bluuebunny/crossref_metadata_2025 huggingface dataset:
Luminal microbial sensing regulates colonic goblet cell associated antigen passages through the activation of p42/p44 MAPK and the EGFR (P3154)
Authors: Knoop Kathryn , McDonald Keely , Miller Mark , Newberry Rodney
Published: January 2023

The intestinal tract is full of proteins ranging in antigenicity from inert food antigens to potential pathogens and bacterial toxins. A major focus of mucosal immunology is to better understand how these antigens are acquired and introduced to the immune system. Using intravital two-photon imaging we recently showed that in conventionally housed mice, small intestinal goblet cells, but not colonic goblet cells, act as a route of antigen delivery to intestinal dendritic cells by forming goblet cell-associated antigen passages (GAPs), in a mechanism linked to goblet cell secretion. In contrast to conventionally housed mice, we observed that GAPs were present in the colon of germfree mice, mice given antibiotics, and, surprisingly, neonatal mice. We found that the activation of p44/p42 MAPK and EGFR in colonic goblet cells correlated with the presence of luminal microbiota and the ability to sense the flora via TLRs. Inhibition of either p42/p44 MAPK or EGFR allowed GAP formation to occur in the colon of SPF housed mice, and colonic GAPs in germfree mice were blocked by activation of the EGFR. These observations indicate that sensing the enteric flora inhibits colonic GAP formation. This regulation of antigen delivery would allow sampling of luminal antigens when the environment is permissive, such as in the homeostatic small intestine, but would prevent sampling luminal antigens in a hostile environment, such as the high microbial load in the colon or during infection.
Microbiota down-regulates intestinal DC expression of miR-107 which targets IL-23p19 and IL-10RB (P3158)
Authors: Yao Suxia , Xue Xiaochang , Cao Anthony , Evans Heather , Elson Charles , Cong Yingzi
Published: January 2023

Commensal flora plays important roles in development of mucosal immune system and maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host-microbiota interaction are still not completely understood. In this study, we observed that lower microRNA-107 (miR-107) expression in the inflamed intestines of colitic mice compared to that in normal control mice. miR-107 was preferably expressed by intestinal epithelial cells and dendritic cells (DC). Consistent with high levels of proinflammatory cytokines and more commensal bacteria translocation across the intestinal epithelium in colitic mice, we demonstrated that IL-6, IFNγ and TNFα down-regulated, whereas TGFβ promoted miR-107 expression. In addition, microbiota down-regulated miR-107 expression in both mouse and human DC in MyD88- and NF-κB-dependent manners. We identified IL-23p19 and IL-10RB, the key molecules in innate immune responses to commensal bacteria, as direct targets of miR-107, in that ectopic expression of miR-107 specifically repressed the expression of IL-23p19 and IL-10RB. We concluded that microbiota regulation of miR-107 could serve as an important pathway for maintaining the intestinal homeostasis to microbiota.
Characterization of allergen-specific T-cell responses in a humanized mouse model relying on a human mugwort-specific T-cell receptor and HLA-DR1 (P6208)
Authors: Neunkirchner Alina , Mager Lukas , Wojta-Stremayr Daniela , Schmetterer Klaus , Leb-Reichl Victoria , Rosloniec Edward , Ronald Naumann , Jahn-Schmid Beatrice , Bohle Barbara , Pickl Winfried
Published: January 2023

While mice expressing an I-Ad restricted murine T-cell receptor (TCR) specific for chicken ovalbumin (Ova) are widely used to mimic allergic diseases induced by aeroallergens in vivo, implications for human allergic diseases remain questionable, since Ova is not a human-relevant aeroallergen. We here created double tg mice expressing a human TCR specific for the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 and HLA-DR1. In tg mice ninety percent of peripheral blood CD4+ T lymphocytes expressed the Art v 1-specific TCR, while CD14+ monocytes and B220+ B lymphocytes revealed HLA-DR1 expression. Splenocytes of tg mice specifically proliferated upon incubation with the human-relevant immuno-dominant Art v 125-36 peptide or whole Art v 1 protein when compared to control mice. In vivo, after allergen-specific sensitization, only tg mice showed enhanced airway-hyperreactivity (AHR) as assessed by methacholine or allergen-specific challenge. Three consecutive exposures to nebulized allergen were found to be sufficient for induction of AHR. The effects on bronchoalveolar lavage fluids, lung histology and specific antibody titers will be described and discussed. Humanized allergy models are amenable to the evaluation of human-relevant allergen formulations or cellular intervention protocols and will thus contribute to the further understanding of allergic diseases and their cure.
Granulysin requires heparin sulphate proteoglycan for the uptake by target cells and moves to mitochondria matrix in CHO cells. (P1320)
Authors: Okada Satoshi , Natsuya Ryosuke , Nishida Jiro
Published: January 2023

