bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crucial role for TACI in TLR activation and macrophage phenotype determination (P1206)
Authors: Akkoyunlu Mustafa , Katsenelson Nora , Uslu Kadriye , Allman Windy 
Published: January 2023

Abstract
Transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) regulates antibody responses to T cell independent type 2 (TI-2) antigens such as polysaccharides. However, it is not known whether TACI is important in generation of Ab response against T cell dependent (TD) antigens. In this study, we investigated the role of TACI in Ab responses against Neisseria meningitidis type C polysaccharide-tetanus toxoid conjugate (MCPS-TT) vaccine. We found that TACI-/- mice had lower amounts of Abs against MCPS and TT compared to WT mice. More importantly, even the potent adjuvant CpG did not augment Ab responses to MCPS-TT vaccine in TACI-/- mice. Stimulation of splenic cells or peritoneal macrophages of TACI-/- mice with CpG, LPS, or PolyI:C led to a significantly impaired production of pro-inflammatory cytokines and lower levels of co-stimulatory molecule upregulation, suggesting that TACI plays a key role in TLR signaling. Significantly lower expression of TLR9, TLR4, TLR3 and CD14 as well as impaired co-localization of the adaptor molecules, MyD88 and TRIF with the TLR receptors is likely to play a role in the impaired TLR response in TACI-/- mice. Further characterization of TACI-/- macrophages revealed that they manifest the characteristics of alternatively activated (M2) macrophages. Thus, M2 macrophage phenotype appears to be responsible for the impaired TLR activation and poor Ab responses to TD antigens in TACI-/- mice.
TLR4 as a TRIF-biased receptor (P1212)
Authors: Kolb Joseph , Casella Carolyn , SenGupta Shuvasree , Mitchell Thomas 
Published: January 2023

Abstract
We reported previously that the vaccine adjuvant monophosphoryl lipid A (MPLA) could behave as a TRIF-biased agonist of TLR4, with mostly intact TRIF-dependent outcomes but less MyD88/TRIF co-dependent activation of proinflammatory cytokines, and proposed that TRIF-bias was an important component of MPL’s success as a clinically safe immunostimulant. Taking a pharmacological approach, we have now found that synthetic MPLA (sMLA) has approximately 10% the potency of synthetic Lipid A (sLipid A) for all TRIF and MyD88/TRIF co-dependent signaling outcomes tested, revealing that sMLA is not a TRIF-biased agonist of TLR4. We observed instead that both agonists’ potencies for TRIF-dependent outcomes were markedly higher than MyD88/TRIF co-dependent outcomes. This suggests that the receptor, not the agonist, is TRIF-biased. TLR4’s TRIF-bias was not restricted to “highly-inducible” chemokines because potency measurements using TRIF-dependent CD86 showed the same result. Tests of tetra-acylated lipid IVa also showed TRIF-biased signaling by TLR4, indicating that the phenomenon was not unique to hexa-acylated structures such as sMLA and sLipid A. Lower potency TLR4 agonists like MPLA may be useful as vaccine adjuvants because they take advantage of a TRIF-biased receptor system, providing enough signal strength to activate TRIF-dependent adaptive priming with little MyD88/TRIF co-dependent inflammation.
Regulation of the expression of MKP-1 and MKP-2 by ERK MAP kinases in macrophages following LPS stimulation (P1214)
Authors: Liu Yusen , Xue Jianjing , Wancket Lyn , Shakibi Yasaman , Nelin Leif , Frazier W. 
Published: January 2023

