eliminated the enhancement of PMN infiltration and IL-6 production and also prevented severe tissue damage in DSS-treated mice. Thus, IL-17A produced at the chronic stage of colitis serves as an essential feedback signal that enhances PMN infiltration and promotes inflammation.
Immunomodulatory effects of Simvastatin in Rheumatoid arthritis synoviocytes are mediated via BNIP3 (119.1)
Authors: Khan Mohammad Saeed , Al-Karakooly Zeiyad , Ali Nawab , Kannan Krishnaswamy
Published: January 2023

We investigated the anti-inflammatory effects of Simvastatin (SMV) in vitro, in fibroblast-like synoviocyte (FLS) cell lines from control and RA subjects, in the presence of an activated TLR2 signaling pathway. Cells (10^5 cells/well) were grown in 12-well culture plates and treated with SMV in triplicates for 4 hours and then treated with TLR2 ligand (Pam3CSK4) for 24-hours. Cytotoxicity was assessed by Alamar-blue assay, interleukin-6 (IL-6) by ELISA, reactive oxygen species (ROS) and apoptosis by flow cytometry. Inositol phosphates (IPs) were measured by labeling IPs with tritium in cell culture, followed by anion exchange chromatography and analyzed in a beta scintillation counter. Gene expression analysis of 84-ROS associated genes was performed using a real time PCR array profiling kit from SABiosciences. SMV caused dose-dependent cytotoxicity in both cell lines and this was due to apoptosis. Basal apoptosis was higher in normal than RA-FLS (14% vs 4.3%). ROS was increased modestly (5% basal to 12%). IL-6 increased and IP-1 and IP-2 decreased with SMV treatment. Gene expression of GSR, TXNRD1, PRDX1, SOD2, MT3 and PTGS2 were significantly upregulated. BNIP3 expression was increased 2200-fold in RA-FLS after SMV/TLR2 treatment. These findings indicate that SMV induce FLS apoptosis in RA by upregulating BNIP3 and FLS react to survive by increasing IL-6, decreasing IP-1 and IP-2 and upregulating anti-oxidant genes.
Defective regulatory T cells function and spontaneous colitis induced by protein phosphatase 4-deficiency (120.14)
Authors: Liao Fang-Hsuean , Lin Yu-Jun , Hsing En-Wei , Hsou Wan-I , Jhou Yi-Jyun , Tan Tse-Hua , Huang Ching-Yu
Published: January 2023

Protein phosphatase 4 (PP4) is essential during early T cells development. To assess the function of PP4 in peripheral T cells, we generated conditional PP4 knockout mice with CD4-cre transgene (CD4cre-PP4f/f). In these mice a partial block of thymocyte development was manifested as a ~40-60% reduction of peripheral T cells. Further studies showed that PP4 deletion resulted in poor T cell proliferation, inefficient Treg polarization, decreased Treg cell number and reduced Treg suppression activity. In vivo, CD4cre-PP4f/f mice exhibited defective T-dependent antibody production. More interestingly, by 5-month old ~50% of the CD4cre-PP4f/f mice developed spontaneous colitis with wasting syndrome, and showed increased percentages of IFNgamma+, IL-6+, or IL-17A+-producing cells in intra-epithelial lymphocytes and lamina propria lymphocytes. When investigating the pathogenesis of colitis, we found that transferring PP4-deficient CD4+CD45RBhigh cells failed to induce colitis in RAG-/- recipients, whereas antibiotics treatment could reverse the wasting syndrome. Combined with the defects in Treg and antibody production, these results are consistent with the possibility that PP4 deficiency reduced the efficiency of mucosal immunity against commensal bacteria, causing uncontrolled gut inflammation that eventually develop into colitis. The spontaneous colitis in CD4cre-PP4f/f mice may thus serve as an unique model for studying the pathogenesis of human inflammatory bowel disease.
EBI2 expression is associated with B cell inflammation in human sinuses (120.35)
Authors: Hulse Kathryn , Zhong Qiu , Mahdavinia Mahboobeh , Norton James , Kern Robert , Conley David , Chandra Rakesh , Tan Bruce , Peters Anju , Grammer Leslie , Harris Kathleen , Seshadri Sudarshan , Carter Roderick , Suh Lydia , Schleimer Robert
Published: January 2023

