bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Foxo3 transcription factor in the susceptibility to central nervous system autoimmunity (P1127)
Authors: Dejean Anne , Stienne Caroline , Carrié Nadège , Saoudi Abdelhadi
Published: January 2023

Foxo proteins control gene expression during many cellular processes including cell cycle progression, reactive oxygen species detoxification, survival and death. During viral infection, we demonstrated that Foxo3 critically down-regulates the magnitude of the anti-viral T cell response by constraining the production of key inflammatory cytokines by dendritic cells (DCs). This impact on the innate immune response suggests that Foxo3 is likely to control the intensity of the adaptive immune response irrespective of its nature. We therefore analyze the implication of Foxo3 in the susceptibility to central nervous system inflammation. We showed that mice deficient in Foxo3 are remarkably resistant to experimental autoimmune encephalomyelitis (EAE), a common mouse model for multiple sclerosis. We showed that the reduced severity to EAE of Foxo3 deficient mice is not the consequence of an impaired priming of CD4 T cells in response to immunization. Rather, Foxo3 appears to play a role in the regulation of Th17 and Th1 differentiation. Thus, the reduced severity of EAE in Foxo3 deficient mice may be related to the inability of Foxo3-deficient CD4 T cells to differentiate into encephalogenic T cells. By dissecting the cellular mechanism involved in this phenotype, we showed that Foxo3 plays crucial role in both DC and T cells. Understanding Foxo3 function will bring new insights into the mechanisms that support immune cell homeostasis during normal and pathologic immune responses.
A role for inactivation of the Foxo1 transcription factor in the differentiation of T follicular helper cells (P1131)
Authors: Stone Erica , Pepper Marion , Katayama Carol , Yang Edward , Goldrath Ananda , Crotty Shane , Hedrick Stephen
Published: January 2023

We have previously shown that mice with a T cell-specific deletion of Foxo1 develop autoimmunity due to reduced Foxp3+ regulatory T cell development and function. These mice also have an increased percentage and absolute number of CXCR5+PD1+ T follicular helper (TfH) cells, a T cell subset that is able to help B cells, and this may help to explain how high levels of auto-antibodies develop in mice in which Foxo1 is specifically deleted from T cells. However, it was unclear if the large number of TfH cells is due to a cell intrinsic role for Foxo1 in restricting TfH differentiation or more indirectly the result of inflammatory autoimmunity. We now report that activated Foxo1-deficient CD4 T cells more readily differentiate into TfH cells. This increased differentiation into TfH cells occurs despite reduced expression of ICOS, a key molecule leading to TfH differentiation, in the absence of Foxo1. Furthermore, Foxo1-deficient CD4 T cells can differentiate into TfH cells in the absence of ICOS signaling. Additionally, ICOS stimulation rapidly decreases Foxo1 protein levels in activated CD4 T cells suggesting a negative feedback control mechanism. In conclusion, our data strongly suggest that ICOS signaling serves to inactivate Foxo1 in the differentiation of TfH.
The role of innate immune adaptor STING in suppressing lupus (P4070)
Authors: Sharma Shruti , Schattgen Stefan , Chan Jennie , Huyler Annie , Rothstein Ann , Fitzgerald Katherine
Published: January 2023