9 kD granulysin (GLN) is one of cytotoxic molecules released by cytotoxic T cells and NK cells. GLN-derived peptide G8 which covers a part of second and third helixes (Q23-R51) is much more potent than GLN in killing target cells. It was previously shown that GLN and G8 enter Jurkat cells and cause mitochondria-mediated cell death. In Jurkat cells, GLN and G8 are diffusely localized inside cells. In the present study, we evaluated the uptake of G8 by CHO, PgsA-745 and PgsD-677 cells. PgsA-745 and PgsD-677 are mutants of CHO and incapable of producing proteoglycan (PG) and heparin sulphate PG (HSPG), respectively. The uptake of G8 was about ten times less efficient in PgsD-677 and PgsA-745, strongly suggesting that HSPG is pivotal in the uptake of GLN and G8 by target cells. Surprisingly, both GLN and G8 eventually became localized in mitochondria in 45 minutes after the uptake by CHO cells although they did not enter the nucleus. A study using isolated mitochondria in sucrose buffer and proteinase K showed that GLN and G8 enter mitochondria matrix. However, replacement of one the cysteine residue of GLN and G8 with serine inhibited the transfer to inter-membrane space and matrix. Our study may help further understanding the mechanism by which internalized protein is sorted to organelles. Furthermore, GLN-derived peptide may provide a clue in developing a therapeutic tool to transfer nucleotides or peptides into mitochondria.
DAP12 impacts trafficking and surface stability of killer immunoglobulin-like receptors on natural killer cells (P1323)
Authors: Mulrooney Tiernan , Hurley Carolyn
Published: January 2023

Killer Ig-like receptors (KIR) aid in the regulation of natural killer (NK) cell activity. In this study, the effect of the interaction between the two domain stimulatory KIR (KIR2DS) and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when co-transfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12 specific siRNA. Treatment of the KIR2DS and DAP12 transfected cells with either cycloheximide or Brefeldin A repressed KIR2DS surface expression revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS indicating the interaction likely initiates within the endoplasmic reticulum. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules.
Identification of gene targets of PLZF in NKT cells (P1289)
Authors: Newsome Courtni , Sant'Angelo Derek
Published: January 2023

The transcription factor PLZF (promyelocytic leukemia zinc finger) is required for the development of NKT cell effector functions. Studies described here investigate the gene targets of PLZF in NKT cells. The aims are: 1) to determine the gene expression in wild type and PLZF deficient NKT cells, and 2) to define direct genetic targets of PLZF. A hurdle to carrying out gene expression analysis has been the need to use CD1d tetramers to identify invariant NKT cells. Crosslinking of the TCR, as a result of binding the tetramer, can cause changes in signaling and gene expression. Therefore, for aim 1, we used a BAC eGFP/PLZF reporter mouse system. In this mouse, PLZF expressing cells are labeled with eGFP, which obviates the need for CD1d tetramer. Importantly, GFP expression is retained in NKT cells that develop in PLZF deficient mice. Therefore, “untouched” NKT cells can be collected from PLZF expressing and deficient mice. RNA was isolated from these cells and subjected to microarray analysis. Microarray analysis indicated many gene changes, of which only some will be direct targets of PLZF. Aim 2 will be to carry out Chromatin Immunoprecipitation assays followed by high-throughput sequencing. We will utilize an NKT cell hybridoma cell line that expresses PLZF and has retained the capacity to produce IL-4 and IFN-γ. By combining the data, we expect to define genes that are regulated by PLZF and are necessary for the development of NKT cells.
Molecular characterization of an alternative model of inflammation (P1347)
Authors: Myers Michael , Deaver Christine , Peters Sharla , Swaim Heidi , Yancy Haile
Published: January 2023

Non-steroidal anti-inflammatory drugs (NSAIDs) for food-producing animals such as swine need to control systemic and local inflammatory responses. However, there are no NSAIDs approved to control inflammation in swine due to an absence of qualified measures of efficacy needed to substantiate those claims. The absence of systems and approaches to substantiate a claim to control inflammation has led to the total absence of approved products for swine. The goal of this study was to develop an in vitro carrageenan model of local inflammation for use in studies of novel non-steroidal anti-inflammatory drug efficacy, and to determine if there are common pathways/biomarkers for local and systemic inflammatory responses. We evaluated carrageenan induced stimulation and its capacity to affect previously identified systemic inflammatory biomarkers in swine, using swine whole blood cultures. We found that lambda carrageenan increases the production of IL-8 and MCP-1 via ELISA. Conversely, TNF-α and SAA demonstrated a no change in production. Additionally, Phosphokinase-3, Monocyte Chemotactic Protein-1, Alveolar Macrophage Chemotactic -II showed increased gene expression. These results suggest lambda carrageenan impacts ubiquitination on immune cells. These results also demonstrate that carrageenan initiates an inflammatory response that differs from that induced by endotoxin in terms of the pathways and gene products altered as a consequence of carrageenan exposure.
Rapamycin-resistant mTORC1 restrains dendritic cell B7-H1 expression that requires IL-1β to enhance regulatory T cell induction (P1349)
Authors: Rosborough Brian , Raïch-Regué Dàlia , Matta Benjamin , Lee Keunwook , Boothby Mark , Turnquist Heth , Thomson Angus
Published: January 2023