Epstein-Barr virus-induced G-protein coupled receptor 2 (EBI2) is important for the migration of B cells and induction of extrafollicular plasma cells within secondary lymphoid tissues. Recent studies from our group have demonstrated that many molecules associated with B cell survival and plasma cell differentiation (e.g. BAFF and immunoglobulins) were elevated in nasal polyps from patients with chronic rhinosinusitis (CRS). To investigate whether B cells or EBI2 played a role in CRS pathogenesis, expression of B cell- and EBI2-related genes in nasal tissue from normal controls and CRS patients with (CRSwNP) or without (CRSsNP) nasal polyps was assessed. EBI2 mRNA was quantitated by qRT-PCR, EBI2 protein was measured by western blot, and B cell subsets were characterized by flow cytometry. Expression of EBI2 mRNA was significantly elevated in polyp tissue (p<0.001) and correlated with mRNA expression of CD20 (r=0.71; p<0.0001) and CH25H, an enzyme responsible for production of an EBI2 ligand (r=0.65; p<0.001). EBI2 protein was also elevated in polyps (p<0.01, n=10). In addition, B cells (CD20+) and plasma cells (CD138+) were significantly elevated in polyps compared to nasal tissue from normal controls (p<0.05). Collectively, these data suggest that elevated EBI2 may facilitate B cell migration and differentiation in the nasal mucosa, especially nasal polyps. These findings provide new insights into the pathogenesis of CRS and may indicate potential new therapeutic targets.
Examining the role of T cell co-receptors CD4 and CD8 in T cell activation by using high-affinity T cell receptors (121.28)
Authors: Marshall Elizabeth , Weber K. Scott , Donermeyer David , Allen Paul , Kranz David
Published: January 2023

The recognition of foreign antigens by T cells is accomplished through the binding of the T cell receptor (TCR) in collaboration with co-receptor (CD4 or CD8) recognition of MHC. It is clear that CD4 and CD8 function by binding to their cognate MHCs and by delivering the kinase Lck to the TCR complex for initiation of signaling, however, the mechanistic details of this process remain unclear. For example, it remains unknown if co-receptors must engage the same MHC as the TCR, or if engagement of a nearby non-cognate MHC molecule is sufficient to initiate signaling. To examine the details of co-receptor function, we used a class II MHC specific TCR system consisting of a wild-type (KD 20 μM) and high-affinity variant (KD 25 nM). A panel of T cell hybridomas was generated expressing either the wild-type or high-affinity TCR paired with either the CD4 or CD8 co-receptor. The low-affinity, but not the high-affinity, TCR required cognate CD4 for activity. Unexpectedly, non-cognate CD8 was able to enhance the activity of the low-affinity TCR, and CD8:class I MHC interactions were required for activity mediated by the high-affinity TCR. Unexpectedly, CD8 also operated by preventing apoptosis of the antigen presenting cells (APCs). These data show that CD8 can act by binding to non-cognate MHC, and that its functions extend to the prevention of apoptosis of APCs. The findings thus provide a framework for a new model of co-receptor function.
Ablation of PDK1 reduces CD4 T cell proliferation and alters cytokine production (121.6)
Authors: Yu Minjun , Ghosh Sankar , Farber Donna
Published: January 2023