Abstract
Although the MKP family plays an important role during host defense, the regulation of MKPs is poorly understood. We studied the expression of MKP-1 and MKP-2 in RAW264.7 cells during LPS stimulation. We found that pretreatment of cells with U0126 (a MEK1/2 inhibitor), substantially decreased MKP-1 and MKP-2 mRNA levels in LPS-stimulated macrophages. In contrast, SB203580 (a p38 inhibitor), had little effect on the levels of MKP-1 and MKP-2 mRNA. The combination of U0126 and SB203580 resulted in greater decreases in MKP-1 and MKP-2 mRNA levels, suggesting that p38 contributes to MKP-1 and MKP-2 induction in response to LPS. Interestingly, in the absence of LPS, SB203580 enhanced ERK and MEK1/2 activities, suggesting that p38 negatively regulates the ERK pathway by inhibiting a signaling event upstream of MEK1/2. LPS potently stimulated CREB phosphorylation mediated by p38, but not ERK, and enhanced phospho-CREB binding to MKP-1 and MKP-2 chromatins. Interestingly, LPS also enhanced the phosphorylation of RNA polymerase II at serine-2 at the MKP-1 chromatin in a manner dependent on CDK9 and ERK. At the protein level, both MKP-1 and MKP-2 underwent robust phosphorylation mediated by ERKs. Phosphorylation of both MKP-1 and MKP-2 substantially increased the stability of these proteins. Taken together, our studies suggest that the ERK pathway plays a predominant role in the regulation of MKP-1 and MKP-2 through both transcriptional and post-translational mechanisms.
Phenotypic and functional characteristic of newly identified CD8+Foxp3-CD103+ regulatory T cells (P1047)
Authors: Zheng Song Guo , Liu Ya , Wang Julie , Horwitz David , Quismorio Francisco , Xu Anping 
Published: January 2023

Abstract
TGF-β is crucial for induction of CD4+Foxp3+ Tregs and maintenance of immunologic tolerance. It is, however, unclear if TGF-β also induces the similar CD8+ Tregs. While CD8+ cells primed with TGF-β (CD8+ iTregs) displayed much lower Foxp3 expression compared with compartment CD4+ cells, their suppression activity in vitro and in vivo was equivalent or even better than CD4+ Tregs. These cells did not express Granzyme A, Granzyme B or Perforin and lacked cytotoxic activity on T response cells. Additionally, CD8+ iTregs generated from Granzyme B and Perforin KO mice still suppressed autoimmunity. Transwell experiments revealed that cell-contact is required for their suppressive activity. Adoptive transfer of the CD8+ iTregs markedly suppressed experimental colitis, EAE and cGVHD. We further found both Foxp3- and Foxp3+ subsets from TGF-primed CD8+ cells had suppressive activities. Among CD8+Foxp3- cells, we identified CD103 expression is crucial for their generation and function since CD8+ but not CD4+ iTreg production decreased on CD103 KO mice. Adoptive transfer of CD8+CD103+Foxp3- subset suppressed colitis, and IL-10 and TGF-β signal seem to be crucial for this therapeutic effect. TGF-β can induce CD8+Foxp3- and CD8+Foxp3+ iTreg subsets that displayed suppressive activity in cell contact-dependent, non-cytotoxic manner and have protective effects on autoimmune diseases. Generation of CD8+ iTregs may have considerable therapeutic potential on patients with autoimmune diseases.
Regulation of the expression of GARP/latent-TGF-β1 complexes on mouse regulatory T cells and their role in Th17 differentiation. (P1033)
Authors: Edwards Justin , Unutmaz Derya , Shevach Ethan 
Published: January 2023

Abstract
GARP/LRRC32 has previously been defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation, but in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4+ T cells results in lack of expression of latent-TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of T conventional cells in vitro. Activated Tregs expressing GARP/latent-TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6. Induction of Th17 producing cells is preferentially induced by Tregs expressing the latent-TGF-β1/GARP complex on their cell surface rather than by secreted latent-TGF-β1.
Characterization of a naturally occurring severe combined immunodeficiency in pigs (P6087)
Authors: Wyatt Carol , Cino Ozuna A. , Rowland Raymond , Dekkers Jack , Waide Emily , Tuggle Christopher 
Published: January 2023

Abstract
Severe combined immunodeficiency (SCID) refers to a group of inherited disorders in which two components of the immune system are defective. We have identified a naturally occurring, SCID in a line of pigs that were bred for increased feed efficiency. Affected piglets remain healthy while on the sow, but succumb to infections soon after weaning. SCID piglets have underdeveloped lymph nodes and spleen with a paucity of lymphocytes, they do not make antibodies to Porcine Reproductive and Respiratory Syndrome (PRRS) virus, and they accept human tumors. Further analyses showed that the defect has an autosomal recessive inheritance pattern. Blood was collected within 2 days after birth to titer serum immunoglobulins, perform lymphocyte counts and identify lymphocyte populations by flow cytometry. Affected piglets had no detectable serum immunoglobulins, and very low numbers of circulating lymphocytes. By flow cytometry, there were Natural Killer cells, but few to no T or B lymphocytes. At necropsy, thymus and small intestine were collected from each affected piglet and from control littermates. Histologic sections, stained with Hematoxylin and Eosin, were evaluated for cellularity. Affected piglet thymic remnants were small, containing Hassall’s corpuscles, but relatively few thymocyte-like cells. There were no detectable Peyer’s Patches in small intestine. These observations are consistent with T-/B- NK+ SCID.
Effect of antibiotic treatments on the expression levels of metabolizing and drug resistance proteins in immunological cells from salmon head kidney (P6071)
Authors: Cárcamo Juan , Arias Luis , Mancilla Edith , Yañez Alejandro 
Published: January 2023