Introduction: The mammalian Target of Rapamycin (mTOR) is a central regulator of dendritic cell (DC) function that performs the catalytic activity of mTOR complex (mTORC)1 and 2. mTORC2 functions independently from mTORC1 and is resistant to inhibition by rapamycin (RAPA); however, mTORC1 has both RAPA-sensitive and -resistant outputs. Our goal was to ascertain the role of RAPA-resistant mTOR in DC. Methods: WT C57BL/6 or B7-H1-/- bone marrow-derived DC were generated with the addition of RAPA or ATP-competitive mTOR inhibitor, which blocks all mTOR signaling. DC lacking rictor, an mTORC2-specific subunit, were generated from conditional rictor KO mice. DC induction of regulatory T cells (Treg) was determined in MLR, using BALB/c CD4+CD25- T cell responders. Results and Conclusion: RAPA and mTORC2 deletion reduced DC B7-H1 expression, but ATP-competitive mTOR inhibitors enhanced B7-H1 expression. Augmented B7-H1 expression was blocked by STAT3 inhibition and correlated with reduced expression of the STAT3 negative regulator, SOCS3. DC exposed to ATP-competitive mTOR inhibitors increased Treg induction, which was dependent on DC B7-H1. IL-1β neutralization additionally reduced Treg induction by B7-H1-/- ATP-competitive mTOR inhibitor-exposed DC, suggesting that IL-1β and B7-H1 act additively to promote Treg induction by these DC. These findings establish a RAPA-resistant mTORC1 pathway that acts through SOCS3 and STAT3 to regulate DC B7-H1 expression and Treg induction.
Transcription factor Ets2 negatively regulates IL-6 production in innate immunity (P1351)
Authors: An Huazhang , Wen Mingyue
Published: January 2023

Transcription factor Ets2 was reported to increase production of proinflammatory cytokine TNF-α. We investigated the regulatory role of Ets2 in LPS-induced inflammatory cytokine production. Upon LPS stimulation, Ets2 was translocated into nucleus. ERK1/2 and p38 inhibitors inhibited LPS-induced Ets2 nuclear translocation, suggesting that Ets2 was activated by ERK1/2 and p38 pathways. Although Ets2 knockdown decreased LPS-induced TNF-α production, it increased LPS-induced IL-6 production in mouse peritoneal macrophages. Similarly, Ets2 knockdown also increased VSV infection-induced IL-6 production in mouse peritoneal macrophages. Upon LPS stimulation, Ets2 was induced to bind the promoter of IL-6 gene, suggesting that Ets2 functions as a negative transcription factor in IL-6 gene transcription in innate immunity.
Sex differences in IRF5 levels and implications for pDC IFNα response (P1365)
Authors: Griesbeck Morgane , Tomezsko Phillip , Doyle Erin , Sirignano Michael , Diefenbach Thomas , Altfeld Marcus , Chang J. Judy
Published: January 2023

Background: Differential sex-dependent immune activation is a common feature in both HIV-1 and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) derived from females have been shown to produce significantly more IFNα in response to HIV-1-encoded TLR7/8 ligands than pDCs derived from males, resulting in stronger secondary T cell activation. Given their crucial role in the regulation of IFNα production by pDCs, we investigated the impact of the interferon regulatory factors (IRF) on the observed differences. Methods: Fresh PBMCs were isolated from healthy donors, and the quantity of IRF7 and IRF5 were measured by flow cytometry directly ex vivo and after TLR7 stimulation. IRF5 nuclear translocation following TLR7 stimulation was confirmed by confocal microscopy and quantified using TissueFAXS. Results: Baseline levels of IRF5 in pDCs were found to be significantly higher in females than in males (p<0.05). No differences between males and females in baseline levels of IRF7 in pDCs were observed. Importantly, levels of IRF5 were significantly higher in pDCs secreting IFNα compared to the non-secreting pDCs after TLR7 stimulation (p<0.05). Conclusion: These data indicate that sex differences in IRF5 levels might account for the described higher IFNα production upon TLR7 stimulation in females, providing new insights into the mechanisms underlying higher inflammation observed in females for both HIV-1 infection as well as autoimmune diseases.
Methionine sulfoxide reductase A attenuates heme oxygenase-1 induction through inhibition of Nrf2 activation (P1377)
Authors: Kim Hwa-Young , Kim Jung-Yeon , Choi Seung Hee , Lee Eujin , Kang Young Jin
Published: January 2023

Methionine sulfoxide reductase A (MsrA) functions as a protein repair enzyme by catalyzing the stereospecific reduction of methionine-S-sulfoxide to methionine. We previously identified that MsrA deficiency inhibits normal cell growth via activation of the p53-p21 pathway. In this study, we report a critical role of MsrA in expression of heme oxygenase-1 (HO-1), a highly inducible enzyme that has an anti-proliferative effect mediated by up-regulation of p21. Down-regulation of MsrA induced HO-1 expression in mammalian cells with increased p21 levels, but MsrA overexpression did not affect HO-1 expression. MsrA depletion activated Nrf2 by increasing its expression and nuclear translocation. Nrf2 activation was associated with increased reactive oxygen species production. MsrA overexpression in MsrA-depleted cells led to the reduction of increased HO-1 expression, and suppressed nuclear accumulation of Nrf2. Taken together, the data suggest that MsrA attenuates HO-1 induction by inhibiting Nrf2 activation.
MicroRNA-17/20a/106a modulate macrophage inflammatory responses through targeting signal regulatory protein α. (P1303)
Authors: Zen Ke , Zhu Dihan , Li Limin
Published: January 2023

