bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin E2 affects T cell responses through modulation of CD46 expression. (178.8)
Authors: Astier Anne , Kickler Karoline , Ni Choileain Siobhan , Stephen Jillian , Hafler David , Jabbour Henry
Published: January 2023

The complement regulator CD46 controls T cell activation and differentiation. CD46-mediated differentiation pathway is defective in several chronic inflammatory diseases, underlying the importance of CD46 in controlling T cell function and the need to understand its regulatory mechanisms. Using an RNAi-based screening approach in primary T cells, we have identified that two members of the G-protein coupled receptor (GPCR) kinase family were involved in regulating CD46 expression at the T cell surface. We have investigated the role of prostaglandin E2 (PGE2), which binds to GPCRs, in the regulation of CD46 expression and function. Conflicting roles of PGE2 in T cell functions have been reported, and the reasons for these apparent discrepancies are not well understood. We show that: i) addition of PGE2 strongly downregulates CD46 expression in activated T cells, ii) PGE2 differentially affects T cell activation, cytokine production and phenotype depending on the activation signals received by the T cells, iii) this was correlated with a distinct pattern of the EP2-EP4 PGE2 receptors induced, with EP4 being preferentially expressed upon CD46, and iv) addition of an EP4 antagonist could reverse the effects observed on cytokine production upon CD46 costimulation. These data demonstrate a novel role of the PGE2-EP4-GRK axis in CD46 functions, which might partly explain the diverse roles of PGE2 in T cell functions.
Induced regulatory T cell-derived interleukin 10 is sufficient for the cure of experimental colitis (179.19)
Authors: Schmitt Erica , Haribhai Dipica , Williams Jason , Jia Shuang , Aggarwal Praful , Yan Ke , Broeckel Ulrich , Simpson Pippa , Salzman Nita , Hessner Martin , Williams Calvin
Published: January 2023

Induced Treg (iTreg) cells are essential for tolerance. Although in vitro derived iTreg cells can be used therapeutically in preclinical models of autoimmune disease, their stability in vivo and their mechanisms of suppression have not been examined. Here, we used a treatment model of lymphopenia-associated colitis to examine the role of IL-10 in iTreg cell function. Monotherapy with IL-10-deficient iTreg cells slowed the onset of colitis and decreased colon infiltration, but did not resolve disease. Mice treated with WT iTreg cells in combination with IL-10-deficient natural Treg (nTreg) cells survived and gained weight, even though iTreg cells were numerically disadvantaged and comprised just ~20% of all Treg cells in treated mice. Notably, ~85% of the transferred iTreg cells lost Foxp3 expression (ex-iTreg) but retained a portion of the iTreg transcriptome which failed to limit their pathogenic potential. The TCR repertoires of iTreg and ex-iTreg cells exhibited almost no overlap, indicating that maintenance of the iTreg pool is based on antigen specificity. These data demonstrate a potent and critical role for iTreg cell produced IL-10 that can supplant the IL-10 produced by nTreg cells and compensate for the inherent instability of the iTreg population.
CD8+CD122+ regulatory T cells contain clonally expanded cells with identical CDR3 sequences of T cell receptor β chain (179.9)
Authors: Suzuki Haruhiko
Published: January 2023

We identified CD8+CD122+ regulatory T cells (CD8+CD122+ Tregs) and reported their importance in maintaining immune homeostasis. The absence of CD8+CD122+ Tregs has been shown to lead to severe systemic autoimmunity in several mouse models including inflammatory bowel diseases (IBD) and experimental autoimmune encephalomyelitis (EAE). Interleukin-10 (IL-10) is an important effector molecule for CD8+CD122+ Tregs to suppress conventional T cells. The T-cell receptors (TCRs) expressed on CD8+CD122+ Tregs recognize the target cells to be regulated. To build a step to identify the target antigen(s) recognized by TCRs of CD8+CD122+ Tregs, we compared the TCR diversity of CD8+CD122+ Tregs with that of conventional naïve and memory T cells in mice. We analyzed the use of TCR-Vβ in IL-10-producing population of CD8+CD122+ Tregs marked by high-level expression of CD49d, and found the significantly increased use of Vβ13 in these cells. Immunoscope analysis of the CDR3 region of the TCR β-chain revealed remarkable skewing in a pair of Vβ regions, suggesting the existence of clonally expanded cells in CD8+CD122+ Tregs. Clonal expansion in Vβ13+ cells was confirmed by determining the DNA sequences of the CDR3s. The characteristic TCR found in this study would become a clue of further study to identify the target antigen recognized by CD8+CD122+ Tregs.
Role of HMGB1 in TLR3-mediated inflammation (180.10)
Authors: Son Mina , Kim Eun-sook , Shin Jeon-Soo
Published: January 2023

Nuclear high mobility group box 1 (HMGB1), a DNA binding protein, belongs to the superfamily of HMG proteins and mediates structural and functional changes to chromatin. HMGB1 can be released either passively by necrotic cells or actively by stimulated cells. The extracellular role of HMGB1 as a cytokine has been intensively studied and shown to be important as a danger-associated molecular pattern protein. HMGB1 co-operates with other pro-inflammatory molecules to induce inflammation. HMGB1 forms complexes with various Toll-like receptor (TLR) ligands of LPS and CpG, enhancing their cytokine-inducing function. In this study, we demonstrated that HMGB1 binds to TLR3 both directly and indirectly in cooperation with poly(I:C), using immunoprecipitation and surface plasmon resonance assays. Exogenous HMGB1 treatment to HEK293/TLR3 cells was co-localized in intracytoplasmic endosomes containing TLR3 followed by NF-κB activation. In conclusion, HMGB1 is a novel ligand of TLR3 and is involved in TLR3-mediated inflammation.
Characterization of a human toll-like receptor 3 antibody designed to block ligand binding and ligand mediated signaling (180.16)
Authors: Singh Sujay , Chitta Sriram , Lee Hyun-Ku , Singh Gita , Rosenberg Jonathan
Published: January 2023