In CD4 T cells, phosphoinositide-dependent protein kinase 1(PDK1) plays a crucial role in activating AGC kinases including Akt, p70 ribosomal S6 kinase, PKCθ, which control downstream proliferation, protein synthesis and NF-κB activation. We used mice with conditional expression of PDK1 (PDK1 floxed) and an YFP reporter (PDKF/FR26YFP) to assess the effect of PDK1 deletion on effector and memory CD4 T cell persistence, homeostasis and function. CD4 T cells were activated by anti-CD3/anti-CD28 antibodies and treated with TAT-Cre fusion protein. PDK1 knockdown was confirmed by PCR and western blotting of YFP+ cells which also showed reduced p70 S6 kinase phosphorylation. Transfer of primed PDK1 deficient effector T cells into Rag2-/- mice resulted in reduced persistence. This reduced persistence was associated with decreased proliferative turnover both in vivo and in vitro, but not an increase in apoptosis, as assessed by annexin V staining. PDK1 deficient effector and memory CD4 T cells reduced IL-2 production but IFN-γ production was intact. These results suggest that the PDK1 selectively controls effector CD4 T cell proliferation and IL-2 production with potential effects on memory CD4 T cells persistence in vivo.
Ikaros regulates naive CD8+ T cell autocrine IL-2 production and differentiation (121.7)
Authors: O'Brien Shaun , Thomas Rajan , Wells Andrew
Published: January 2023

Naive CD8+ T cell responses are regulated by signals from antigen, co-stimulatory molecules, and cytokines such as IL-2. CD8 T cells produce IL-2 only transiently after activation, and subsequently become dependent upon CD4+ T cells for their differentiation. The mechanism by which CD8 cells extinguish autocrine IL-2 production is unclear. We find that Ikaros, a DNA binding protein that negatively regulates IL-2 production by CD4+ T cells, also controls autocrine IL-2 production by CD8+ T cells. In an in vitro model devoid of CD4-derived signals, co-ligation of TCR and CD28 led to initial activation and expansion of naive CD8+ T cells, but wild-type cells failed to differentiate into IFN-γ-producing CTL unless exogenous IL-2 was added. Conversely, naive CD8+ T cells with reduced Ikaros activity due to loss of one copy of the ikzf1 gene exhibited sustained autocrine IL-2 secretion and could differentiate in response to TCR and CD28 alone. The IL-2 produced by ikzf1+/null CD8+ cells was required for their CD4-independent differentiation, as neutralizing IL-2 Ab abrogated IFN-γ production, and was sufficient to drive the differentiation of co-cultured wild-type CD8+ cells in a cell-extrinsic manner. These data show that Ikaros negatively regulates autocrine IL-2 production by CD8+ T cells as means to ensure that their differentiation remains dependent upon CD4+ T cell help. Ikaros may therefore be a crucial regulator of autoimmunity and tumor immunity.
Signaling via neurokinin 1 receptor is necessary for the development of efficient Th1 and Th2 immunity (122.2)
Authors: Ho Chin , Sumpter Tina , Tkacheva Olga , Shufesky William , Morelli Adrian , Larregina Adriana
Published: January 2023

The outcomes of the immune response depend on a delicate interaction of the immune and nervous systems. Although pro- and anti-inflammatory neuropeptides are secreted in lymphoid and peripheral organs, and immune cells secrete neuropeptides and express their receptors, the role of these mediators on Th1 and Th2 biased immunity is lacking. It has been postulated that substance P and hemokinin-1, the prototypic pro-inflammatory neuropeptides, promote Th1 biased autoimmunity by agonistic binding of the neurokinin-1 receptor (NK1R). We hypothesized that agonistic binding of NK1R favors Th1 as well as Th2 mediated immunity. To test our hypothesis, we compared delayed type hypersensitivity responses following in vivo treatment of C57BL/6 wild type (WT) or NK1RKO mice with either Th1 (2-4-dinitrochlorobenzene) or Th2 (fluorescein isothiocyanate) sensitizers. We show that NK1RKO mice had impaired development of both Th1 and Th2 immunity. Our results were further confirmed in WT mice treated with either of the NK1R antagonists L733,060 or RP67,580 prior to sensitization. Mechanistic analyses demonstrated that NK1R signaling was necessary to promote acute inflammation in skin and skin-draining lymph nodes as well as for the recall of effector immunity following elicitation. We conclude that NK1R signaling is required for efficient development of both Th1 and Th2 effector immune responses.
Inflammatory cytokines in the pathogenesis of autoimmune type 1 diabetes (123.21)
Authors: Ramanathan Sheela , Bobbala Diwakar , Chen Xi Lin , Mayhue Marian , Leblanc Chantal , Tanaka Toshiyuki , Chen Yi-Guang , Ilangumaran Subburaj
Published: January 2023