Myeloid cell-specific signal regulatory protein α (SIRPα) is an essential signaling molecule that modulates leukocyte inflammatory responses. The regulation of SIRPα expression and its dynamic changes in leukocytes, however, remains incompletely understood. Here we study the role of microRNAs in regulating SIRPα expression at posttranscriptional level and macrophage inflammatory responses. We identified SIRPα as a common target gene of miR-17, miR-20a and miR-106a. During SIRPα induction in promyelocytic cells by retinoic acid or phorbol-12-myristate-13-acetate, the levels of these three miRNAs were all reduced and their downreulation occurred through suppression of the c-Myc signaling pathway. All miR-17, miR-20a and miR-106a specifically bound to the same seed sequence within the SIRPα 3’-untranslated region and correlate inversely with SIRPα protein level in various cells. In macrophages, up-regulation of miR-17, miR-20a and miR-106a by lipopolysaccharide (LPS) served as the mechanism underlying LPS-induced SIRPα reduction and macrophage activation. Both in vitro and in vivo assays demonstrate that miR-17, miR-20a and miR-106a regulate macrophage infiltration, phagocytosis and proinflammatory cytokine secretion through targeting SIRPα. In summary, these findings demonstrate for the first time that miR-17, miR-20a and miR-106a regulate SIRPα production and SIRPα-mediated macrophage inflammatory responses in a redundant fashion.
The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of CD74 (P1108)
Authors: Beisner Daniel , Langerak Petra , Parker Albert , Dahlberg Carol , Otero Francella , Poirot Laurent , Barnes Whitney , Young Michael , Niessen Sherry , Wiltshire Tim , Bodendorf Ursula , Martoglio Bruno , Cooke Michael
Published: January 2023

B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. Here we report, using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T-dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis (RIP) of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.
The gene related to anergy in lymphocytes regulates stat-mediated differentiation of CD4 T cells via ubiquitination and degradation of the kinase chaperone CDC37 (P1128)
Authors: Schartner Jill , Su Leon , Fathman C. Garrison , Whiting Chan
Published: January 2023

GRAIL, an E3 ligase, is necessary and sufficient for the induction of T cell anergy. GRAIL expression is rapidly diminished following T cell activation. Recent data demonstrated naïve CD4+ T cells from GRAIL KO mice are more efficient than WT T cells in skewing toward a Th1 or Th17 phenotype. The molecular basis by which GRAIL regulates T cell differentiation is not known. Using microarray analysis of naïve CD4 T cells we identified dysregulated signaling pathways in GRAIL KO cells. Ingenuity Pathway Analysis of dysregulated genes in GRAIL KO CD4 T cells revealed activation of multiple pathways downstream of STAT. We found that naïve CD4 T cells from KO mice hyper-phosphorylated Stat3 and 5 in response to stimulation with IL-6 and IL-2 respectively. To identify GRAIL substrates that contribute to the hyperSTAT status in KO T cells, we used a bacterial genetic screen to identify CDC37 as a target for ubiquitination and degradation by GRAIL. CDC37/Hsp90 serve as co-chaperones that stabilize expression of kinases including Jak1/2, key regulators of STATs in cytokine signaling. Loss of GRAIL may result in increased CDC37-mediated enhancement of Jak1/2 activity allowing more efficient cytokine signaling. Conversely, increased GRAIL expression in anergic and regulatory T cells may limit cytokine signaling. These data suggest a role for GRAIL in regulating global response to cytokine and is the first report on the role of an E3 ligase in modulating T cell differentiation.
Rapid recall ability of memory T cells is associated with epigenetic gene poising (P1134)
Authors: Barski Artem , Cuddapah Suresh , Kartashov Andrey , Imamichi Hiromi , Lane Clifford , Zhao Keji
Published: January 2023

While association of histone modifications with expressed or silent genes has been established, it remains unclear how changes in chromatin environment relate to changes in gene expression. We used ChIP-Seq to analyze the genome-wide changes in chromatin modifications during short-term activation of total human CD4+ T cells by T cell receptor (TCR) signaling. In resting and activated T cells, expressed genes were strongly associated with “active” modifications (e.g. H3K4me1/2/3, H2A.Z) and RNA Polymerase II (Pol II), while silent genes were typically associated with repressive marks. However, we found that about 20-30% of silent genes were poised - they possessed positive modifications and sometimes even Pol II at their promoters. Interestingly, majority of genes induced during T cell activation were poised in resting cells even before the TCR signaling was initiated. Similarly, genes that were silenced upon T cell activation retained active chromatin modifications even after being silenced. This suggested that poising might result from memory of past transcription. We next proceeded to compare histone modification profiles of naïve and memory T cells and discovered that ability of memory cells to produce characteristic cytokines is correlated with the presence of poising marks at the enhancers and promoters of these genes. This led us to propose a model of T cell memory in which immunological memory state is determined epigenetically - by transcriptional memory and poising.
The role of TRAF3 in B cell specific regulation of survival and malignancy (P1112)
Authors: Lin Wai , Stunz Laura , LeBeau Robert , Mambetsariev Nurbek , Bishop Gail
Published: January 2023