Toll-like receptors (TLRs) play key roles during pathogen invasion to initiate innate immune responses. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (microbial and endogenous) and transmits signals to activate NF-kappa B. TLR3 is restricted to acidic pH+ intracellular compartments of innate immune cells (viz immature DC). Ligand binding and formation of multimeric signal complexes, to activate down-stream signaling, are facilitated by acidic pH in these compartments. Mutational and co-crystallization studies determined the interaction of TLR3 & ligand binding at the molecular level and also individual residues important in ligand recognition and signal transduction. Besides, neutralizing antibodies were utilized as tools for better understanding of ligand binding and TLR3 signaling. In the present study, we report results of a TLR3 antibody which blocks ligand (poly IC) binding and its' effect on functional activity. We tested multiple TLR3 antibodies, generated through use of synthetic peptides designed to include various regions of protein and also the residues playing important role in ligand binding. The binding and blocking (ligand) activities were analyzed by flow cytometry using TLR3 transfectant cells in presence/absence of poly IC. We also tested blocking activity in a functional assay by measuring secreted alkaline phosphatase (SEAP) in cultures of TLR3-NF-kB-SEAP co-transfectants stimulated wih poly IC in the presence of antibody.
Genetic screen reveals CYLD as a regulator of the balance between inflammation and cell death (180.18)
Authors: Schworer Stephen , Smirnova Irina , Poltorak Alexander
Published: January 2023

During the innate immune response to infection, a critical balance exists between the production pro-inflammatory cytokines and immune cell death. Cell death through RIP1-mediated regulated necrosis is emerging as an important mechanism through which cells respond to certain viral pathogens. To determine novel regulatory mechanisms of this signaling pathway, we studied cellular responses to TLR agonists in evolutionarily divergent wild-derived mice (MOLF/Ei) in a forward genetic screen of TLR-induced necrosis. In contrast to C57BL/6 macrophages, which are susceptible to TLR-induced necrosis, MOLF macrophages are resistant to death induced through this pathway. Using an N2 panel and forward genetic analysis, we found a locus on chromosome 8 that confers resistance to TLR-induced necrosis. Through gene expression analysis, allelic bias studies, and siRNA knockdown in peritoneal macrophages, we concluded that a genetic difference in CYLD confers the differential susceptibility to TLR-induced necrosis between B6 and MOLF macrophages. This difference was revealed through analysis of the regulation of CYLD mRNA splicing following TLR stimulation. The molecular defect that results in greater survival of MOLF cells appears to be mutually exclusive splicing of CYLD mRNA in activated or quiescent cells. Through our use of wild-derived mice in forward genetic analysis, we were able to uncover a key function of naturally occurring CYLD splice isoforms in a physiologic setting.
Regulation of TLR-triggered innate immune response by ubiquitin-specific protease 25 (180.8)
Authors: Zhong Bo , Liu Xikui , Wang Xiaohu , Liu Xindong , Chang SeonHee , Li Hongxiu , Darnay Bryant , Lin Xin , Sun Shaocong , Dong Chen
Published: January 2023

Protein ubiquitination plays a critical role in Toll-like receptor (TLR) signaling and innate immunity. Although several E3 ubiquitin ligases have been identified downstream of TLRs, the deubiquitination regulation in innate immunity is poorly understood. Here, we identified ubiquitin-specific protease 25 (USP25) as a novel negative regulator for TLR signaling. Deficiency of USP25 potentiated TLR2- and TLR4-induced activation of NF-kB and MAPKs and pro-inflammatory cytokine expression. Consistently, Usp25-/- mice exhibited increased susceptibility to LPS-induced septic shock than the control littermates. USP25 was found recruited to the TLR4 signaling complex and associated with TRAF3 and TRAF6 after LPS stimulation. Notably, USP25 specifically removed cIAP2-induced K48-linked ubiquitin conjugation of TRAF3 and loss of USP25 resulted in enhanced K48-linked ubiquitination and accelerated degradation of TRAF3 after TLR2/4 activation. Our findings thus identify USP25 as a novel, critical negative regulator in innate immunity via regulating TRAF3 degradation during TLR2/4 activation.
A genome guardian role for RAG-mediated restriction of repair pathway choice (42.10)
Authors: Coussens Marc , Wendland Rebecca , Deriano Ludovic , Arnal Suzzette , Roth David
Published: January 2023

The V(D)J recombinase, which includes the RAG 1/2 proteins, retains broken DNA intermediates in a post-cleavage complex, restricting repair to the canonical nonhomologous end joining (NHEJ) pathway. Previously, we described a C-terminal RAG2 truncation mutant that ablates repair pathway restriction, allowing RAG-generated DNA double strand breaks (DSBs) to access alternative NHEJ, an error-prone pathway characterized by junctional microhomologies and associated with oncogenic chromosome translocations. In “core” RAG2 knock in mice, which lack RAG2’s dispensable C-terminus, V(D)J junctions show characteristics of alternative NHEJ and thymocytes display genomic instability. RAG2 core/core p53-/- mice rapidly succumb to thymic lymphomas bearing complex chromosome translocations involving antigen receptor loci. Because the C-terminus of RAG2 contains multiple regulatory motifs, it remained unclear whether pathway choice restriction per se contributes to genomic stability. We now show that deleting or altering a C-terminal acidic “hinge” region destabilizes the post-cleavage complex, allows repair by alternative NHEJ, and promotes genomic instability. These data identify a novel regulatory region in RAG2 and show that the post-cleavage complex plays a genome guardian role by restricting repair pathway choice.
Interaction of the humoral immune system with peptides presented by class I major histocompatibility (MHC) complexes (42.4)
Authors: Held Gerhard , Luescher Immanuel , Neumann Frank , Sester Martina , Guillaume Philippe , Hoth Markus , Pfreundschuh Michael
Published: January 2023