Inflammatory cytokines are implicated in the pathogenesis of different autoimmune diseases like Type 1 diabetes, Rhumatoid arthritis and inflammatory bowel disease in humans as well as in animal models. IL-21, and IL-15 are pro-inflammatory cytokines and they exert their biological effects at various stages of the immune responses. We have shown previously that IL-21 can synergize with IL-15 or IL-7 to reduce the threshold of TCR signaling, while simultaneously increasing the production of effector cytokines such as TNFα and IFNγ. IL-21 maps to one of the shared autoimmune disease susceptibility loci in humans and mice. In Non Obese Diabetic (NOD) mouse that develops spontaneous autoimmune type 1 diabetes (T1D), IL-21 is a candidate gene in the Idd3 locus. C57BL/6 derived Idd3 allele protects NOD mouse from T1D. As IL-2 also maps to the same locus, the relative contribution of IL-2 and IL-21 to the disease process is not well understood. It has been shown previously that NOD mouse lacking the receptor for IL-21 does not develop autoimmune diabetes. Here we report that the absence of IL-21 protects the 8.3 TCR transgenic NOD mice from diabetes. Similarly we observed that NOD mice that lack the expression of IL-15 show reduced incidence of type 1 diabetes. Our results suggest that IL-21 and IL-15 may be implicated at different stages of disease development in the NOD mouse model.
CD3 zeta chain deficient T cells instigate multi-organ tissue inflammation (123.35)
Authors: Deng Guo-Min , Beltran Jessica , Mizui Masayuki , Terhorst Cox , Tsokos George
Published: January 2023

A population of T cells that lack the CD3 zeta (ζ) chain exists in systemic lupus erythematosus (SLE), rheumatoid arthritis, cancer, and infectious diseases. SLE is a chronic autoimmune disease characterized by high production of autoantibodies and multi-organ tissue inflammation. However, the role of CD3ζ chain-deficient T cells in the pathogenesis of these disorders remains unknown. Here we show the role of CD3ζ chain deficient T cells in multi-organ tissue inflammation. We found that CD3ζ -deficient mice spontaneously developed multi-organ tissue inflammation that can be promoted following the injection of poly: (ic) or allogeneic cells. Interestingly, a humoral response did not accompany the cellular response. CD3ζ chain deficient T cells were found defective in activation-induced apoptosis, and to display aggregated lipid rafts in which CD44, and CCR2 colocalized and to produce IFN-γ. Anti-IFN-γ treatment of CD3ζ deficient mice inhibited spontaneous multi-organ tissue inflammation. Promotion and resolution of lipid raft aggregation resulted in enhanced and decreased IFN-γ production suggesting a clear role of aggregated lipid rafts in the production of IFN-γ. Our results demonstrate that CD3ζ chain deficient T cells instigate multi-organ tissue inflammation by enabling lipid raft aggregation. Thus we demonstrate that decreased CD3ζ chain expression in autoimmunity, cancer and infection, contributes independently to inflammation and damage.
Regulation of T cell metabolism and autoimmunity by the orphan nuclear receptor ERRγ (123.47)
Authors: Yin Yiming , Perry Daniel , Perl Andras , Morel Laurence , Telarico Tiffany
Published: January 2023