TNF-receptor associated factor 3 (TRAF3), a negative regulator of CD40 and BAFF-receptor- mediated signaling pathways, also serves as a potentially important tumor suppressor in B cell malignancies. Deletion or loss of function mutations of the traf3 gene are highly associated with multiple myeloma (MM) and different subtypes of B cell lymphoma. Current dogma has attributed TRAF3’s role of restraining cell survival to its negative regulatory role in activating the non-canonical NF-κB2 pathway. However, studying cell-type-specific TRAF3-deficient mouse strains we produced, we found that TRAF3 deletion in B cells, T cells or dendritic cells all results in constitutive NF-κB2 activation, while enhanced cell survival is unique to TRAF3 deficient B cells. This indicates that TRAF3 must negatively regulate additional survival or anti-apoptotic pathways in a B-cell-specific manner. Our recent results show that TRAF3 deficient B cells, but not other TRAF3-deficient cell types, highly express the pro-survival kinase Pim2. Pim2 expression levels also correlate with copy number and loss of function mutations in the traf3 gene in human MM cell lines. TRAF3 deficient B cells also display higher basal levels of the activated form of the B cell-specific kinase Btk, a kinase implicated in MM pathogenesis. These findings reveal B cell-specific mechanisms by which TRAF3 can exert its cell-specific impact on regulation of survival, and are being further explored in ongoing work.
Kinase-independent feedback of the TAK1/TAB1 complex on BCL10 proteolysis and NF-κB activation in lymphocytes (P1114)
Authors: Moreno-Garcia Miguel , Sommer Karen , Rincon-Arano Hector , Brault Michelle , Ninomiya-Tsuji Jun , Matesic Lydia , Rawlings David
Published: January 2023

Somatic mutations resulting in constitutive activation of the CARMA1 scaffolding protein occur frequently in poor prognosis diffuse large B cell lymphoma (DLBCL). Such mutations directly promote assembly of the CARMA1, BCL10, MALT1 (CBM complex) thereby leading to activation of the IKK signalosome. These events lead to sustained NF-κB signaling, which is essential for lymphocyte survival. Thus, understanding the events that down-regulate CBM complex assembly is predicted to identify novel therapeutic targets in DLBCL. Notably, stimulation of antigen receptors simultaneously triggers both CARMA1-dependent BCL10 activation and CARMA-dependent-BCL10 degradation, which ultimately limits CARMA1 signaling. Using mutant B cell models, and genetic as well as biochemical approaches, we identify a kinase-independent requirement for the kinase, TAK1, and its adaptor, TAB1, in antigen receptor-induced BCL10 proteolysis. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 over-expression restrains CARMA1-dependent NF-κB activation by reducing BCL10 levels. TAK1 also promotes counter-selection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counter-balances NF-κB and JNK signals essential for activation and survival of lymphocytes and CARMA1-addicted lymphoma types.
Phenotypic and functional analysis of double negative T cells in kidney. (P1002)
Authors: Martina Maria , Dai Hong , Uddin Sophia , Bandapalle Samatha , Chiu Yen-Ling , Rabb Hamid , Hamad Abdel
Published: January 2023

Double negative T cells (DNT cells) are the main subset in kidney lymphocytes. In peripheral blood represent less than a 5%, but they gradually accumulate in lymph nodes and spleens of deficient FasL mice (gld). The role of these cells in kidney and how they mediate suppression/regulation are unknown. We performed a comprehensive phenotypic and functional analysis of this population in kidney. Hypothesis: DNT cells modulate immune responses in kidney. They have a suppressive function on cytokine production, playing a role in protection cells from injury. Methods: Purified T lymphocytes from mice were analyzed for expressing surface markers and cytokines secretion. B6 wild type, gld mice and ischemic reperfusion injury mice model were used. Surface and intracellular staining were done and analyzed with flow cytometry. Results: There are more DNT cells in the kidney (B6 and gld) than renal draining lymph nodes or inguinal lymph nodes. The percentage decreases in kidney after reperfusion ischemic injury. DNT cells showed an increased expression of activation markers such as CD44/CD69/CD28 compared to CD4+ and CD8+ in kidney. DNT cells exhibit a low secretion of cytokine like IL-17, IL4 or IFN gamma. Conclusion: Our data shows that double negative T cells have the potential to modulate immune responses in kidney. This is promising for better control of kidney injury or autoimmunodiseases.
T lymphocyte responses to RBC-derived microparticles are APC-dependent (P1040)
Authors: Danesh Ali , Inglis Heather , Heitman John , Norris Philip
Published: January 2023

Microparticles (MPs) generated in stored blood are thought to cause immunomodulatory effects in blood recipients. MPs are small double membrane vesicles derived from leukocytes, platelets or other cells. We hypothesized that RBC-derived MPs would inhibit T cell responses. MPs were isolated from stored RBCs using differential centrifugation. PBMCs were cultured +/-MPs, supernatants were collected on day one, and PBMC survival was tested on day seven. PBMCs or purified T cells were stained with CFSE and cultured for seven days with PHA+/-MP. We found that MPs alone induced pro-inflammatory cytokine secretion and enhanced survival of T cells, B cells, and NK cells to a similar degree, but did not induce T cell proliferation. MPs augmented PHA-induced proliferation of CD4+ and CD8+ T cells in PBMC cultures, and blockade of CD40 or CD40L accessory molecules prevented the augmentation effect of MPs on T cell proliferation. Furthermore, stimulation of purified T cells with PHA+/-MP did not induce T cell proliferation, and addition of MP-exposed and washed monocytes to purified T cells recapitulated augmentation of T cell proliferation. In summary, addition of RBC-MPs to PBMCs improved survival of unstimulated cells and augmented mitogen driven T cell responses in an APC-dependent manner. These results suggest that, contrary to our hypothesis, MPs found in transfusion products may bias the recipient immune system in a pro-inflammatory fashion.
Different Munc13 isoforms function as priming factors in lytic granule release from murine cytotoxic T lymphocytes (P1044)
Authors: Krause Elmar , Dudenhöffer-Pfeifer Monika , Schirra Claudia , Marshall Misty , Halimani Mahantappa , Ulf Matti , Pattu Varsha , Becherer Ute , Dirks Jan , Jung Martin , Lipp Peter , Hoth Markus , Sester Martina , Rettig Jens
Published: January 2023

