bluuebunny/crossref_metadata_2025 huggingface dataset:
The dysfunction of the E3 ubiquitin ligase TRIM21 in systemic lupus erythematosus (HUM7P.301)
Authors: Kamiyama Reikou , Yoshimi Ryusuke , Tsukahara Toshinori , Ueda Atsuhisa , Takeno Mitsuhiro , Ozato Keiko , Ishigatsubo Yoshiyuki
Published: January 2023

Although the increased expression of type I interferon (IFN)-inducible genes, called “IFN signature”, has been suggested to have important roles in pathogenesis of systemic lupus erythematosus (SLE), its mechanism still remains unclear. Recent studies in vitro or with mice suggest that TRIM21, an autoantigen also called Ro52 or SSA1, is involved in the regulation of type I IFN production as an E3 ubiquitin ligase for IFN regulatory factors (IRFs). Here, we investigated the pathological role of TRIM21 in SLE. We collected peripheral blood mononuclear cells (PBMCs) from 20 patients with SLE and 24 healthy controls (HC). The mRNA levels of TRIM21 and other type I IFN-inducible genes were higher in PBMCs from patients with SLE as compared to HC. Thus, TRIM21 mRNA levels correlated positively with other type I IFN-inducible genes. On the other hand, type I IFN mRNA levels showed a negative correlation with TRIM21 mRNA levels in HC but not in patients with SLE. Consistent with higher TRIM21 mRNA levels, TRIM21 protein levels were higher in SLE patients as compared to HC. After treating with MG-132, the protein levels of IRFs were increased in PBMC from HC, but not in the PBMC from patients with SLE. These results suggest that dysregulation of TRIM21 to function as an E3 ubiquitin ligase for IRF family may be associated with the increased expression of type I IFNs in SLE.
Aberant expansion of CXCR5+ memory CD4 T cells in SLE patients (HUM7P.307)
Authors: Choi Jinyoung , Kim Sang Taek , Kang Insoo , Craft Joe
Published: January 2023

Autoreactive B cells in SLE undergo autoantigen selection, suggesting a requirement for germinal center follicular helper T (Tfh) cells in their maturation. However, evidence for dysregulation of Tfh cells in SLE and their potential contribution to disease remains unclear. Recently, blood CXCR5+ CD4 T cells, a heterogeneous pool consisting of functionally distinct Th1-, Th2-, and Th17-like subsets, have been proposed to be the circulating (blood) memory Tfh cells. We hypothesized that expanded CXCR5+ memory cells in the blood of human lupus patients promote B cell helper function reflecting the abnormal T-B cell responses in secondary lymphoid organs. We have characterized such cells in SLE patients by flow cytometry and T-B coculture studies. SLE patients had significant expansion of CXCR5+ICOShiPD-1hi CD4 T cells compared to controls. Such cells were Bcl6-, but robustly expressed IL-21 with a portion Ki-67+, indicating their functional activity. The blood cells were capable of providing blood-born memory B cells with survival and differentiation signals to secrete isotype switched Igs, including anti-nuclear antibodies. We speculate that therapies that alter T-B collaboration in lupus will abrogate their expansion, accompanied by reduced autoantibody titers and improved disease activity in SLE patients. Our results suggest that aberrant T-B collaboration in lupus is critical to disease pathogenesis and its blockade is likely to be important therapeutically.
Use of humanized mouse models for the study of idiopathic CD4 lymphocytopenia (HUM7P.313)
Authors: Perez-Diez Ainhoa , Liu Xiangdong , Sheikh Virginia , Sereti Irini
Published: January 2023

Patients with Idiopathic CD4 Lymphocytopenia (ICL) have low circulating CD4 T-cell counts and develop opportunistic diseases and papillomavirus-related dysplasia. The etiology of ICL is unknown but the low numbers of CD4 T-cells could be explained by decreased thymic production, increased cell death, and/or increased migration or retention of the T cells in secondary lymphoid organs or tissues. To distinguish between these three possibilities we set up a humanized mouse model by transferring either peripheral blood mononuclear cells (PBMC), or Hematopoietic Stem Cells (HSC) from ICL patients or healthy controls (HC) into immune-compromised mice. First, we transferred HC cells into three different strains of mice to identify the most efficient and reproducible host for the human cells. We found that NOD-RAGKO-γcKO (NRG) mice represented the best model, as one to four weeks after transferring PBMC into NRG mice, we reproducibly found human T cells in blood, spleen, lymph node and bone marrow with no graft versus host disease. We next transferred PBMC from either HC or ICL patients into NRG mice to compare survival, expansion, phenotype and migration properties of the human cells. Preliminary results show that mice that received ICL cells developed lymphocytopenia comparable to ICL patients. These data encourage further use of this humanized mouse model to identify the etiology and pathogenesis of ICL and perhaps other human immune-deficiencies that affect T-cell homeostasis.
Expression and function of a novel isoform of Sox5 in malignant B cells (HUM7P.314)
Authors: Edwards Shanique , Desai Anand , Liu Yan , Moore Carissa , Xie Ping
Published: January 2023