Antibodies usually bind to unprocessed antigens, while cytotoxic T cells react with peptides derived from intracellular antigens when presented by class I major histocompatibility (MHC) complexes. We screened human sera for antibodies reacting specifically with the influenza matrix protein (IMP) derived peptide (58-66) displayed by HLA-A*0201 complexes by ELISA. Among blood donors, high-titered HLA-A*0201/IMP (58-66) complex-specific IgG antibodies were detected in HLA-A*0201- females with a history of pregnancies. Extended analyses of specificity indicated that these antibodies interacted peptide-specific with the MHC complex. No antibodies were detected in HLA-A*0201+ female or male blood donors. In another cohort of 218 females on delivery, only HLA-A*0201- mothers had HLA-A*0201/IMP (58-66) antibodies, which were also detected in the cord blood of the newborns, demonstrating that HLA-A*0201/ IMP58-66 antibodies are produced in HLA-A*0201- mothers and enter the fetal blood system. Refolding the peptide IMP (58-66) with all HLA-A, -B and -Cw alleles of the mothers and newborns revealed, that these antibodies are allo-reactive and are binding in some cases to the peptide displayed by a MHC allele of an offspring. Therefore allo-MHC/IMP (58-66) antibodies might provide immunity in HLA-disparate pregnancies. These antibody responses specific to a peptide displayed by a class I MHC complex opens a new dimension of interactions between the cellular and humoral immune system.
Retinoic acid regulates α-galactosylceramide-mediated B cell activation and differentiation in CD19hi and CD19lo B cell subsets (42.9)
Authors: Chen Qiuyan , Ross A. Catharine
Published: January 2023

B cells are professional antigen-presenting cells and effector cells for antibody production. Besides the B-cell receptor (BCR), B cells may possess BCR coreceptors and other antigen receptors, such as CD1d. Previously, all-trans retinoic acid (RA) has been shown to promote the differentiation of B cells, and increase CD1d expression and CD1d-mediated antibody production in vivo. The current study focused on the characterization of B cell differentiation regulated by RA and the CD1d ligand, α-galactosylceramide (αGalCer). Treatment of purified murine B cells with αGalCer and RA enriched a CD19hi B cell subpopulation with higher CD1d expression; these cells exhibited a more differentiated phenotype in terms of expression of costimulatory molecules CD40 and CD86; surface IgG1; the plasmacytic marker CD138; and Fas/PNA, markers of germinal center formation. RA and αGalCer also regulated the gene expression of transcription factor Pax-5 and IRF-4, as well as γ1-germline transcript, activation-induced cytidine deaminase (AID, Aicda), genes that control B cell proliferation and plasmacyte differentiation. Moreover, whereas αGalCer increased the expression of CD40, CD86 and Pax-5 that are critical factors for B cell activation, RA mainly enriched CD138+ and Fas+/PNA+ cells that represent the later stage of B cell differentiation. Our data demonstrate that αGalCer and RA are potent B cell regulators that play complementary roles to promote B cell differentiation.
Cellular basis for clearance of the protozoan parasite Babesia microti in immunocompromised hosts (43.13)
Authors: Vannier Edouard , Silver Zachary , Wilson Carrie , Su Jia-Shyuan , Chiam Jake , Laurie Sonia , Telford Sam , Gelfand Jeffrey , Krause Peter , Wortis Henry
Published: January 2023

Babesia microti is the primary etiologic agent of human babesiosis, an emerging infectious disease in the US. In healthy individuals, babesiosis typically causes a mild flu-like illness or is asymptomatic. In immunocompromised patients, particularly those with B cell lymphoma and treated with rituximab, babesiosis is severe, persistent, and sometimes fatal. The mechanisms by which the host controls this obligate parasite of erythrocytes are poorly understood. In C57Bl/6 mice, parasitemia is marginal and transient. Lack of mature B cells due to igh6 deficiency does not hinder resistance. In rag1-/- mice, however, parasitemia is intense and persists for at least three months. This pattern also is observed in athymic (nude) mice, in mice lacking TCRαβ cells and in MHCii-/- mice. Surprisingly, parasitemia is intense but transient in cd4-/- mice. In the spleens of these mice, CD8+ T cells and CD4-CD8- T cells greatly expand during the course of infection. Parasitemia resolves in cd4-/- cd8-/- mice, but persists in cd4-/- igh6-/- mice. Likewise, parasitemia persists in cd4-/- mice depleted of B cells by repeated administration of anti-CD20. Resolution of parasitemia in cd4-/- mice is concomitant with the rise in circulating Babesia specific IgG, but does not require Fc receptors as demonstrated in cd4-/- fcegr1-/- mice. Our studies establish that, in the absence of CD4, B cells are critical for resolution of babesiosis. The role of complement and cytokines will be discussed.
Phagocytosis, Interferon-γ, IL-21, and CD4+ MHC class II-restricted T cells in resistance to Babesia microti infection (43.14)
Authors: Laurie Sonia , Vannier Edouard , Wortis Henry
Published: January 2023