Autoreactive T cells and abnormal cytokine production are key features of human and mouse systemic lupus erythematosus (SLE). The SLE susceptibility locus Sle1c2 is associated with CD4+ T cell hyperactivation, hyperproliferation and hyperproduction of IFNγ in the NZM2410 mouse model. Sle1c2 contains two genes, only one of which, Esrrg, is expressed in mouse CD4+ T cells. qRT-PCR and Western blot analysis showed that Esrrg is expressed in CD4+ T cells, albeit at a low level. Esrrg expression is significantly decreased in B6.Sle1c2 CD4+ T cells as compared to B6, and negatively correlates with T cell activation. In vitro, Esrrg expression is dramatically decreased after CD4+ T cells are stimulated with anti-CD3/CD28. These data suggest that Esrrg is required to maintain T cells in a quiescent state. Esrrg is a key regulator of cell metabolism. Consistently, Sle1c2 is associated with increased expression of genes involved in the glycolytic pathway, such as MCT4 and Hif1a, which have been previously associated with enhanced T cell effector functions. Sle1c2 is also associated with a decreased mitochondrial mass and functions, which is consistent with Esrrg playing a key role in these processes. Furthermore, treatment of CD4+ T cells with ERRγ agonist and reverse agonist affects T cell survival, proliferation, Foxp3 expression, and IL-2 and IFNγ production. Our study suggests that decreased Esrrg expression contributes to T cell autoimmunity through regulating cell metabolism.
The H1-isoform of the cell surface receptor Slamf6 suppresses expansion of PD-1+ OPN+ CD4+ T Follicular Helper cells in the lupus-prone B6.Sle1b mice. (123.5)
Authors: Keszei Marton , Detre Cynthia , Castro Wilson , Magelky Erica , Terhorst Cox
Published: January 2023

A major role for the Slamf6 gene in the pathogenesis of lupus-related autoimmunity has been revealed by the analysis of a number of congenic mutant mouse strains. Importantly, upon introduction of one copy of a BAC transgene expressing the protein isoform Slamf6-H1 markedly ameliorates T cell-dependent lupus in the Sle1b mouse, which lacks H1. Here we report that a PD-1+ subset of memory CD4+ T cells dramatically expands in Sle1b mice, but not in Sle1b mice, which express Slamf6-H1. Further analysis showed that this PD-1+ population mainly consists of CXCR5+ T follicular helper cells the expansion of which is prevented by Slamf6-H1 as well as by the absence of the adapter SAP. Surprisingly, these T follicular helper cells express the cytokine osteopontin, which is known to drive polyclonal B cell proliferation. As the human osteopontin gene have been linked to the development of SLE, the outcomes of the present studies could suggest novel therapeutic modalities for SLE patients.
Sexually dimorphic control of regulatory T cell function and autoimmunity protection through B7-H1 and estrogen signals (123.50)
Authors: Curiel Tyler , Kious Mark , Daniel Benjamin , Lin Pei , Thibodeaux Suzanne , Vadlamudi Ratna , Wierman Margaret , Turka Laurence , Zhang Ruan , Sharpe Arlene , Francisco Loise , Lishi Sun , Pandaswara Srilakshmi , Tekmal Rajeshwar , Hurez Vincent
Published: January 2023

CD4+CD25hi regulatory T cells (Tregs) mediate peripheral tolerance and defend against autoimmunity. Among immune sexual dimorphisms, autoimmunity is more common in females but underlying mechanism(s) are poorly understood. We show that B7-H1 signals dampen estrogen-driven Treg mTOR activation through estrogen receptor beta, reducing Treg function independent of PTEN. Poorly functional Tregs from B7-H1 deficient hosts appeared phenotypically normal and did not produce IL-17. B7-H1-deficient female mice (but not males or WT of either sex) were prone to anti-CTLA-4-induced autoimmunity. Autoimmune propensity was rescued by estrogen withdrawal or B7-H1-sufficient dendritic cell transfer. B7-H1-mediated effects on estrogen signals were also mediated by B or T lymphocytes or monocytes, and occurred during induced Treg generation, with no effect on differentiated Tregs. CD80 and PD-1 were not ligands for these effects nor was Treg B7-H1 expression required. Estrogen affected male Tregs in vitro and in vivo provided B7-H1 was blocked. Strikingly, B7-H1 and estrogen did not affect thymic Treg differentiation or function, suggesting differential control of natural Tregs and a potential role for induced Tregs in autoimmunity protection. Similar Treg results were seen in BL6 and Balb/c mice. We thus show a mechanistic basis for the female propensity to autoimmunity, and data are consistent with proposed roles for B7-H1 in female-predominant autoimmune disorders, particularly lupus.
A novel method for kinetic measurements of rare immune cell proliferation using Cellometer image-based cytometry (124.1)
Authors: Lin Bo , Chan Leo , Zhong Xuemei , Pirani Alnoor
Published: January 2023