Using a mouse model with the tumor suppressor TRAF3 deleted from B cells, we identified Sox5 as a gene strikingly up-regulated in B lymphomas. Sox5 proteins were not detected in normal or premalignant TRAF3-/- B cells even after treatment with B cell stimuli. We cloned the full-length cDNA of Sox5 from B lymphomas spontaneously developed in 4 different individual B-TRAF3-/- mice. Surprisingly, we found that the sequence of Sox5 expressed in TRAF3-/- B lymphomas represents a novel isoform of Sox5, which has not been previously reported. This new isoform of Sox5, Sox5-BLM, contains a 35 aa deletion in the N-terminal region in front of the leucine zipper domain. Sox5-BLM protein was localized in the nucleus of TRAF3-/- mouse B lymphomas and transduced human multiple myelomas. Overexpression of Sox5 inhibited cell cycle progression, and up-regulated the protein levels of p27 and β-catenin in human multiple myeloma cells. Taken together, our findings indicate that aberrant expression of Sox5 regulates the proliferation of malignant B cells.
T cell-mediated immunopathologic mechanism of liver injury in acute viral hepatitis (HUM8P.336)
Authors: Kim Jihye , Chang Dong-Yeop , Shin Eui-Cheol
Published: January 2023

CD8+ T cells play an important role in immune responses against intracellular infection. They recognize and lyse infected cells, and secrete anti-viral cytokines to control infection. However, excessive immune responses may lead to tissue damage during infection. In particular, severe CD8+ T cell-mediated liver injury is known as a primary cause of tissue damage in hepatitis A virus (HAV) infection. In this study, we demonstrate that during acute viral hepatitis, CD8+ T cells specific to HAV-unrelated viruses such as influenza virus, Epstein-Barr virus (EBV), or cytomegalovirus (CMV) became activated even in the absence of those viral reactivations, which means that non-HAV-specific CD8+ T cells are able to be activated even without cognate antigen. Interestingly, the severity of liver injury correlated with the activation of these non-HAV-specific T cells, not with activation of HAV-specific T cells. HAV-infected cells produced IL-15, which induced antigen-nonspecific activation of CD8+ T cells. IL-15-activated CD8+ T cells exerted NK-like cytolytic activity by NKG2D dependent manner, even without T cell receptor engagement. HAV-infected hepatocytes overexpressed NKG2D ligands and, thus, could be cytolytic targets during HAV infection. We report a human viral disease in which host injury is attributed to NK-like cytolytic activity of IL-15-activated CD8+ T cells not specific to the infecting virus.
Severity of infectious mononucleosis (IM) correlates with the frequency of crossreactive influenza A virus (IAV)-M1 and Epstein Barr virus (EBV)-BMLF-1-specific CD8 T cells (HUM8P.345)
Authors: Selin Liisa K. , Aslan Nuray , Watkin Levi , Gil Anna , Luzuriaga Katherine
Published: January 2023

During EBV-associated IM IAV-specific crossreactive memory T cells are activated and play a role in disease severity. In HLA-A2+ IM patients, influenza M1-58 (IAV-M1)-specific CD8 memory T cell responses crossreacted with two different EBV lytic epitopes, BMLF1-280 (17/29) and BRLF1-109 (19/20). Furthermore, 11/22 IM patients demonstrated some intra-viral crossreactivity between EBV-BRLF1 and -BMLF1 responses. Disease severity of IM directly correlated with significantly increased frequencies of crossreactive IAV-M1/EBV-BMLF1, IAV-M1, and EBV-BMLF1 specific CD8 cells, and with mean viral load over the first 5 weeks of infection. Disease severity did not correlate with BRLF1 or M1/BRLF1 crossreactive responses. When severity of IM was scored and patients were assigned to either mild or severe groups, disease severity correlated with specific TCR Vb usage in IAV-M1 population suggesting that TcR selection is driving disease outcome. Consistent with IAV-M1 and EBV-BMLF1 responses driving increased immunopathology was the observation that patients with severe disease had significantly more IAV-M1 and EBV-BMLF1 cells producing IFNg/MIP1-b in response to antigen as compared to patients with mild disease. These results suggest that T cell crossreactivity impacts T cell selection and function and ultimately disease outcome. Insights on these issues are important for the intelligent design of vaccines and to develop therapeutic interventions for virally induced disease.
Selectivity of Toll-like receptor stimulation in human B cells (HUM8P.350)
Authors: Simchoni Noa , Cunningam-Rundles Charlotte
Published: January 2023

B cells activated by nucleic-acid sensing Toll-like receptor 7 and TLR9 proliferate and secrete immune globulin. Memory B cells are more responsive due to higher TLR expression levels, but further selectivity remains largely unknown. In this study, human B cells stimulated by TLR ligands, with or without IFNα, or by T cell mimic CD40L plus IL-21 were examined to identify differentially responsive subsets, defined phenotypically or through BCR characteristics. Both stimuli induced CD27hiCD38hi plasmablasts, with TLRs inducing more IgM+ plasmablasts and IgM+ CD27hiCD38lo pre-plasmablasts than CD40L/IL-21. TLR stimulation also increased the relative proportion of IgM+ memory B cells and fostered higher IgM secretion, while CD40L/IL-21 expanded IgM- memory cells and fostered higher IgG secretion. Ig heavy chain deep sequencing showed V and J preference among TLR responsive cells, clustering TLR stimulated conditions apart from CD40L/IL-21 and control conditions. TLR stimulation expanded BCRs containing V3 at the expense of V1, and particularly expanded V3 to J4 rearrangements. More somatically hypermutated sequences were seen following TLR stimulation as compared to CD40L/IL-21. Additionally, complementarity determining region 3 segments from TLR activated cultures were shorter, more hydrophilic, and less negatively charged compared to CD40L/IL-21 or control CDR3s. Human B cells show selective sensitivity to TLR stimulation, with distinctive proliferative and genetic signatures.
Functional polymorphisms of TLR8 gene contribute to Mycobacterium tuberculosis infection (INC7P.401)
Authors: Eng Hock-Liew , You Huey-Ling , Wang Chiou-Huey , Su Pei-Yi , Lin Tsun-Mei
Published: January 2023