Babesiosis is an emerging infectious disease caused by the hematoprotozoan parasite Babesia microti. The mechanisms of resistance to B. microti are not fully understood. The laboratory has developed a murine model of babesiosis with many of the features of the human disease that is used to examine the genetic determinants of resistance. The goal of the present study was to identify cellular processes that convey resistance to this parasitic infection. We established that the depletion of macrophages by chronic administration of clodronate liposomes resulted in early (0 to 30 days post-infection) hyperacute phase parasitemia (quantified as percent parasitized erythrocytes) but did not affect late phase (days 35-50) clearance of B. microti. In contrast, the absence of MHC-ii or the αβ T cell receptor prevents both early and late resistance. We find that early resistance requires the production of the cytokine IL-21. We also observed that the dual loss of CD4 and IL-21 results in the complete loss of resistance. We therefore hypothesize that IL-21 activates CD4+ T lymphocytes to synthesize IFN- γ that, in turn, activates macrophages to phagocytose and destroy B. microti infected red blood cells or B. microti itself. A model of resistance that incorporates these observations will be presented.
MyD88 acts directly on CD4+ T cells to promote resolution of Chlamydia muridarum genital tract infection (43.18)
Authors: Frazer Lauren , Zurenski Matthew , Mintus Margaret , Darville Toni
Published: January 2023

MyD88-dependent signaling is required for resolution of Chlamydia muridarum genital tract infection. We hypothesized that MyD88 was required for development of a robust chlamydiacidal CD4+ T cell response. Examination of chlamydia-specific cytokine production by CD4+ T cells in the genital tract revealed a 2-fold decrease in the frequency of CD4+ T cells producing IFNγ in the oviducts of MyD88-/- mice, but this difference was not statistically significant and was accompanied by an increased frequency of TNFα producing CD4+ T cells. In addition, the absolute number of CD4+ T cells in the oviducts, uterine horns, and lymph nodes did not differ between the strains. These data indicate that impaired trafficking of T cells to the genital tract is not responsible for the deficits in resolution of infection and that the defective chlamydiacidal capacity of MyD88-/- T cells may be IFNγ-independent. To determine if MyD88 plays a direct role in promoting T cell chlamydiacidal capacity, MyD88-/- or WT CD4+ T cells were transferred into Rag1-/- mice. Infection was significantly increased (p<0.01, two-way RM ANOVA) and prolonged (p<0.03 via log-rank test) when T cells lacked MyD88, with a median time to clearance of 23 days for mice receiving WT CD4+ T cells and 47 days for mice receiving MyD88-/- CD4+ T cells. MyD88 directly promotes the chlamydiacidal function of CD4+ T cells in vivo, but the precise requirement for this molecule remains to be determined.
IL-25 mediates the induction of IL-9-producing T cell responses against T. spiralis infection (43.20)
Authors: Angkasekwinai Pornpimon , Srimanote Potjanee , Wang Yui-Hsi , Sakolvaree Yuwaporn , Pattanapanyasat Kovit , Chaicumpa Wanpen , Chaiyaroj Sansanee , Dong Chen
Published: January 2023

IL-9-producing T cells were shown to be important in immunity against helminth infection, however, the occurrence and regulation of this distinct cell subset during T. spiralis infection remain elusive. Here, we showed that the occurrence of both IL-9-producing T cells (Th9 cells) and Th2 cells increased significantly at early stage of infection. While the frequency of induced Th2 cells within mesenteric lymph node sustained, Th9 cells declined sharply after 14 days of infection. Notably, we observed that the transient expression of IL-25 transcript preceded those of IL-9 and IL-4 genes in the intestine during T. spiralis infection. Administration of anti-IL-25-neutralizing antibody into T. spiralis-infected mice resulted in the reduction of antigen specific Th9 cell development, which was correlated with the increased worm burden. In addition, the expression of intestinal IL-9-regulated genes, including mast cell and paneth cell-specific genes such as mast cell protease 1, and angiogenin 4 (Ang4), cryptdins, and phospholipase A2 were markedly reduced in the intestine of T. spiralis-infected mice after IL-25 blockade, but strongly up-regulated after IL-25 treatments. Our study reveals a temporal expression pattern of Th9 development during helminth infection and suggests that IL-25 may play key roles in regulating the induction of protective IL-9 immune response against parasitic infection.
Experimental tuberculosis in the humanized mouse (43.28)
Authors: Endsley Janice , Calderon Veronica , Valbuena Gustavo , Goez-Rivillas Yenny , Judy Barbara , Johnston Rebecca , Estes Don , Cirillo Jeffrey
Published: January 2023

Currently there is no animal model of HIV/Mtb co-infection, limiting our understanding of how HIV-induced defects in immune function promote the dramatic susceptibility to Mtb. The humanized mouse is contributing to discoveries relevant to HIV/AIDS, but the model has not been further developed for opportunistic infection. The goal of this project was to develop the humanized mouse as a small animal model of Mtb infection to determine the potential use for HIV/Mtb co-infection research. NOD-SCID/γcnull mice were engrafted with human fetal liver and thymus tissue, and injected intravenously with CD34+ fetal liver cells. At 12 weeks following engraftment in this model we observe that on average, 40% of the total leukocytes in mouse peripheral blood are human CD45+ cells. These human leukocytes include human T cells (CD4 and CD8), as well as NK cells, and antigen presenting cells (macrophages, DC). Importantly, the circulating human T cells and NK cells respond to specific antigen and/or positive stimuli as measured by proliferation and expression of effector molecules (e.g. IFN-γ, granulysin). Animals infected intranasally with Mtb have progressive bacterial infection in the lung, dissemination of bacteria to spleen and liver, and formation of organized granulomatous lesions at sites of infection. The results demonstrate the utility of the humanized mouse as a model of experimental TB and suggest promise as a small animal model of HIV/Mtb co-infection.
Abnormal distribution of peripheral B lymphocyte subpopulations in chronic human Trypanosoma cruzi infection (43.7)
Authors: Postan Miriam , Fernandez Esteban , Gonzalez Mariela , Hernandez Vasquez Yolanda , Sirena Natalia , Moran Maria , Ledesma Oscar , Olivera Gabriela
Published: January 2023