Measuring cell proliferation is important for studying the effects of various treatments on cultured primary cells or cell lines. Current proliferation analysis methods employ flow cytometry for fluorescence detection of CFSE-labeled cells. However, conventional flow cytometers require a considerable amount of cells per reading, which becomes an issue for kinetic measurements with rare cell population due to the lack of samples for flow cytometric analyses at multiple time points during proliferation period. Here we report the development of a novel cell proliferation kinetic detection method for low cell concentration samples using the new Cellometer Vision image-based cytometry system. Since the Cellometer system requires only 20 µl of sample, cell proliferation can be measured at multiple time points over the proliferation period, whereas typically, flow cytometry is only performed at the end of the proliferation period. To validate the detection method, B1 and B2 B cells were treated with a B cell mitogen and proliferation was measured on day 1, 3, 5, and 6. To demonstrate the capability, B1 B cells were treated with a panel of TLR agonists and proliferation was measured on day 2, 4, 6, and 7. Cellometer was able to obtain proliferation results on each day, which were comparable to flow cytometry. This novel method allows for kinetic measurements of the rare cell samples such as B1 B cell, which has the potential to revolutionize kinetic analysis of cell proliferation.
A novel method for autophagy analysis using statistical microscopy (124.4)
Authors: Pugsley Haley , Hall Brian , Mordecai Scott , Friend Sherree , DeMarco Richard , Kong Raymond , George Thaddeus
Published: January 2023

Autophagy is a process in which normal cellular components that accumulate during growth and differentiation are degraded via the lysosome; it is a survival mechanism that reallocates nutrients from unnecessary processes to more vital processes in the cell. Basal levels of autophagy are usually low but can be up-regulated by numerous stimuli including starvation, physiological stress, pharmacological agents and infections. In addition, autophagy suppression has been associated with cancer, neurodegenerative disorders, infectious diseases and inflammation. During autophagy, cytoplasmic LC3 is processed and recruited to the outer membrane of forming autophagosomes, and cells undergoing autophagy can be identified by visualizing LC3 puncta by immunofluorescence microscopy. While manual microscopy allows visual per-cell identification of autophagy, objective statistical analysis of autophagy can be challenging with this method owing to its time-consuming and generally subjective nature. To overcome these problems, we used the ImgeStreamX imaging cytometry platform, which objectively collects and quantifies statistically large numbers of images per sample, to measure autophagy in K562 cells, a human CML line. In this study, we demonstrate a method for determining the best image-based parameter for quantifying LC3 puncta in these cells, and apply that parameter to large data sets to measure compound-induced autophagy in an objectively and statistically significant manner.
A simple and rapid method for the isolation of untouched human memory CD8 T cells (124.9)
Authors: Fairhurst Maureen , Posarac Vesna , McQueen Karina , Thomas Terry
Published: January 2023