Innate immunity is important for antimycobacterial defence through senses various pathogen-associated molecular patterns of Mycobacterium tuberculosis (MTB). Davila et al. first reported the evidence of association of TLR8 polymorphisms with tuberculosis (TB) susceptibility in males, but the mechanism through which TLR8 recognizes MTB and intracellular signaling remains unknown. In this study, we demonstrated that TLR8-129C /+1 A allele was present at higher frequency in males TB infected patients as compared to the healthy controls (24.1% vs. 6.8%, p=0.001) in Taiwan. Ex vivo phagocytosis assay of the functional effects of these polymorphisms revealed significantly lower TLR8 expression (p=0.002) but higher phagocytosis ability (p=0.03) in monocytes of male individuals of TLR8 -129C/+1A genotype than those of wild type, TLR8 -129G/+1G. Since Mycobacteria within monocytes can escape the immune destruction, individuals with the TLR8 -129C/+1A might thus be higher susceptible to TB infection due to higher phagocytosis ability. In addition, ex vivo analyzing mRNA expression and cytokine production in whole blood stimulated with inactivated MTB, TLR7 expression (p=0.05) and TNFα production (p=0.03) were significantly downregulated in TLR8 -129C/+1A subjects. In conclusion, these findings provide an evidence of association between TLR8 gene polymorphisms and susceptibility to MTB infection which is related to the effectiveness of MTB phagocytosis and impaired TLR signaling.
Differential cytokine response by macrophages to ManLAM of Mycobacterium tuberculosis under various culture conditions (INC7P.419)
Authors: Bagriacik Emin , Albayrak Nurhan
Published: January 2023

Lipoarabinomannan, a lipoglycan is a major virulence factor for Mycobacterium. Mannosylated form of Lipoarabinomannan (ManLAM) is commanly found in pathogenic Mycobacterium tuberculosis. It has been shown that ManLams inhibit production of TNFα and IL-12 by macrophages under in vitro culture conditions. In this study, we tested cytokine responses of macrophages stimulated with ManLAM under various culture conditions. ManLAM was isolated from M. tuberculosis strain H37Rv (ATCC 25618). Macrophages (J744.1; murine macrophage cell line) were stimulated with ManLAM in three different conditions, either alone or in co-cultures with fibroblasts (3T3; ATCC, CRL1658) in contact-culture and non-contact culture systems. Cytokines were measured using commercially available ELISA kits. In comparison to unstimulated controls, TNFα, IL-6 and IFNγ levels increased significantly in co-culture systems after stimulation with ManLAM while a slight increase occurred for IL-12 in the same cultures. However, we observed no change in TNFα, IL-6 and IL-12 for stimulated macrophages when they were cultured alone in the absence of fibroblasts. These results suggested that macrophage activity to ManLAM changed in the presence of the fibroblast in co-culture systems that might be an in vitro model for granuloma formation in tuberculosis infections.
Neutrophils in granulomas from Mycobacterium tuberculosis-infected macaques express granzyme B in response to mycobacterial products and pro-inflammatory signals (INC7P.406)
Authors: Mattila Joshua , Flynn JoAnne
Published: January 2023

Inflammation is a critical component of tuberculosis (TB), and may contribute to a poor prognosis. The factors responsible for this inflammation are poorly studied. In addition, the role of neutrophils in TB, and whether neutrophils express granzyme B (grzB), a cytotoxic pro-apoptosis enzyme, is controversial. We examined neutrophils in peripheral blood (PB) and granulomas to determine if mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. PB neutrophils did not express grzB. In contrast, granuloma neutrophils expressed grzB and these cells contained more grzB than T cells. Elevated neutrophil, but not T cell, grzB expression correlated with higher tissue bacterial load. Perforin is required for T cell-mediated cytotoxicity but was not expressed by neutrophils. PB neutrophils upregulated grzB expression when infected with M. tuberculosis or stimulated with M. tuberculosis culture filtrate protein or LPS from E. coli, but not peptide antigens associated with T cell stimulation. Moreover, stimulated PB neutrophils secreted grzB in a perforin-independent process. GrzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration or regulating activation in other cell types. These data indicate M. tuberculosis-specific factors in granulomas upregulate neutrophil grzB expression and suggest a previously unappreciated aspect of neutrophil biology in TB.
NLRP3 inflammasome activation in Alzheimer's disease (INC9P.446)
Authors: Tzeng Te-Chen , Golenbock Douglas
Published: January 2023

Neuroinflammation is now recognized as a fundamental response in Alzheimer’s pathology. Prominent activation of inflammatory responses has been observed in the brains of patients with Alzheimer’s disease, as well as in mouse models, however the role of neuroinflammation in Alzheimer’s disease pathogenesis is still not clear. We have reported that the fibrillar form of beta amyloid is capable of stimulating the production of the inflammatory cytokine IL-1β from microglia cells through the activation of the NLRP3 inflammasome. NLRP3 mediated IL-1β production, in turn, induced neurotoxic factors from microglia cells and resulted in neuronal cell death. More importantly, we have recently bred the APP/PS1 mouse into the NLRP3 knock out mice and showed that NLRP3 deficient APP/PS1 mice were completely protected from learning and memory deficits and had decreased Amyloid beta plaques. NLRP3 activation induces both IL-1β and IL18 production. However the role of IL-1β or IL-18, both downstream of NLRP3 inflammasomes, in neuronal cell death are not clear. We used microglia cells and mouse neuronal cell line co-culture system to show the increased neuronal cell death was dependent on the activation of caspase-1 when stimulating microglia cells with Amyloid beta. In addition, IL-1β or IL-18 have synergistic effect in inducing neuronal cell death.
MicroRNA-302b is a new regulator of inflammatory responses via feedback to TLR/IRAK4 circuits during bacterial infection (INM9P.459)
Authors: Wu Min
Published: January 2023