Abstract
The massive use of drugs on fish farming could have an important effect on immunological status of these organisms. Here we showed the effect of antibiotics used in salmon farming on the protein expression levels involved in metabolism and drug resistance in immunological cells from salmon head kidney (SHK-1 cells). The protein expression levels of CYP1A, CYP3A, FMO, GST, Pgp and MRP1 were measured by Western blot after antibiotic treatments. Cell viability was measured by MTT assays. Significant fluctuations of expression in the most proteins analyzed as response to antibiotic treatments were evidenced, while no effect on cell viability were observed. Therefore, the antibiotic treatment changes the expression levels of metabolizing and drug resistance proteins in these salmonid cells, which may be associated with the induction of drug resistance phenomena and a immunological response diminished. This information allows a deeper understanding of the mechanisms involved in the immunological status from a fish specie belonging to one of the most important economic sectors in Chile.
Endogenous tumor-reactive CD8+ T cells are differentiated effector cells with high expression of CD11a and PD-1 but are unable to control tumor growth (P2089)
Authors: Gibbons Rachel , Liu Xin , Harrington Susan , Krco Christopher , Markovic Svetomir , Kwon Eugene , Dong Haidong 
Published: January 2023

Abstract
We utilized CD11a, an integrin up-regulated on effector and memory CD8+ T cells, as a biomarker to identify, measure, and monitor endogenous tumor-reactive cytotoxic lymphocytes (CTLs) in two mouse tumor models and in melanoma patients. CD11ahigh CD8+ T cells were detected in primary and metastatic tumor sites after tumor cell injection, as well as in spontaneous murine breast cancer tissues. In the peripheral blood of melanoma patients, tumor antigen-specific T cells were detected within the population of CD11ahigh CD8+ T cells that express high levels of PD-1. Infusion of lethally irradiated tumor cells failed to stimulate the expansion of CD11ahigh CD8+ T cells, suggesting that the access of tumor cells to target tissues is required to induce CD11ahigh CD8+ T cell responses. Phenotypic analysis revealed that the CD11ahigh CD8+ T cells detected in our studies were proliferating (Ki67+) activated T cells (CD62L-, CD69+). Although CD11ahigh CD8+ T cells were differentiated effector cells with CTL effector functions, they were typically unable to control tumor growth in our mouse models, possibly due to high expression of PD-1. Our results demonstrate that CD11a can be used as a biomarker to identify and track endogenous tumor-reactive CTLs.
The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation (P2107)
Authors: Poojary Venuprasad , Ahmed Neesar , Zeng Minghui , Polin Lisa , Sinha Indrajit , Rathinam Chozhavendan , Flavell Richard , Massoumi Ramin 
Published: January 2023

Abstract
Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. We demonstrated that ubiquitin ligase Itch and deubiquitinase Cyld formed a complex via interaction through the ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved Lys63-linked ubiquitin chains and catalyzed Lys48-linked ubiquitination on TGF-beta activated kinase 1(Tak1) to terminate inflammatory signaling. Itch or Cyld deficiency resulted in sustained Tak1 activation and elevated production of proinflammatory cytokines by bone marrow derived macrophages (BMDMs). Tak1 selective inhibitor, (5Z)-7-Oxozeaenol abrogated Tak1 activation and production of proinflammatory cytokines by Itch-/- and Cyld-/- BMDMs. Reconstitution of wild-type Cyld but not the mutant Cyld (Y485A), which cannot associate with Itch, blocked sustained Tak1 activation and proinflammatory cytokine production by Cyld-/- BMDMs. Deficiency in Itch or Cyld led to chronic production of tumor-promoting cytokines by tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have identified an Itch-Cyld-mediated regulatory mechanism in innate inflammatory cells.
Identification and expansion of an endogenous polyclonal population of tumor antigen specific T cells with high affinity TCRs (P2109)
Authors: Moran Amy , Weinberg Andrew 
Published: January 2023