In previous studies we have shown that human chronic T. cruzi infection is associated with abnormalities of CD8+ and CD4+ T cell compartments, probably as a consequence of chronic antigenic stimulation by persisting parasites. In this study, we measured the levels of circulating B cell subsets in 25 patients chronically infected with T. cruzi and 19 non-infected controls (mean age±SD=41.0±12.2 and 34.9±10.6 y respectively). The results showed that T. cruzi-infected patients had higher percentages of CD19+CD27-IgD+ (naïve; mean%±SD=54.1±16.1) and lower percentages of CD19+CD27+ (Ag-experienced; mean%±SD=35.8±16.5) B lymphocytes compared to controls (mean%±SD=33.8±15.8 and 55.7±18.0, respectively, p<0.05), with similar levels of CD69 or CD86 activation markers. The observed memory B cell reduction comprised switched (IgD-) and no-switched (IgD+) subsets (mean%±SD=17.9±11.2 and 17.9±12.6, respectively) in relation to controls (mean%±SD=30.9±17.8 and 24.8±16.4, respectively; p<0.05). The proportion of CD19+CD27+CD138+ plasma cells in patients was lower than in the controls (mean%±SD=5.0±3.6 and 26.4±27.1 respectively; p<0.05), with no differences in CD19+CD10+ cell levels (immature; mean%±SD=5.6±4.7 and 4.3±3.3, respectively). The accumulation of naïve and loss of memory B cell and plasma cell subsets in chronic Chagas disease patients indicate that T. cruzi infection affects the phenotypic profile of the peripheral blood B cell-compartment. Support: FONCyT PICT 2007 N°1732
TNF-alpha modulates alveolar bone repair under homeostatic conditions in mice (44.1)
Authors: Garlet Gustavo , Vieira Andreia , Repeke Carlos Eduardo , Francisconi Carolina , Silveira Elcia , Assis Gerson , Taga Rumio , Campanelli Ana Paula , Trombone Ana Paula
Published: January 2023

Cytokines have been implicated in the pathogenesis of bone diseases, but its role in bone repair process remains unknown. Our objective was to characterize the role of TNF-alpha in alveolar bone healing after tooth extraction in mice. Following the extraction of the right upper incisor of C57Bl/6(WT) and TNFp55KO strains, the maxilla containing the remaining alveolus was collected for histomorphometric and molecular (RealTimePCR) analysis. In WT mice the initial formation of clot was followed by the transient inflammatory infiltrate and a granulation tissue, gradually substituted by bone. In WT mice TNF-a mRNA was detected with an initial peak at 7 days, followed by a decreasing expression over time and a second peak at 28 days. Bone markers expression presented an initial high expression of COL-I, followed by CBFA-1 and ALP and subsequently by OCN and PHEX. TNFp55KO strain presented a delayed angiogenesis and osteogenesis processes, increased inflammatory reaction, despite of a decrease in CXCL1, CCL-2 and CCL3 levels; and increased COL-I mRNA levels and decreased CBFA-1, ALP, OCN and PHEX expression. The results presented here demonstrate that the absence of TNF-alpha interferes in alveolar bone repair through mechanisms that involve the modulation of inflammatory cell migration and osteogenic markers expression during the course of alveolar bone repair.
Different GATA factors dictate CCR3 transcription in a cell type-specific manner of allergic inflammatory cells (44.10)
Authors: Chung Il Yup , Kong Su-Kang , Kim Byung Soo , Uhm Tae Gi , Hwang Sae Mi , Kang Jin Hyun
Published: January 2023

CCR3 is expressed in prominent allergic inflammatory cells, including eosinophils, mast cells, and Th2 cells, which preferentially express GATA-1, GATA-2, and GATA-3, respectively. We have previously demonstrated GATA-1-mediated CCR3 transcription with functional mapping of a GATA element in the regulatory region of CCR3 gene. Herein, we investigated whether GATA factors other than GATA-1 play a major role in CCR3 transcription in these cell types. A CCR3 reporter plasmid, but not a mutant CCR3 reporter lacking the functional GATA element, was activated in all three cell types, EoL-1 eosinophilic cells, HMC-1 master cells, and Jurkat T cells, suggesting a critical involvement of the GATA element regardless of cell types expressing different GATA factors. Knockdown assay showed that siRNAs directed to GATA-1 and GATA-2 significantly reduced CCR3 transactivation with an additive effect in EoL-1 and HMC-1 cells, while GATA-3 siRNA exclusively abrogated CCR3 transcription in Jurkat cells. In parallel, EMSA analysis showed that the functional GATA element preferentially bound GATA-1, GATA-2, and GATA-3 with nuclear extracts from EoL-1, HMC-1, and Jurkat T cells, respectively. These results highlight that different GATA factors are engaged in CCR3 transcription in a cell type-specific fashion, depending on the major allergic inflammatory cells.
Analyzing the in vivo contribution of G-protein coupled receptors for naive B cell trafficking in lymph nodes (44.23)
Authors: Stein Jens , Coelho Fernanda , Soriano Silvia , Natale Daniela , Swoger Jim , Mayer Juergen , Chai Qian , Hons Miroslav , Pieczyk Markus , Zerwes Hans-Guenter , Junt Tobias , Sailer Andreas , Ludewig Burkhard , Sharpe James , Figge Marc Thilo
Published: January 2023