MicroRNAs (miRNAs) have been implicated in a spectrum of physiological and pathological conditions, including immune responses. miR-302b has been implicated in stem cell differentiation and cell proliferation; however its role in infection and immunity remains to be defined. In this study, we showed that miR-302b was induced by TLR2 and TLR4 through ERK-p38- NF-κB signaling upon Gram-negative bacterium Pseudomonas aeruginosa infection. Suppression of inflammatory responses to bacterial infection is mediated by targeting IRAK4, a protein required for the activation and nuclear translocation of NF-κB. Through negative feedback, enforced expression of miR-302b in mice with its “mimics” (or IRAK4 siRNA silencing) inhibited downstream NF-κB signaling and airway leukocyte infiltration, thereby alleviating lung injury and increasing survival in P. aeruginosa-infected mice. Similarly, IRAK4 siRNA silencing blocked airway leukocyte infiltration and inflammatory cytokine production, thereby increasing survival after infection in mice. On the other hand, miR-302b inhibitors exacerbated inflammatory responses and decreased survival in both P. aeruginosa-infected mice and lung epithelial and macrophage cells. Collectively, our findings revealed that miR-302b is a novel inflammatory regulator of NF-κB activation in respiratory bacterial infections through negative feedback to TLRs-mediated immunity.
Development of microbial-derived inhibitory peptides using structural studies of microbial TIR proteins TcpB, TcpC and host adapters TIRAP and MyD88. (INM9P.449)
Authors: Snyder Greg , Deredge Daniel , Waldhuber Anna , Fresquez Theresa , Smith Patrick , Duerr Suri , Cirl Christine , Jiang Jiansheng , Jennings William , Luchetti Timothy , Snyder Nathaniel , Sundberg Eric , Wintrode Patrick , Miethke Thomas , Xiao Tsan
Published: January 2023

Microbial pathogens have evolved mechanisms to regulate and evade innate immunity. One such mechanism involves the subversion of Toll-like receptor (TLR) signaling by bacterial TIR Interacting Proteins (TIPs). TIPs are thought to function by disruption of host Toll/IL-1 receptor (TIR) signaling proteins. For example, the TIP proteins TcpB from Brucella and TcpC from E. coli inhibit TLR signaling through direct interaction with host adapter proteins TIRAP and MyD88. We have previously reported the crystal structure of MyD88 and characterized TcpC peptides capable to inhibit TLR4 and MyD88 signaling. We now report the X-ray crystal structure of the Brucella TIR protein TcpB and characterize its interactions with TIRAP using hydrogen/ deuterium (H/D) exchange mass spectrometry, co-immunoprecipitation and NF-ΚB reporter assays. The crystal structure of TcpB reveals the BB loop microtubule-binding site as well as a symmetrical dimer involving the DD and EE loops. The dimer interface is further characterized through H/D exchange mass spectrometry, which identifies a set of candidate potential TcpB blocking peptides. A comparison between the microbial TcpB, TIRAP and MyD88 crystal structures reveal differences in the region that encompasses the BB loop. These findings lend insight into the molecular mechanisms of microbial mimicry of host signaling adapter proteins and provide a framework for identification and development of novel microbial-derived therapeutics.
CD4+ T cell memory is limited by cellular competition for sub-threshold ligands (IRC9P.704)
Authors: Singh Nevil
Published: January 2023

The requirements for the maintenance of CD4+ memory T cells are not clear. While memory CD4+ T cells are relatively short lived in hosts with an intact repertoire of T cells, their lifespan is dramatically prolonged in T-deficient hosts. Accordingly, MCC-specific 5C.C7 T cells primed in intact hosts briefly expand before clonally contracting - while those in T-deficient hosts do not undergo significant contraction. As a result, 6 weeks after immunization, T-deficient hosts support >200 fold more 5C.C7 memory T cells than intact hosts. Pre-populating T-deficient hosts with polyclonal CD4+ T cells from an intact host was sufficient to restore the contraction phase to a subsequent 5C.C7 response. However neither CD8s nor other monoclonal CD4+ TCR transgenic T cells had an analogous impact. We had previously reported a similar principle regulating naïve and tolerant 5C.C7 T cell survival; and in that case a single TCR (Vα2+/Vβ3) screened from over 6000 polyclonal T cells was able to replicate the inhibitory activity of the complete repertoire. Surprisingly, naive Vα2+ T cells also had only a modest impact on memory 5C.C7 T cell frequencies. However, pre-activating the Vα2+ T cells enabled them to robustly control the size of the memory 5C.C7 T cell population. As the Vα2+ T cell shares an endogenous sub-threshold ligand (STL) with 5C.C7, but not agonist-specificity, these data suggest that the CD4+ memory pool is limited by non-clonal competition for STLs between memory T cells.
Program for memory differentiation is CD8 T cell intrinsic and not determined by CD4 help. (IRC10P.467)
Authors: Kim Juhyun , Oh Keunhee , Ryu Su Jeong , Ju Ji Min , Jeon Ji Yeong , Sproule Thomas , Roopenian Derry , Choi Eun Young
Published: January 2023

To elucidate the fundamental role of CD4 help in CD8 T cell response, the dynamics of the CD8 T cells participating in the help-deficient response was tracked longitudinally via in vivo imaging and phenotypic analyses. Help-deficient CD8 T cells underwent intact effector differentiation with fast kinetics, albeit insignificant burst expansion. However, they failed to effectively eliminate the antigen, which rendered them to be activated continuously, defective in memory cell differentiation, and exhausted eventually. Although PD-1-deficiency rescued the exhaustion, it could not restore the memory differentiation. Instead, simply increasing the numbers of effector CD8 T cells or accelerating antigen clearance enabled efficient memory generation even in the absence of help. These results demonstrate that memory program is intrinsic to CD8 T cells, not encoded by CD4 help, and provide insight into the role of CD4 help, permitting a better understanding of CD8 T cell differentiation and immunity.
Mir-15/16 antagonizes c-Myb to control natural killer cell maturation (LYM7P.714)
Authors: Sullivan Ryan , Schneider Stephanie , Leong Jeff , Fehniger Todd
Published: January 2023