Abstract
Previously, the field of tumor immunology has relied on T cell receptor (TCR) transgenic mice and model tumor antigens to study the T cell and tumor cell interaction. However, these models have limitations. Namely, they do not reflect the natural affinity or precursor frequency of tumor antigen specific T cells. Recently, a novel Nur77GFP mouse was generated that allows us to study the antigen receptor signal strength and study the tumor immune response to endogenous tumor antigens. This mouse has a green fluorescent protein regulated by antigen receptor signaling. The stronger the antigen:antigen receptor interaction, the higher the Nur77GFP. Importantly, the reporter is sensitive to even very weak antigen receptor engagement as is speculated to be the nature of endogenous tumor antigens. Our preliminary data suggests that the co-expression of Nur77GFP and PD-1 identifies a subpopulation of polyclonal endogenous tumor infiltrating lymphocytes that are exclusively tumor antigen specific; produce more IFNγ upon re-stimulation with syngeneic tumors, and increases with anti-OX40 therapy. We have previously shown that treatment of tumor bearing hosts with anti-0X40 can promote tumor regression, therefore we propose that one mechanism of OX40 mediated immunotherapy is via the expansion of high affinity tumor antigen specific T cells within the tumor microenvironment.
Tumor-derived exosome alters the proliferation and function of T lymphocytes in nasopharygneal carcinoma (P2111)
Authors: Li Jiang , Ye Shu-Biao , Tang Xiao-Feng , Mo Hao-Yuan , Zeng Yi-Xin 
Published: January 2023

Abstract
Background: Tumor-derived exosomes take an important role in the inflammatory responses and anti-tumor immune responses in tumor microenvironments. Here, we used exosomes from NPC tumor cell line TW03-EBV+ (exosome1) and TW03-EBV- (exosome2) to evaluate the impact of exosome1 and exosome2 on the proliferation of CD4+ and CD8+ T lymphocytes, the differentiation of CD4+ naïve T cells mediated by NPC tumor cells, and the lysis function of tumor-infiltrating lymphocytes (TILs) to the tumor cells from NPC. Results: Exosome1 and exosome2 all could promote the proliferation of CD4+ and CD8+ lymphocytes stimulated by OKT3 or PHA through up-regulated the phosphorylation of Erk-1,2; however exosome1 and exosome2 inhibited the proliferation of CD4+ and CD8+ lymphocytes in IL-2 medium; Exosome1 and exosome2 inhibited the Th1 and Th17 cell differentiation mediated by NPC tumor cells through changing the phosphorylation of STAT1 and STAT3; Exosome1 and exosome2 inhibited the lysis function of TIL to EBV-positive NPC tumor cell line TW03. Conclusions: The tumor-derived exosomes can alter the cross talk between lymphocytes and tumor cells in NPC, and maybe be a potential tool for NPC immunotherapy in the near future.
p53-dependent chemokine expression by tumor cells supports NKG2D-dependent elimination of senescent tumors by natural killer cells (P2115)
Authors: Iannello Alexandre , Thompson Thornton , Ardolino Michele , Raulet David 
Published: January 2023

Abstract
The cellular senescence is a key mechanism by which p53 suppresses tumorigenesis. Using a mouse model in which p53 expression can be regulated in tumor cells, we confirmed that NK cells participate in the elimination of senescent tumors. We further demonstrate that tumor elimination is dependent on the function of NKG2D. Surprisingly, however, p53 induction did not increase the expression of any of the NKG2D ligands. Furthermore, tumor cells grown in vitro were sensitive to NK lysis, in an NKG2D-dependent fashion, but p53 induction did not increase the sensitivity of the cells to NK lysis. Instead, our data demonstrate that p53 restoration enhanced the recruitment of NK cells into the tumor. We found that p53 restoration in vitro and in vivo induced the secretion of various chemokines and cytokines. Among the chemokines induced in senescent tumor cells was CCL2. Blockade of CCL2 in vivo with antibodies inhibited NK cell recruitment to the tumor and reduced the rate of elimination of the senescent tumors. Our findings suggest that expression of NKG2D ligands occurs largely independently of p53 or senescence, but that recruitment of NK cells to the tumor, and consequently the efficient elimination of the tumor by NK cells, is dependent on chemokine expression associated with the induction of senescence. Thus, p53-induced immune cell recruitment acts cooperatively with other signals in tumor cells that induce NKG2D ligands to promote the elimination of tumors.
Tumor infiltrating lymphocytes are enriched for highly suppressive CD4+ Foxp3+ regulatory T cells compared to peripheral blood: a flow cytometric analysis of two distinct cancer types (P2123)
Authors: Kovacsovics-Bankowski Magdalena , Chisholm Lana , Vercellini Jonna , Newell Philippa , Ma Jun , Tseng Paul , Vetto John , Wolf Ronald , Hansen Paul , Weinberg Andrew 
Published: January 2023