B cell motility within lymphoid tissue is precisely orchestrated by G-protein-coupled receptors (GPCRs) of the chemokine receptor family and other GPCRs, including the recently described 7α,25-dihydroxycholesterol receptor Ebi2. Here, we performed an exhaustive twophoton microscopy (2PM) analysis of the contribution of the chemokine receptors CXCR4, CXCR5 and CCR7, as well as Ebi2, during the dynamic migration of naïve B cells inside peripheral lymph nodes (PLNs) and the kinetics of B cell accumulation from the T cell area in B cell follicles. Using the intravital 2PM model of murine popliteal PLNs paired with genetic and pharmacological inhibition of GPCRs and their ligands, we observed a non-redundant role for CXCR5 for dynamic B cell motility in the T cell area and B cell follicles, while the functional absence of CXCR4 and CCR7 did not influence B cell migration parameters. Although CXCR5 was central for efficient B cell accumulation in B cell follicles, we did not find evidence for directed migration of T cell area-borne B cells towards B cell follicles, supporting a chemokinetic, and not chemotactic, role for the CXCR5 ligand CXCL13 during this process. Lack of Ebi2 confined B cells to the central part of B cell follicles, resulting in higher average migration velocities as compared to wild type B cells. In summary, our data provide novel insight into the contribution of GPCRs during the dynamic migration of naïve B cells in non-inflamed and inflamed PLNs.
The role of interleukin (IL)-22 on the proliferation of human keratinocyte cell line, HaCaT by Ultraviolet B (UVB) irradiation (44.27)
Authors: Kim Yejin , Bae Seyeon , Kim Hyemin , Yu Yeonsil , Kim Hang-Rae , Hwang Young-il , Kang Jae Seung , Lee Wang Jae
Published: January 2023

Interleukin 22 (IL-22), a member of IL-10 family, is a potent mediator of inflammatory responses. It is produced by activated CD4+ T cells and natural killer (NK) cells, but its receptor expression is restricted to nonhematopoietic cells in the skin, pancreas, intestine, liver, lung and kidney. IL-22 suppresses IL-4 production from Th2 cells and induces acute phase proteins in liver and pancreas. It is recently found that IL-22 plays a critical role in the maintenance of epidermal homeostasis by controlling cell cycle of keratinocytes. Therefore, we investigated that role of IL-22 on the proliferation of UVB-irradiated human keratinocytes, HaCaT. First, we found the increase of IL-22 mRNA expression was increased in HaCaT by UVB irradiation (150J/m2). When UVB-irradiated HaCaT was cultured in the presence of recombinant IL-22, the suppressed proliferation of HaCaT by UVB was rescued. It was closely related with the progression of cell cycle in UVB-irradiated HaCaT. We confirmed that the hyperproliferation and cell cycle progression of UVB-irradiated HaCaT by the addition of activated T cells or its culture supernatant. Taken together, IL-22 plays a critical role in the deterioration of epidermal hyperplasia by UVB irradiation. And it suggests that UVB-induced skin inflammation could effectively be controlled by the regulation of IL-22 production and its action on keratinocytes.
Visualization of IL-22 in mouse intestinal inflammation with an IL-22 tdTomato reporter mouse Wei Shen1, Wenqing Li1, Lionel Feigenbaum2, Julie A. Hixon1 and Scott K. Durum1. 1 Laboratory of Molecular Immunoregulation, CIP, CCR, NCI. 2 Laboratory Animal Sciences Program, CCR, NCI, Frederick MD. (44.5)
Authors: Shen Wei , Li Wenqing , Feigenbaum Lionel , Hixon Julie , Durum Scott
Published: January 2023

Interleukin (IL) 22 is produced by innate and adaptive immune cells and has activities in host defense, barrier function, tissue repair and inflammation. Previous studies of IL-22-producing cells have relied on ex-vivo restimulation, a method which assays the capability of cells to produce the cytokine, but not the actual production in vivo. To visualize IL-22 producing cells during immune responses in vivo, a bacterial artificial chromosome (BAC) was modified to introduce the fluorescent reporter gene (tdTomato) into the Il-22 locus. In vitro, the tdTomato reporter was visualized in splenic T cells cultured under Th17 conditions, and in cells from lamina propria following stimulation with IL-23. In colitis induced by either DSS or T cell (CD4+CD45RBhigh) transfer, intestinal intraepithelial cells were isolated and showed expression of the IL-22 reporter by quantitative real-time PCR and flow cytometry analysis. Visualized in histological sections, most of the tdTomato-positive cells in these induced models of inflammatory bowel disease were first present in the mesenteric lymph nodes, and then accumulated inside lamina propria of distal small intestine and colon tissues. In conclusion, this novel IL-22 reporter was expressed in the intestine and draining lymph nodes in several colitis models and will be useful in identifying the cell types, their site of commitment and sites of production of this key cytokine in homeostasis and models of infection of intestine and lung.
Interleukin-22 drives endogenous thymic regeneration in mice (44.6)
Authors: Dudakov Jarrod , Hanash Alan , Jenq Robert , Young Lauren , Ghosh Arnab , Singer Natalie , West Mallory , Smith Odette , Holland Amanda , Tsai Jennifer , Boyd Richard , van den Brink Marcel
Published: January 2023