NK cells develop in the bone marrow and complete maturation in peripheral organs, but the factors controlling maturation are incompletely understood. The miR-15/16 family is highly expressed in NK cells, and to study its role in NK cells, we generated a novel mouse model of NK-specific miR-15/16 deficiency (15aKO). 15aKO NK cells have a deficiency of mature (CD11b+CD27-) NK cells, with no alterations in total NK cell numbers or death, consistent with a block in maturation. The transcription factor Myb is expressed highly in immature NK cells, nearly absent in mature NK cells, and has been implicated in NK maturation. We found that miR-15/16 represses Myb, and Myb levels are increased in 15aKO NK cells. To directly investigate the role of Myb in NK cells, we developed a novel NK maturation system in which immature primary NK cells are transduced, adoptively transferred, and matured in vivo. As in the 15aKO mouse, transferred 15aKO NK cells had defective maturation. However, both 1) restoration of miR-15/16, or 2) knockdown of Myb, rescued maturation of the 15aKO NK cells, demonstrating that miR-15/16’s repression of Myb is critical to proper NK cell maturation. Furthermore, WT NK cells overexpressing Myb did not mature as well as control (WT NK+GFP) cells. Thus, the regulation of Myb by miR-15/16 is essential for NK cell maturation, revealing a novel mechanism controlling NK cell development.
Conservation of TB epitope sequences within the MTB complex and beyond, dictates the epitope specificity and associated phenotypes (MPF6P.732)
Authors: Lindestam Arlehamn Cecilia , Paul Sinu , Huang Charlie , Greenbaum Jason , Sidney John , Peters Bjoern , Sette Alessandro
Published: January 2023

We have performed a detailed analysis of epitopes identified in an unbiased genome-wide analysis of CD4 MTB recognition using PBMCs from individuals latently infected with MTB (LTBI) or non-exposed individuals. This analysis revealed that surprisingly, some reactivity was detected in non-exposed individuals. The identified MTB epitopes could be divided into LTBI-specific, mixed reactivity and non-TB-specific categories. Reactivity of MTB sequences recognized by both LTBIs and non-exposed individuals is explained by sequence conservation in non-tuberculous mycobacteria (NTM), and suggests environmental exposure to NTMs. Reactivity to conservation-specific peptide pools showed that non-MTB donors reacted only to pools containing MTB/NTM conserved epitopes, whereas LTBI donors showed reactivity to MTB and MTB/NTM pools, thus suggesting that a more potent diagnostic assay might be developed. Interestingly, sequences broadly conserved across bacterial genera are significantly less recognized and result in IL-10 production, which could be explained by tolerance. Additionally, we have defined a set of NTM-specific epitopes which can be utilized as a tool to characterize T cell responses to NTMs. Exposure to NTM might provide some preexisting immunity or occasional boosting of responses. Together, these results provide important insight into future vaccine design and evaluation.
Anti-fungal memory Tc17 cells persist as bona fide cells without plasticity (MPF6P.745)
Authors: Nanjappa Somashekarappa , Klein Bruce
Published: January 2023

The rising rates of fungal infections in immunocompromised hosts stress the need for fungal vaccines. We have shown that experimental fungal vaccine-induced CD8+ T cells are maintained as memory cells in the absence of CD4+ T-cell help and antigen. We also showed that IL-17A producing CD8+ T cells (Tc17) are indispensable for fungal vaccine immunity. Although we found that Tc17 cells evince long-lasting, memory signature molecules, any plasticity towards Tc1 may not be fruitful as IL-17A producing CD8+ T cells were necessary for fungal vaccine immunity. The persistence of memory Th17 cells is debated, but some work suggests the cells are maintained and undergo plasticity to Th1 cells and expressed T-bet. We investigated similar reprogramming of IL-17A producing CD8+ T (Tc17) cells after fungal vaccination. Using IL-17A fate mapping mice and adoptive transfer methods, we report that vaccine-induced Tc17 cells persisted as bona fide memory cells without plasticity in the absence of antigen and CD4+ T-cell help. Elicitation of Tc17 cells required IL-1 and IL-23 signaling in addition to IL-6. IL-23 was essential for memory Tc17 responses in the lung after pulmonary challenge despite the normal recruitment of global CD8+ T cells. Tc17 memory precursors expressed low levels of Eomes and Bcl-2 but not Bcl-xL compared to Tc1 cells suggesting an alternative survival program. We conclude that fungal vaccine-induced Tc17 cells persist as bona fide memory cells without plasticity.
Inflamed gingiva derived CD103 negative dendritic cell are differentiate to Foxp3+ Treg cell in periodontal disease mouse model (MUC7P.761)
Authors: Kobayashi Ryoki , Yamamoto Masafumi , Fujihashi Kohtaro , Ochiai Tomoko
Published: January 2023