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues like brain and neuroendocrine glands this process is mediated by members of the Munc13 protein family. In human CTLs mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3 (FHL3) suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs. We demonstrate on both mRNA and protein level that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Furthermore mobility studies of LGs performed with TIRF micrscopy indicate that Munc13-4 as well as Munc13-1 are responsible for the priming process of LGs. Even more the domains of the Munc13 protein which are responsible for functional fusion could be identified. We conclude from these data that both isoforms of the Munc13 family are functionally redundant and identify Munc13-1 as an alternative target for immunotherapy in patients suffering from FHL3.
Stunned invariant NKT cells are not anergic, but acquire IL-10-dependent regulatory characteristics (P1052)
Authors: Wingender Gerhard , Sag Duygu , Krause Petra , Tyznik Aaron , Hedrick Catherine , Kronenberg Mitchell
Published: January 2023

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that after activation produce copious amounts of cytokines, impacting a dazzling variety of different immune reactions. However, it has been reported that strong activation of iNKT cells with the synthetic model antigen α-galactosylceramide (αGalCer) induces a hypo-reactivity that was proposed to be related to anergy. In contrast, here we report, that αGalCer pretreated iNKT cells, referred to here as ‘induced stunned’ iNKT (isNKT) cells, retain their cytotoxic activity and their response to TCR-independent, cytokine-mediated stimulation. Additionally, isNKT cells acquire characteristics of regulatory cells, marked by the expression of PD-1, Neuropilin-1, CD152 and the ability to produce IL-10. Through the production of IL-10, isNKT cells were able to modulate the tumor load of mice challenged with B16 melanoma and to ameliorate the outcome of Experimental Autoimmune Encephalomyelitis (EAE). Furthermore, a subset of iNKT cells with a stunned phenotype and function were present even in untreated mice, and were enriched in adipose tissue, indicating that natural stunned iNKT cells are present in vivo under steady state conditions. Preliminary data suggest that such stunned iNKT cells can also be found in human PBMCs. These data demonstrate that stunned iNKT cells are not anergic but rather acquire a novel functional state with regulatory potential and that they likely represent a novel, distinct iNKT cell subset.
Sprouty-2 and the MAPK/ERK pathway regulate polyfunctionality of virus-specific human T cells (P1006)
Authors: Chiu Yen-Ling , Haupt Carl , Oelke Mathias , Powell Jonathan , Griffin Diane , Schneck Joanthan
Published: January 2023

The ability for a single T cell to upregulate multiple effector functions, polyfunctionality, is crucial for protective immunity. Loss of polyfunctional T cells during chronic viral infections is associated with pathogen persistence and results in poor memory T cell formation and progressive T cell exhaustion. However, the molecular mechanisms determining the generation and maintenance of polyfunctional T cells remain unclear. We found that CD8+ T cell polyfunctionality can be directly regulated by antigen dose. High antigen dose stimulation induced robust T cell proliferation but, in contrast, only poorly polyfunctional T cells. Microarray analysis revealed that poorly polyfunctional T cells induced by high dose antigen had a unique transcription profile similar to exhausted T cells observed during chronic viral infections including upregulation of inhibitory receptors. However, the poorly polyfunctional T cell state was independent of inhibitory receptor signaling. Instead, biochemical and genetic experiments showed that the inhibition of MAPK/ERK pathway through upregulation of spry2, a negative regulator of the MAPK/ERK pathway, directly modulated polyfunctionality by selectively inhibiting cytokine secretion. Our findings reveal novel mechanisms controlling human T cell polyfunctionality and are directly relevant to effective vaccine development and T cell immunotherapy.
Opposing roles of PD-1 and PD-L in modulating CNS inflammation and infarct volume following experimental stroke in mice (P1012)
Authors: Bodhankar Sheetal , Chen Yingxin , Murphy Stephanie , Offner Halina
Published: January 2023

Stroke severity is worsened by recruitment of inflammatory immune cells into the brain. This process depends in part on T cell activation, in which the B7 family of co-stimulatory molecules plays a pivotal role. Previous studies demonstrated more severe infarcts in mice lacking Programmed Death-1 (PD-1), a member of the B7 family, thus implicating PD-1 as a key factor in limiting stroke severity. The purpose of this study was to determine if this protective effect of PD-1 involves either of its ligands, PD-L1 and PD-L2. Thus, CNS inflammation and stroke severity were evaluated in male PD-L1 and PD-L2 knockout (KO) mice undergoing 60 min middle cerebral artery occlusion (MCAO) followed by 96 h reperfusion. Unexpectedly, PD-L1 KO and to a lesser extent PD-L2 KO mice had reduced levels of activated microglia and infiltrating monocytes and total infarct volume compared to wild-type mice, as well as reduced splenic atrophy and MHC class II expression, suggesting a pathogenic rather than a regulatory role for both PD-ligands. Both positive and negative regulatory roles have been suggested for the PD-ligands in context of various diseases and our novel observations indicate that PD-L1 and PD-L2 might have proinflammatory functions independent of PD-1 binding that worsen stroke outcome. These results suggest the possibility of using antibodies to PD-L1/2 as a potential treatment for human stroke patients.
mCMV alters the airway inflammatory milieu regulating protective and pathogenic heterologous immunity to influenza A virus infection (P6137)
Authors: Sharma Shalini , Duan Susu , Thomas Paul
Published: January 2023