Thymic regeneration is a crucial function that allows for renewal of immune competance after stress, infection or immunodepletion, however the mechanisms governing this rejuvenation remain poorly understood. Here we detail a framework of endogenous thymic regeneration centred on IL-22, a recently identified cytokine primarily implicated in maintenance of epithelial barrier function. Although IL-22 was redundant for steady-state thymopoiesis, thymic recovery was impaired in IL-22-deficient mice and intrathymic levels of IL-22 were significantly increased in WT mice following thymic damage. IL-22, which signalled through thymic epithelial cells and promoted their proliferation and survival, was upregulated by radio-resistant RORγ(t)+NKp46-CCR6+ lymphoid tissue-inducer cells after thymic injury under the control of dendritic cell-derived IL-23 and triggered by the depletion of CD4+CD8+ double positive (DP) thymocytes. Importantly, administration of IL-22 enhanced thymic recovery following irradiation damage. These studies reveal a network of endogenous thymus regeneration where 1) the depletion of DP thymocytes triggers 2) upregulation of IL-23 by DCs that induces 3) the production of IL-22 by thymic LTi. This cascade of events leads to regeneration of the epithelial microenvironment and, ultimately, to rejuvenation of thymopoiesis; presenting an innovative strategy for improving immune competence in patients whose thymus has been damaged from infection or cytoreductive therapy.
Chemokine guidance of central memory T cells is critical for anti-viral recall responses in lymph nodes (44.8)
Authors: Sung Jung Hwan , von Andrian Ulrich
Published: January 2023

A defining feature of vertebrate immunity is the capacity to acquire immunological memory against recurrent pathogens. During a viral infection, virus-specific memory T cells accelerate anti-viral effector responses and promote faster viral clearance than naive T cells (Tn), but the mechanism for this enhanced protection has been unclear. To address this question, mice were subcutaneously injected with either live virus or antigen (Ag)-pulsed dendritic cells (DCs) and the response of Tn and central memory T cells (Tcm) was visualized in draining lymph nodes (LNs). Upon challenge with Ag-pulsed DCs that carried Ag to deep T cell area, Tn and Tcm interacted similarly with Ag-pulsed DCs. Upon viral challenge, however, Tcm relocalized much more efficiently than Tn toward regions of initial viral infection in the medullary, interfollicular and subcapsular area. This redistribution depended upon chemokine receptor CXCR3 and occured in two sequential steps driven by distinct cytokines and chemokines. Initially, type I interferon-induced CXCL10 promoted peripheralization of Tcm independently of Ag specificity. Subsequently, upon encountering cognate Ag, Tcm produced interferon-γ, which in turn induced a second CXCR3 ligand, CXCL9. Upon viral infection, in vivo recall responses by CXCR3-deficient Tcm were markedly compromised, whereas in vitro responses were intact, suggesting that CXCR3-dependent Tcm relocalization is essential for intranodal anti-viral Tcm function.
Inhibition of iNKT cells by adenosine A2A receptor activation in patients with sickle cell disease (45.1)
Authors: Linden Joel , Lin Gene , Neuberg Donna , Nathan David , Field Joshua
Published: January 2023

Invariant natural killer T (iNKT) cells comprise a minor subset of lymphocytes that are activated by CD1d-restricted lipid antigens produced by pathogens or by the host in response to ischemia reperfusion-injury (IRI). Sickle cell disease (SCD) causes IRI due to chronic widely disseminated vaso-occlusion. IRI and SCD cause iNKT cell activation and release of IFN-γ that triggers an inflammatory cascade with recruitment and activation of other leukocytes to propagate tissue inflammation and injury. In mice with SCD, deletion of iNKT cells or inhibition of their activation with anti-CD1d antibodies or adenosine A2A receptor (A2AR) agonists inhibits tissue inflammation and injury at steady state or during evoked disease exacerbations. People with SCD have increased numbers of a subset iNKT cells that express cellular activation markers including activated NFκB (phospho-p65). The activated iNKT cell-subset concordantly displays elevated A2ARs that cause activated iNKT cells to become hypersensitve to inhibition by adenosine. Infusion of very low doses (1.5 nM in blood) of the A2AR agonist regadenoson into SCD patients results in no cardiovascular effects, but a rapid inhibition of iNKT cell NFκB and a reduction in A2AR immunoreactivity. We plan to initiate a clinical trial to determine if regadenoson infusion reduces the severity and duration of vaso-occlusive pain crises or acute chest syndrome in SCD patients.
INF-γ production of NK cells is suppressed in cancer patients. (45.10)
Authors: Lee Hyo Jun , Koh Jiae , Kim Jongsun
Published: January 2023

Natural killer (NK) cells are the innate immune system that play an important role in killing viral infected and transformed cancer cells by direct cell to cell interaction. NK cells also secrete cytokines such as interferon (IFN)-γ and TNF-α which primes subsequent adaptive immune responses. However, tumor cells employ many different strategies to evade NK cells. Moreover, tumor cells secrete molecules to directly inhibit the function of immune cells of both innate and adaptive immunity. Here, we designed to investigate whether NK cells from cancer patients exhibit an altered ability to produce INF-γ. The results showed that peripheral blood mononuclear cells (PBMC) and purified NK cells of the cancer patients had suppressed ability to produce IFN-γ. Therefore, we propose that impaired IFN-γ production in peripheral blood mononuclear cells and in purified NK cells reflects immunosuppression and inadequate antitumor immune response in cancer patients. Thus, the measurement of INF-γ producing ability of NK cells in clinical blood samples may allow to assess the impact of immunosuppression on these effector cells, and this information may be helpful in understanding cancer and pathogenesis.
Human BDCA3+ dendritic cells (mDC2) recognize HCV-infected hepatoma cells and produce IFN-λs (45.14)
Authors: Zhang Shuye , Kodys Karen , Szabo Gyongyi
Published: January 2023