Memory CD8 T cells are long-lived antigen-specific cells that persist after clearance of infection. Functionally, they are distinguished from naïve CD8 T cells by their less extensive signal requirement for activation and their ability to respond quickly to recall antigens and secrete a broad repertoire of cytokines. In addition, the two populations are phenotypically distinct, with naïve cells expressing CD45RA, and memory cells expressing CD45RO, a marker indicative of previous activation. Current protocols for the isolation of memory CD8 T cells are time-consuming and require the use of columns. We have developed a new kit for easy and rapid isolation of memory CD8 T cells from PBMCs by immunomagnetic, column-free cell separation (EasySep™). Non-CD8 T cells and CD45RA positive CD8 T cell subsets are targeted for depletion by bispecific tetrameric antibody complexes crosslinked to dextran-coated magnetic particles. The labeled cells are separated using an EasySep™ magnet and the desired fraction is poured off. The procedure is performed in 30 minutes and can be fully automated using RoboSep™. The mean enrichment purity and recovery of CD45RO+ CD45RA- CD8+ T cells is 86 ± 4, and 23 ± 11, respectively. The kit provides a simple and efficient means of isolating untouched human memory CD8 T cells that are ideal for studies in signal transduction, activation, cytokine expression, and response to infectious disease.
Allergen specific IgG augments Th2 mediated inflammation in the lungs through FcγRIII signaling and upregulation of IL-33 (125.1)
Authors: Tjota Melissa , Williams Jesse , Lu Tiffany , Byrd Tiara , Clay Bryan , Sperling Anne
Published: January 2023

Asthma is a chronic inflammatory disease of the lungs marked by mast cell activation, eosinophilia, and airway remodeling. It has been seen that primary sensitization to inhaled allergens leads to increased production of allergen specific IgE and IgG1. Several studies have shown a critical role for IgE in allergic lung disease, but it is unclear how IgG1 might contribute to the initiation and perpetuation of this disease. Previous work in our lab identified a key role for FcγRIII on dendritic cells (DC) in the development of optimal Th2-dependent airway inflammation. As FcγRIII is efficiently engaged by IgG1-immune complexes, we hypothesized that sensitized individuals could form allergen-specific immune complexes upon secondary exposure which could contribute to the augmented Th2 responses found in allergic asthma. We used two different models of immune complex mediated Th2 inflammation through either immune complex formation in vivo or local installation of preformed immune complexes. Both models showed that this process was mediated through FcγRIII. Utilizing both in vitro and in vivo approaches, we then identified IL-33 as a gene downstream of FcγRIII signaling on DCs. Notably, immune complex mediated Th2 inflammation in the lungs was decreased in ST2-/- mice with a specific loss of eosinophilia suggesting a critical role for IL-33. Our findings that previous sensitization can direct the response of DCs could provide a new paradigm for the development of Th2 inflammation.
Environmental exposures alter T cell differentiation and aggravate airway disease through the Aryl Hydrocarbon Receptor (125.6)
Authors: Mezrich Joshua , Zhang Xiaoji , Fechner John
Published: January 2023

Environmental exposures can aggravate airway disease. 10% of World Trade Center disaster responders developed asthma. Polycyclic aromatic hydrocarbon (PAH) levels were the highest ever reported. The Aryl Hydrocarbon Receptor (AHR) directs T-cell differentiation between FoxP3+ Regulatory T-cells and Th17 effector cells, and is the primary receptor for PAHs. Exposure to specific inhaled PAHs found in diesel exhaust particles (DEP), urban dust particles (UDP), and coal fly ash (CFA) has a direct effect on T-cell differentiation in an AHR-dependent manner, that enhances Th17 differentiation and hastens the development of asthma. Naïve CD4+ CD25- T cells from C57BL/6 mice were cultured under Th17 conditions. We utilized standard reference materials (SRM) representing PAHs from UDP, DEP, CFA in cultures and mixed lymphocyte cultures. Human epithelial cells were treated, and then examined for Epithelial to Mesenchymal Transition (EMT), in the presence of PBMC and SRMs. Finally we exposed lungs of mice in vivo to UDP intratracheally (IT). SRMs increased IL-2, IL-17, and IL-22 in the supernatant of naïve T-cells by bead array. SRMs increased IL-17 expression in MLCs. Both IL-17 and UDP were able to cause significant EMT. In lungs exposed to UDP, the AHR was activated and IL-17 was upregulated in tissue by qPCR. PAHs lead to a TH17 deviation in the lung through the AHR. This may play a role in aggravating airway disease in susceptible people, and will be a target for future treatment.