Prior immunity to apparently unrelated pathogens can affect the course of subsequent acute infections. The mechanisms of heterologous regulation in most circumstances remain poorly defined. In mice co-infected with murine cytomegalovirus (mCMV) and Influenza A virus (IAV), we show that prior infection with mCMV results in significantly reduced weight loss upon acute challenge with IAV as well as improved recall responses. Tetramer and ex-vivo ICCS assay showed an early recruitment of mCMV specific CD44hi CD62Llo IFNγ+ TNFα+ CD8 T cells in the bronchoalveolar lavage (BAL) of both acutely and latently infected animals following IAVx31 challenge that was absent in mice infected with CMV-alone. This protection was dependent on IFNγ production by these mature anti-CMV CD8 T cells, the protective phenotype was completely ablated in co-infected IFNγ-/- animals or in animals co-infected simultaneously. However, no cross-reactive T cells were detectible. Furthermore, comparative kinetics of CMV specific CD8 T cell responses showed that IAV infection resulted in the attrition of the CMV specific CD8 T cell response in both acutely and latently infected animals. Finally, analysis of the IAV-specific T cell receptor repertoire by multiplex nested PCR suggested that prior CMV infection altered the recruited IAV-specific T cell repertoire. These data demonstrate that CMV co-infection can determine the outcome of subsequent IAV infection by altering the airway microenvironment.
The PB1-F2 protein of influenza A virus induces danger molecule accumulation in infected lungs, priming for an enhanced inflammatory response to bacterial pneumonia (P6162)
Authors: Brown Nicholas , Zheng Pan , Liu Yang
Published: January 2023

We recently described a signaling pathway, CD24-Siglecg G/10, that suppresses the inflammatory response to danger-associated molecular patterns (DAMPs). Here we report on the role of DAMPs in secondary bacterial pneumonia following influenza A virus (IAV) infection. During IAV infection, two prototypical DAMPs, HSP70 and HMGB1, accumulated in the lungs of infected mice. IAV PB1-F2, a protein encoded by highly-pathogenic strains, such as 1918 “Spanish flu,” contributed to this accumulation, as infection with PB1-F2-deficient IAV led to a decrease in DAMPs. To model bacterial superinfection, we administered sublethal LPS intranasally 7 days after IAV infection. While all mice were more sensitive to the LPS challenge after IAV infection, CD24-/- and siglecg-/- mice did not recover from LPS challenge as quickly as WT mice. Indeed, this regimen was lethal for a subset of knockout mice, while all WT mice survived. Furthermore, CD24- and Siglec G-deficient mice were more susceptible to superinfection with the gram-negative bacterium Haemophilus influenzae. Together these results demonstrate that the ability of inflammatory PB1-F2 protein of IAV to prime for lethal, secondary bacterial pneumonia is due to the accumulation of DAMPs in the lungs of infected animals, and provide new targets for the treatment of this often fatal disease.
Partial MHC class II constructs inhibit MIF/CD74 binding and downstream effects (P5221)
Authors: Benedek Gil , Meza-Romero Roberto , Andrew Shayne , Leng Lin , Burrows Gregory , Bourdette Dennis , Offner Halina , Bucala Richard , Vandenbark Arthur
Published: January 2023

Macrophage migration inhibitory factor (MIF) and its receptor, CD74, are pivotal regulators of the immune system. Here we demonstrate for the first time that partial MHC class II constructs comprised of linked beta 1 alpha 1 domains with covalently attached antigenic peptides (also referred to as recombinant T cell receptor ligands - RTL) can inhibit MIF activity by not only blocking the binding of rhMIF to immunopurified CD74, but also down-regulating CD74 cell-surface expression. This bi-functional inhibition of MIF/CD74 interactions blocks downstream MIF effects, including enhanced secretion of proinflammatory cytokines, anti-apoptotic activity and inhibition of random migration that all contribute to reversal of clinical and histological signs of experimental autoimmune encephalomyelitis (EAE). Moreover, we demonstrate that enhanced CD74 cell surface expression on monocytes in mice with EAE and subjects with multiple sclerosis (MS) can be down-regulated by humanized RTL, resulting in reduced MIF binding to the cells. Thus, binding of partial MHC complexes to CD74 blocks both the accessibility and availability of CD74 for MIF binding and downstream inflammatory activity.
Activation of monocytes and dendritic cells through TLR4: modulation by dendrimer ABP as a potential therapeutic agent. (P5226)
Authors: Davignon Jean-Luc , Degboé Yannick , Fruchon Severine , Turrin Cédric-Olivier , Caminade Anne-Marie , Poupot Rémy , Cantagrel Alain
Published: January 2023

Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease whose etiology is unknown. Monocytes (Mo) and dendritic cells (DC) are major mediators of inflammation and immune regulation. We have previously shown that aza-bisphosphonate (ABP) dendrimer targets Mo and reduces inflammation and bone erosion in mouse arthritis. However, the potential effect of ABP on inflammatory human Mo and DC is not well understood. We have used ABP to treat human Mo and DC that were stimulated in vitro with the TLR4 agonist LPS and with IFN-γ. Mo were purified from PBMC collected from healthy blood donors. DC were obtained by 6-day culture of purified Mo in the presence of GM-CSF and IL-4. Mo and DC were then preincubated alone or with 10µM ABP dendrimer for 1h, followed by LPS and IFN-γ. We quantitated the production of pro-inflammatory cytokines and IL-10, as well as markers of DC activation and maturation. ABP significantly decreased the production of TNF-α, IL-12 and IL-23. Furthermore, treated DC exhibited a tolerogenic profile, as suggested by an increase in IL-10, decrease in IL-12, and inhibition of maturation as assessed by inhibition of the costimulatory molecules CD80 and CD86 and of CD83. Our data extend our previous observations on the anti-inflammatory properties of ABP on human Mo and demonstrate the induction of a tolerogenic profile on DC. These observations should elucidate the in vivo response of the monocytic lineage to ABP as a novel compound for arthritis.
Inhibition of VEGF-R2 reverses type 1 diabetes in NOD mice by abrogating insulitis and restoring islet function. (P5181)
Authors: Villalta S. , Lang Jiena , Kubeck Samantha , Kabre Beniwende , Szot Gregory , Calderon Boris , Brekken Rolf , Pullen Nick , Arch Robert , Bluestone Jeffery
Published: January 2023

The dysregulation of receptor tyrosine kinases (RTKs) in multiple cell types during chronic inflammation is indicative of their pathogenic role in autoimmune diseases. Among the many RTKs, vascular endothelial growth factor receptor (VEGFR) stands out for its multiple effects on immunity, vascularization and cell migration. Herein, we examined whether VEGFR participated in the pathogenesis of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. We found that RTK inhibitors (RTKIs) and VEGF or VEGFR-2 antibodies reversed diabetes when administered at the onset of hyperglycemia. Increased VEGF expression promoted islet vascular remodeling in NOD mice, and inhibition of VEGFR activity with RTKIs abrogated the increase in islet vascularity, impairing T cell migration into the islet and improving glucose control. Metabolic studies confirmed that RTKIs worked by preserving islet function as treated mice had improved glucose tolerance without affecting insulin sensitivity. Finally, examination of human pancreata from patients with T1D revealed that VEGFR-2 was confined to the islet vascularity, which was increased in inflamed islets. Collectively, this work reveals an unappreciated role for VEGFR-2 signaling in the pathogenesis of T1D by controlling T cell accessibility to the pancreatic islets, and highlights a novel application of VEGFR-2 antagonists for the therapeutic treatment of T1D.
Dietary supplements of curcumin attenuate scurfy-induced immune disorder with interaction on CD4 T cells (P5183)
Authors: Lee Gihyun , Kim Hyunseong , Park Soojin , Joo Hwansoo , Song Joohyun , Shin Minkyu , Hong Moochang , Bae Hyunsu
Published: January 2023

Background/Aims: Curcumin has been used in Asian traditional medicine for its medicinal properties. Recent studies have demonstrated that curcumin has antioxidant, anti-tumor, and anti-inflammatory activities. The aim of this study is to investigate the effects of curcumin on the the scurfy-induced immune disorder, in particular its interaction on CD4+ T cells. Methods: Starting at 3 weeks of age, mice were fed a standard diet or a diet containing 1% curcumin for 7 days. The survival period of mice were measured. Additionally, the total CD4+ T cells population and th1, th2 and th17 population and absolute number were compared between curcumin treated mice and control mice. Results: A dietary curcumin delays lethality in scurfy mice. The population of CD4+ T cells in spleen was reduced in curcumin treated mice. Furthermore, Th1, Th2 and Th17 percentage and cell count were decreased after curucumin treatment. Conclusion: Curcumin exerted a protective effect in scurfy mice. We speculate that the protective effects of curcumin in scurfy may involve, at least in part, its interaction on CD4 T cells.
Pharmacological inhibition of PI3Kδ opposes the progression of autoimmune diabetes in non-obese diabetic mice. (P5149)
Authors: Puri Kamal , Durand Caylib , Richer Martin , Brenker Kathrin , Graves Marcia , Shanina Iryna , Choi Kate , Horwitz Marc , Gold Michael
Published: January 2023

B-lymphocytes have been implicated in many autoimmune diseases, and non-obese diabetic (NOD) mice that lack B cells do not develop spontaneous autoimmune diabetes. The catalytic activity of PI3Kδ is important for B cell migration, activation, proliferation, and antigen presentation. We tested whether a PI3Kδ inhibitor could delay the onset or reduce the incidence of autoimmune diabetes in NOD mice. We found that preventative treatment of pre-diabetic NOD mice with GS-548202 (IC87114), a highly selective small molecule inhibitor of PI3Kδ, reduced the infiltration of inflammatory cells into the pancreatic islets and, accordingly, delayed and reduced the loss of glucose homeostasis. Diabetes developed in about 75% of vehicle-treated NOD mice by 18 weeks of age but in only 33% of GS-548202-treated animals at this same time point. Moreover in a therapeutic treatment mode, GS-548202 treatment conferred prolonged protection from progression to overt diabetes in a number of animals. Within 28 days, all of the NOD mice that received only the vehicle developed overt diabetes, with blood glucose levels rising substantially above 300 mg/dl. In contrast, 4 of the 7 GS-548202-treated NOD mice maintained their blood glucose levels below 300 mg/dl for at least 60 days after initiating treatment when they first exhibited blood glucose levels of 200-300mg/dl. These findings suggest that PI3Kδ inhibitors could be useful for managing autoimmune diabetes.