Abstract
Characterizing TIL composition allows monitoring of immunotherapies and can be a prognostic factor for clinical outcome. Here we analyze the phenotype and function of lymphocytes collected from peripheral blood and tumor infiltrating lymphocytes, immediately following resection, from 15 patients with colorectal cancer liver metastases (CRLM) and 21 ovarian cancer primary tumors (OVC). Samples were analyzed using a multi-color flow panel and by qPCR for cytokine production. The percentage of different lymphocyte subpopulations was similar in both patients. Highly suppressive Tregs expressing HLA-DR were significantly increased in TIL compared to PBL in CRLM: 69.4% vs 31.7% (p=0.0002) and in OVC: 74.8% versus 38.6% (p&lt;0.0001). Enhanced expression of CD39, CTLA-4 and Helios on the surface of the Treg, confirmed that TIL are enriched for a highly suppressive subpopulation. The cytokine profile showed that IL-6, is uniquely detected in CD4+ T cells in TIL samples. Both TIL populations also contained a significantly higher percentage of activated CD8+ T cells (HLA-DR+/CD38+) compared to PBL (CRLM: 30.2% vs 7.7%, (p=0.0012), OVC 57.3% vs 11.9%, (p&lt;0.0001)). This study demonstrates that multi-color flow of fresh tumor samples is an effective method to understand the activation state of TIL and may reveal subpopulations not observed in fixed tissue samples. TIL composition were remarkably similar and enriched for highly suppressive Tregs and activated CD8+ T cells.
Extracellular Proliferating Cell Nuclear Antigen is a novel marker for cancer stem cells and facilitates evasion of Natural Killer cell effector function (P2133)
Authors: Horton Nathan , Mathew Porunelloor 
Published: January 2023

Abstract
Natural Killer (NK) cell function is strictly regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. NKp44, originally discovered as an activating NK cell receptor, was recently found to elicit inhibitory effects on NK cell effector function through recognition of cell surface Proliferation Cell Nuclear Antigen (PCNA). Other reports have pointed to potential associations between NKp44 and Human Leukocyte Antigen (HLA) I molecules. In this report, we have identified novel interactions between HLA I and PCNA on the surface of human tumor cells by confocal microscopy and immunoprecipitation. In addition to previous reports, we show PCNA on the cell surface where novel association with HLA I does not require the presence of NKp44 expressing NK cells and occurs with endogenous PCNA. The association of HLA I and PCNA forms the inhibitory ligand for NKp44, resulting in inhibition of NK effector function. Furthermore, extracellular PCNA was only found on tumor cells exhibiting a cancer stem cell phenotype. Thus extracellular PCNA is a novel marker for cancer stem cells, derived from conventional growth of tumor epithelial cell lines in tissue culture, and a mechanism by which cancer stem cells evade NK cell effector functions.
The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation. (P2135)
Authors: Das Rupali , Bassiri Hamid , Peng Guan , Wiener Susan , Nichols Kim 
Published: January 2023

Abstract
The adaptor molecule SAP plays a critical role during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. Using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is dispensable for T cell receptor (TCR)-induced iNKT cell cytokine production and iNKT cell-dependent activation of bystander cells. In contrast, SAP is essential for TCR-induced iNKT cell cytotoxicity against T- and B-cell targets in vitro and for iNKT cell-mediated control of T-leukemia growth in vivo. These findings are not restricted to the murine system, as siRNA-mediated silencing of SAP in human iNKTs also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP is required within iNKTs to enable formation of conjugates with targets and support polarization of the microtubule-organizing center (MTOC) to the immunological synapse (IS). Collectively, these studies highlight a novel and essential role for SAP-associated signals during iNKT cell cytotoxicity and formation of a functional iNKT cell IS.