As a most professional antigen presenting cells, dendritic cells (DCs) can rapidly access and sample the environment for antigens and danger signals to protect the host against invading pathogens. A recent report has identified the CD103 (αE), which is the α-chain of the αEβ7 integrin, expressing (CD103+) DCs in mice as a major DCs population residing in the mucosa. We already reported that, Foxp3+ Treg cell were induced in inflamed gingiva at late stage of inflammation period. Moreover, purified DCs from inflamed gingiva were differentiate to Foxp3+ CD4+ T cell when co-cultured with splenic naïve CD4+ T cell. In this study, we demonstrate the characterization of CD11c+ DCs in inflamed gingival tissue. A murine periodontal disease model with alveolar bone loss was established by oral infection with 109 cfu of Porphylomonas gingival is (P. gingival is), which suspended in 0.1ml PBS with 2% carboxymethylcellulose 15 times over three weeks. Significant inductions of ICAM-1+ DCs were continuously increased in inflamed gingiva, with enhanced levels of both TGF-β and RALDH-2 specific transcripts. However, these DCs dose not express CD103 molecules. These results suggest that inflammation derived CD103 negative DCs have a potential to induce Treg cells at the site of inflamed gingiva.
Role of food antigens on development and function of IELs (MUC8P.811)
Authors: Jung Jisun , Yang Bogie , Kim Kwang-Soon , Surh Charles
Published: January 2023

Intestinal IELs reside within the epithelial cell layer of the gut and are composed of several subsets of T cells, including induced TCRαβ+CD4+, TCRαβ+CD8αβ+, and TCRαβ+CD4+CD8αβ+ IELs, and natural TCRαβ+CD8αα+ and TCRγδ+CD8αα+ IELs. Their close proximity to the gut lumen and their intimate association with enterocytes presumably allow IELs to serve as the first line of defense against invading pathogens and to maintain homeostasis of the intestinal epithelial cells. Despite the fact that IELs have been studied for a long time, their exact biological function remains largely unknown. Early studies have revealed that intestinal colonization with commensal microbiota leads to expansion of TCRαβ+ IELs and their enhanced cytotoxic activity, indicating that a role for bacterial Ags in development and function of IELs. To investigate the role of food Ags in IELs, we analyzed IELs in offspring of germ-free B6 mice raised with elemental diet, designated as antigen-free mice. In antigen-free mice, we found a severe reduction in development of induced IELs, i.e., TCRαβ+CD4+, TCRαβ+CD8αβ+, and TCRαβ+CD4+CD8αβ+ IELs, and a partial depletion of natural TCRαβ+CD8αα+ IELs. The TCRγδ+CD8αα+ IEL population was relatively unaffected in antigen-free mice. We also observed a significant reduction in the ability of IELs to display effector function in antigen-free mice. Our results suggest that development and effector function of many subsets of IELs are strongly dependent on dietary antigens.
Immunoregulatory mechanisms of microRNAs during Clostridium difficult infection (MUC9P.819)
Authors: Viladomiu Monica , Hontecillas Raquel , Michalak Katarzyna , Philipson Casandra , Schiff Eric , Carbo Adria , Michalak Pawel , Bassaganya-Riera Josep
Published: January 2023

MicroRNAs are small non-coding RNA molecules that play pivotal roles in the development and functionality of innate and adaptive immune cells, making them essential during the regulation of bacterial, viral and other immune infections. The role of miRNAs during host-pathogen interactions has recently been highlighted. However, the mechanisms by which miRNAs exert immunoregulatory actions are poorly understood. Our aim is to investigate the impact of Clostidium difficile infection (CDI) on host regulatory and effector pathways by focusing on the disruption of mucosal homeostasis by miRNAs. Sequencing studies on colonic samples and mucosal CD4+ T cells revealed a consistent upregulation of miR146b during CDI in mice, which correlated with worsened disease severity and upregulated MCP-1, IL-1β, IL-6 and IL-17. Moreover, upregulation of miR146b correlated with increased levels of Th17 cells in the spleen, MLN and colonic lamina propria of CDI mice, while abrogating anti-inflammatory responses characterized by IL-10 production. In vivo miR146b inhibition by using locked nucleic acid resulted in accelerated recovery as well as a significant induction of IL-10 production by CD4+, CD8+ and NK T cells and reduced expression of IL-17 by CD4+RORγt+ T cells. C. difficile infection induces upregulation of miR146b at the gut mucosa that contributes to pathogenic Th17 responses and impared immunoregulation.
A critical role for Slc5a8 in the suppression of colonic inflammation by commensal bacteria-derived metabolites (MUC9P.821)
Authors: Gurav Ashish , Singh Nagendra , Ganapathy Vadivel
Published: January 2023

Presence of trillions of bacteria in healthy colon presents an immunological challenge. Colonic bacteria ferment dietary fibers into short-chain fatty acids (butyrate, propionate, and acetate; SCFAs); among which, butyrate and propionate are inhibitors of histone deacetylases (HDACs). Depletion of SCFAs correlates with increased incidence of intestinal inflammation. Slc5a8 is a high-affinity transporter for SCFAs, which is essential for intracellular actions of SCFAs such as HDAC inhibition. Among various immune cells in colon, dendritic cells (DCs) are unique in having access to colonic lumen, thus getting exposed to bacterial antigens. It is known that colonic DCs exhibit tolerogenic properties with ability to suppress conversion and proliferation of naïve T cells into pro-inflammatory T cells. However, the mechanisms involved in the generation of tolerogenic DCs in colon are poorly understood. We hypothesized that Slc5a8-mediated transport of SCFAs into DCs and consequent inhibition of HDACs induces tolerogenic phenotype in these cells. In support of our hypothesis, we found that butyrate and propionate induce the immune-suppressive enzyme Indoleamine 2,3-dioxygenase (IDO) in DCs in an Slc5a8-dependent manner. Further, these DCs are able suppress conversion of naïve T cells into interferon-γ (IFNγ) producing Th1 cells. Together, our data identify the SCFA transporter Slc5a8 as a key determinant of SCFA-mediated protection against intestinal inflammation.
Sequestering HMGB1 using DNA-conjugated beads ameliorates murine colitis (THER5P.840)
Authors: Yang Huan , Ju Zhongliang , Dancho Meghan , Li Jianhua , Tamari Yehuda , Chavan Sangeeta , Tracey Kevin
Published: January 2023