IFN-lambda (IFN-λ) including IL-28a, IL-28b and IL-29 are found closely associated with outcome of HCV-infected patients, however, the mechanism of their induction and cellular sources during HCV-infection are poorly understood. Using co-cultures between normal human peripheral blood mononuclear cells (PBMCs) and JFH-1 HCV virus infected Huh7.5 (HCVcc/Huh7.5) cells, we found that human PBMCs recognized HCVcc/Huh7.5 cells and in response produced all three types of IFNs (IFN-α, IFN-γ and IFN-λ). We determined that a rare dendritic cell (DC) subset, the mDC2 cells (also referred to as CD141+ myeloid DCs), were the major producers of IL-28 and IL-29 in co-cultures with HCVcc/Huh7.5 cells. TLR3 and not TLR7, 8 or 9 stimulation induced IL-28 and IL-29 in mDC2 and endosomal maturation was required for IFN-λ induction by HCVcc/Huh7.5 cells. We further revealed that mDC2 is activated through a cell-cell contact and endocytosis mediated pathway in response to HCVcc/Huh7.5 cells. Finally, we found that IFN-α and IFN-λ production was inter-related as IFN-λ augmented IFN-α production from pDCs and IFN-α significantly increased IFN-λ production. In conclusion, our novel data identifies mDC2 as a major source of IFN-λs in human PBMCs in response to HCV-infected cells and implies that mDC2 is activated through the TLR3 pathway. These observations indicate human DCs can efficiently initiate immune response against HCV infection and highlight the role of IFN-λ in HCV-infection.
Rapid activation and migration of lymph node resident dendritic cells in response to Influenza vaccination. (45.18)
Authors: Woodruff Matthew , Carroll Michael
Published: January 2023

While the existence of lymph node resident dendritic cell (LNDC) populations is widely acknowledged, little is known about their contribution to the generation of an active immune response. Following up on earlier studies describing the importance of LNDCs in humoral immunity, we have interrogated the role of these cells in response to lymph borne antigen. Using UV-inactivated influenza as a model, we have observed direct capture of viral antigen by LNDC populations within 10 minutes of viral injection. Live imaging of viral capture using multi- photon microscopy reveals novel global activation of LNDCs within 60 minutes of viral arrival. Four-dimensional tracking, as well as whole-node fluorescent reconstruction, suggests a mass migration of LNDCs from the T cell cortex into the medullary compartment to areas of high antigen density, likely in response to innate signaling from the node periphery. These data indicate a novel mechanism whereby LNDCs rapidly respond to innate signaling through activation and homing to areas of high antigen density. We believe that this event may represent an early link between innate and adaptive immunity whereby activated DCs within the draining node have access to antigen well prior to the described migration of skin resident DCs from the periphery. We expect that a better understanding of these responses will be highly relevant to both lymph borne diseases, as well as future vaccine development.
T-cell education of dendritic cell responsiveness (45.2)
Authors: Abdi Kaveh
Published: January 2023

LPS-activated Dendritic Cells (DCs) are thought to follow a set program: first secreting inflammatory cytokines (e.g. IL 12p75) and then becoming refractory to further stimulation (i.e. “exhausted”). Here we show that these concepts need to be reconsidered. LPS-activated mouse DCs produce ample IL-12p40, but no p75 without additional stimulating signals from T cells or their products (i.e. IFN-γ, CD40L plus GM-CSF and IL-4). The prevailing view that LPS induces the production of p75 is largely due to the fact that many early studies used detection antibodies that do not distinguish between p40 and p75; or used DC populations containing small numbers of contaminating T cells. Second, although LPS-stimulated DCs do indeed lose their responsiveness to LPS about 24 hours after stimulation, they nevertheless remain perfectly capable of making inflammatory cytokines in response to signals from activated T cells, and to a combination of CD40-L plus soluble T-cell products. In addition, far from being rigidly programmed by the original activating stimulus, the DCs retained sufficient plasticity to respond differentially to interactions with TH0, TH1, TH2 and TH17 T cells, showing that T cells can change the subsequent activity of the DCs with which they interact. These data indicate that DCs are not programmed to respond to stimulation with a pre-determined series of events, but remain sensitive to their environment and can change their differentiation accordingly.
SLC11A1 expression in innate lymphocytes (N)RAMPs up activation (45.5)
Authors: Hedges Jodi , Snyder Deann , Jerome Maria , Holderness Jeff , Jutila Mark
Published: January 2023

SLC11A1 is a divalent ion transporter also known as natural resistance-associated macrophage protein 1 (NRAMP1) and formerly Bcg/Lsh/Ity locus, as mutations to this locus in mice result in susceptibility to Mycobacterium bovis (BCG), Leishmania and Salmonella infections. SLC11A1 was thought to be exclusively expressed in monocyte/macrophages and dendritic cells and to function in cell activation through iron-dependent interference with protein tyrosine phosphatase (PTP) activity. Expression of functional SLC11A1 in vivo has been consistently associated with increased IFNγ expression. We have detected SLC11A1 protein in the majority of γδ T cells, in NK cell subsets and in CD3+CD45RO+ T cells from cattle and humans. SLC11A1+ lymphocytes were more prone to activation and IFNγ expression than were SLC11A1- cells. Chemical blockade of PTPs and iron excess induced lymphocyte activation, underscoring their capacity for activation by PTP inhibition and a potential role for iron, the primary SLC11A1 substrate. MOLT14, a naturally SLC11A1- human γδ TCR+ cell line was transfected to express SLC11A1 which rendered the cells more prone to activation. Commonly used inbred laboratory mouse strains express a non-functional SLC11A1 protein, thus, innate T cell activation may be underestimated in models using these mice. These data support a novel role for SLC11A1 in activation of innate lymphocytes that are important early sources of IFNγ.