Inflammatory Bowel Disease (IBD) is a chronic inflammation of the gastrointestinal tract that affects millions of people worldwide. Although the etiology of IBD is not clear, it is known that mediators from stressed cells and enteric microbes cause intestinal inflammation. High mobility group box 1 (HMGB1) is a cytokine implicated in the pathogenesis of IBD and other inflammatory diseases. HMGB1 is secreted by inflamed human intestinal tissues, and abundantly found in stools of IBD patients, and mouse models of colitis. Neutralizing HMGB1 activity by anti-HMGB1 antibodies or antagonist improves clinical outcomes in animal models of colitis. Since HMGB1 binds DNA with high affinity, here we developed a novel strategy to sequester HMGB1 using DNA immobilized on sepharose beads. Screening of different types of DNA-bead constructs identified B2 beads that bind to HMGB1 with high affinity, captures HMGB1 ex vivo from activated RAW 264.7 cell supernatant and from fecal material obtained from dextran sodium sulfate (DSS)-induced colitis MICE. Oral administration of B2 DNA-beads improved body weight, reduced colon injury and suppressed colonic and circulating cytokine levels in mice with spontaneous colitis (IL10 knock out) or with DSS-induced colitis. Our data suggest that DNA beads sequester HMGB1 and reduce inflammation, and have a therapeutic potential for the treatment of IBD.
Pharmacological doses of vitamin A deteriorate lupus-like disease in the MRL/lpr mouse model (THER5P.829)
Authors: Liao Xiaofeng , Ren Jingjing , Wei Cheng-Hsin , Ross A. , Cecere Thomas , Jortner Bernard , Ahmed S. , Luo Xin
Published: January 2023

The critical roles of retinoic acid (RA), a metabolite of vitamin A (VA), in both tolerogenic and immunogenic immune responses have been documented. However, how VA affects the development of autoimmunity is still unclear. Here we demonstrate that pharmacological doses of VA had adverse effects on the development of autoimmune lupus in the MRL/lpr mouse model. We administered, orally, RA (6 mg/kg body weight/day) or retinyl palmitate combined with 10% RA (VARA, a total of 6.6 mg/kg/day) to the mice starting from 6 weeks of age. At this age, the mice do not exhibit overt signs of lupus. However, the immunogenic environment preceding disease onset has been established that is evidenced by an increase of total IgM/IgG in the plasma and expansion of lymphocytes and dendritic cells in secondary lymphoid organs. After 8 weeks of RA or VARA treatment, significantly higher pathological scores in kidney, lung, and brain were observed, which were correlated with a marked increase in B-cell responses. In addition, although the number of T cells in the mesenteric lymph node decreased with RA that led to reduced tissue weight, the proportions of T-cell subsets did not change. Importantly, neither RA nor VARA affected regulatory T cells in these mice. Altogether, our results suggest that under the immunogenic environment in autoimmune lupus, VA can generate more severe disease rather than inducing tolerance. Thus, administration of VA in lupus treatment should be approached with caution.
Activation of the stimulator of interferon genes (STING) adaptor attenuates experimental autoimmune encephalitis (THER6P.848)
Authors: Mohamed Eslam , Lemos Henrique , Huang Lei , Chandler Phillip , Souza Guilherme , Li Lingqian , Pacholczyk Gabriela , Barber Glen , Hayakawa Yoshihiro , Munn David , Mellor Andrew
Published: January 2023

Activation of Stimulator of Interferon genes (STING) adaptor causes the release of IFN αβ in most of the cells, an activity that is tightly controlled to avoid continuous immune activation that could lead to autoimmunity. Here we show that STING activators can be used systemically to suppress experimental autoimmune encephalitis following myelin oligodendrocyte (MOG) immunization through STING-IFN αβ pathway. DNA nanoparticles (DNPs) attenuated MOG-specific effector responses in CNS and spleen. Therapeutic responses to DNPs are dependent on STING adaptor and IFN αβ receptors as they were abolished in mice lacking those genes. Additionally, indoleamine 2,3 dioxygenase enzyme (IDO) is crucial for therapeutic responses to DNPs as the loss of IDO1 gene in hematopoietic cells and IDO inhibition by 1-methyl-D-tryptophan had ablated those responses. Cyclic diguanylate monophosphate (c-diGMP), a direct STING activator also attenuated EAE progression and disease severity. Thus, Systemic administration of DNPs and c-diGMP can attenuate EAE through STING-IFN αβ-IDO regulatory pathway that is activated in certain immune cells subsets to overcome CNS specific autoimmunity. These findings emphasize a previously unappreciated role of STING activators to attenuate autoimmunity.
Cell-based kidney transplantation in adult Danio rerio (TRAN3P.879)
Authors: Vahey Delia , Delaney Tetyana
Published: January 2023

With the increasing incidence of kidney disease and the growing list of patients in line for kidney transplantation, new methods to restore renal function are critical. An alternative method to whole-organ transplant involves the transplantation of individual cells. Here we show a new approach to the transplantation of kidney cells in zebrafish, Danio rerio. This method involves the transplantation of kidney cells from the donor fish by direct injection into the recipient kidney. The donor zebrafish were transgenic, their cells labeled with green fluorescent protein. Both renal and hematopoetic progenitor cells were isolated from the donor kidney and prepared for transplantation in an injection solution. The recipient fish were treated with gentamicin and immunosuppressed with 10 μg/ml dexamethasone for 2 days. Upon injection of 0.1-0.5 million allogeneic cells via the dorsum of the fish into the head kidney, anesthetized Danio rerio regained complete swimming abilities within 5 min. Seven out of nine fish survived the procedure. The fish were allowed to recover for 10 days, leaving time for the transplant cells to implant. Then the fish were euthanized and a renal biopsy was performed and visualized using a fluorescent microscope. The formation of new fluorescent renal tubules was observed and analyzed. Although further research must be done to refine this technique, we demonstrated that cell-based kidney transplant is a promising alternative to whole-organ transplant.
Cross dressing by donor dendritic cells maximizes T cell responses to indirectly presented alloantigen after BMT by promoting immune synapse formation (TRAN3P.903)
Authors: Markey Kate , Koyama Motoko , Gartlan Kate , Leveque Lucie , Kuns Rachel , Lineburg Katie , Teal Bianca , MacDonald Kelli , Hill Geoff
Published: January 2023

The role of trogocytosis in generating naïve T cell responses is controversial and highly relevant to transplantation. We transplanted B6.CD11cGFP donor bone marrow (BM) grafts (H2Db, IAb, GFP off the CD11c promoter) into irradiated B6D2F1 (H2Db/d, IAb/d,) and tracked donor DC. Using standard and imaging flow cytometry, we noted that all donor cells become “cross-dressed” in high levels of recipient hematopoietic cell-derived MHC early after BMT. To assess the functional consequences of cross-dressed MHC, we transplanted B6.MHCII-/- BM into WT B6 recipients to generate DC cross-dressed with IAb. WT DC induced proliferation in BALB/c CD4 T cells transferred into B6.MHCII-/- recipients but no responses were seen to B6.MHCII-/- DC bearing cross-dressed Ag. Next, DC were generated that were either cross-dressed (B6.MHCII-/-→B6D2F1), bore cross-dressed and indirectly presented Ag (B6→B6D2F1), or could present indirect Ag only (B6→B6.MHCII-/-xD2F1) to the allospecific TEa Tg T cell. T cell proliferation was maximal in response to processed recipient alloAg presented via the indirect pathway in the presence of cross-dressed Ag, markedly reduced to indirect Ag when cross-dressing was absent, and absent to cross-dressed Ag alone. Imaging flow cytometry confirmed that cross-dressing promotes formation of the immunologic synapse. Thus, cross-dressing serves to maximize naïve T cell responses to indirectly presented alloAg and exerts this effect after BMT via the immunologic synapse.
Tools for optimization of adoptive cellular therapy (TRAN3P.908)
Authors: Brunstedt Katja , Wallace Paul , Pan Dalin , Halgreen Charlotte , Pedersen Henrik , Brix Liselotte
Published: January 2023

The transfusion of T lymphocytes is a promising treatment for viral infections in immune-compromised patients, and has induced regression of cancer in early-stage clinical trials. Adoptive T cell therapy can be optimized in several ways: -Patient stratification - only those that benefit from therapy are treated -Measurement of T-cell immunity pre and post treatment - has the desired immune response been induced and is additional therapy needed -Quantitative quality control of the cellular product prior to infusion Multiple adoptive cell therapy trials are investigating the benefit of transferring CMV-specific T cells to transplant patients to avoid reactivation of CMV. We used the MHC multimer based Dextramer CMV assay, to quantitate CMV-specific T cells in whole blood and processed cell samples. The reconstitution of CMV immunity was followed in >90 transplant patients and it was shown that induction of CMV-specific immunity upon adoptive transfer of CMV-specific T cells could be reliably measured. We showed that CMV Dextramers can be used to characterize cellular products with respect to CMV-specific T-cell composition upon in vitro expansion. The CMV Dextramer assay is a valuable tool that may improve CMV-specific adoptive cellular therapy by allowing: -Stratification - Identification of patients with low CMV-specific immunity -Immune monitoring - Determination of the induced cellular response in patients -Quality control - QC of manufactured cellular products
Age-associated increase of IL-6 dampens anti-tumor immune responses through attenuating Th1 differentiation of tumor-specific CD4 T cells (TUM7P.928)
Authors: Tsukamoto Hirotake , Senju Satoru , Swain Susan , Nishimura Yasuharu
Published: January 2023

With age immune function progressively decreases and pro-inflammatory status is concomitantly developed. We focused on the impact of this inflammatory environment on tumor-specific CD4 T cells. We demonstrated that in contrast to tumor elimination in young hosts, adoptive transfer of OVA-specific young CD4 T cells and vaccination with OVA peptide-pulsed DC failed to induce efficient regression of OVA-expressing tumor cells in aged hosts. This was consistent with the result that in aged mice generation of tumor-specific Th1 cells was significantly attenuated. Interestingly, a blockade of IL-6 signaling partially restored Th1 differentiation of donor CD4 T cells and elicited tumor regression in aged hosts, but did not alter their expansion, IL-2 or IL-17A production. Furthermore, in vitro analyses revealed that IL-6 directly acted on CD4 T cells to attenuate Th1 differentiation. IL-6 blockade also restored the defect in an ability of tumor-specific CD4 T cells to promote anti-tumor CD8 T cells induction in aged mice. These results were confirmed by aged IL-6-deficient mice. However, the Th1-mediated help for tumor-specific CD8 T cells was not observed in aged mice when IFN-γ-deficient CD4 T cells were transferred into anti-IL-6 Ab-treated mice. These results imply that systemic increase of IL-6 is an extrinsic factor counteracting tumor-specific Th1 differentiation, and is responsible for the age-associated defect in mounting protective anti-tumor immunity in aged individuals.