The ability of innate immune receptors such as the TLRs to sense both microbial products and self-molecules released from stressed, damaged or dying cells is well accepted. The discovery of TLR-independent DNA/RNA sensing pathways raises the possibility that additional mechanisms besides TLRs regulate self-DNA/RNA driven autoimmune diseases such as systemic lupus erythematosus (SLE). Both TLR-dependent and independent pathways lead to the induction of type I IFNs, which can promote autoimmunity. Here, we examined the role of a key inducer of type I IFNs, the cytosolic DNA-sensing stimulator of interferon genes (STING) pathway in SLE pathogenesis. We generated STING-/- mice on a lupus prone MRL/lpr background and compared disease progression to lpr/lpr littermates. Unexpectedly, STING/lpr mice display accelerated onset and progression of disease. These animals display gross increases in lymphocytic and plasma cell populations, class-switched Abs and exacerbated nephritis. Significant expansion of the myeloid compartment is concomitant with increases in serum cytokines TNF-α and IFNγ. Further analysis of STING/lpr mice shows a surprising increase in anti-SmRNPs, RNA specific Abs and an interferon stimulated gene (ISG) signature. Given the central role of type I IFNs and TLR7 in MRL/lpr pathogenesis, and the increases we see in ISGs and anti-RNA Abs, we propose an unprecedented role for STING in limiting lupus pathogenesis by regulating TLR and type I IFN signaling pathways.
Gene analysis of brain in aged-experimental autoimmune encephalomyelitis mice reveals over-expression of stress response pathway-related genes (P4104)
Authors: Seo Ji-Eun , Hasan Mahbub , Sarma Sailendra , Kang Min-Jung , Jung Byung-Hwa , Cho Mi-La , Nam Suk Woo , Park Won Sang , Kim Ho-Youn , Kwon Oh-Seung
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of multiple sclerosis. In our previous works, two different age groups of mice (6 weeks; Y vs. 15 months; O) were used to compare the severity of EAE. Significant decreases of body weight and increases of clinical score were observed in O-EAE, indicating a more severe form of disease was induced at old age. With the purpose to identify genes that contribute to this pathology, we investigated gene expressions in brain using gene microarray. As a result, 32 genes including MHC class II molecules antigen, complement component and lysozyme in Y-EAE, and 4 genes including circulatory system, cell adhesion and migration functions in O-EAE were up-regulated compared to their controls. Expression of 44 genes was significantly different between O-EAE and Y-EAE. O-EAE showed 27 up-regulated genes with related pathways including response to stress and acute inflammatory responses, and 17 down-regulated genes with related pathways including immune development, hemopoiesis and homeostatic process, compared to Y-EAE. Over-expression of the stress response pathway-related genes in O-EAE such as argine vasopressin, cyclin-dependent kinase inhibitor 1A and hypoxia-induced factor 3α were validated by qRT-PCR. Our data suggest that the highly expressed genes related to stress responses in O-EAE affected serious disease process, and these genes may be prior targets for susceptibility of EAE.
Shared and distinct immunogenetic pathways in myocarditis and type 1 diabetes (P4108)
Authors: Lv HuiJuan , Smith Ross , Shankar Jyotsna , Lipes Myra
Published: January 2023

The transgenic expression of the human type 1 diabetes (T1D) susceptibility allele, HLA-DQ8, in I-Aβ-/- NOD - but not in C57BL/6 - mice results in fatal autoimmune myocarditis. We have found that mortality from myocarditis in each of three lines of DQ8+NOD mice paralleled the incidence of T1D in the corresponding DQ8+/-I-Ag7+/-NOD lines, indicating that these two disease processes, although clinically distinct, share common DQ8-mediated pathways. To study the impact of non-MHC T1D susceptibility genes on myocarditis development, we crossed our highest-incidence myocarditis line with congenic NOD.B6 Idd3/B10 Idd5 mice that are protected from insulitis and T1D, and NOD.B10 Idd9 mice that develop insulitis but no T1D. The DQ8+NOD.Idd3/5 mice harbored the expected B10-derived splice variant of CTLA-4 (Idd5.1) and were strongly protected from myocarditis with a mortality of 9% (3 of 35 mice) compared to 80% in control mice (33 of 41 mice; P < 0.0001), and the majority of DQ8+NOD.Idd3/5 mice showed normal heart histology. In contrast, the Idd9 locus had minimal impact on myocarditis, with similar mortality in DQ8+NOD.Idd9 and control mice. These results show that susceptibility to myocarditis arises from the combined effects of MHC and non-MHC genes, some of which are shared with T1D. Since the human homologue of the Idd5.1 locus confers increased risk for human T1D and other autoimmune diseases, these findings have implications for the pathogenesis of human myocarditis.
MicroRNA-146a acts as a guardian of the quality and longevity of hematopoietic stem cells (P4467)
Authors: Zhao Jimmy , Rao Dinesh , O'Connell Ryan , Baltimore David
Published: January 2023

During inflammation and infection, hematopoietic stem and progenitor cells (HSPCs) are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of miR-146a produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to exhaustion of the HSCs and hematopoietic neoplasms. In the absence of miR-146a, LPS-mediated serial inflammatory stimulation accelerates the effects of aging. The chronic inflammatory stress on HSCs in deleted mice involves a molecular axis consisting of upregulation of TRAF6 leading to excessive activity of NF-κB and overproduction of the cytokine IL-6. At the cellular level, we show that the defects are attributable to both an intrinsic problem in the miR-146a-deficient HSCs and extrinsic effects of miR-146a-deficient lymphocytes and non-hematopoietic cells. This study has identified a microRNA, miR-146a, to be a critical regulator of HSC homeostasis during chronic inflammatory challenge in mice and has provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloid malignancies. This may have implications for human hematopoietic malignancies, such as myelodysplastic syndrome, which frequently displays downregulated miR-146a expression.
New implications of ADAMs in TNFα related diseases: ADAM10 deficient B cells exhibit increased ADAM17 and TNFα (P4388)
Authors: Folgosa Lauren , El Shikh Mohey , Conrad Daniel
Published: January 2023

ADAM10 is required for the development and maintenance of proper secondary lymphoid tissue architecture and optimal Ig class-switching; however, the underlying mechanisms remain unclear. TNFα is cleaved by ADAM10 and ADAM17; and, the aberrant lymphoid tissue architecture noted in B cell specific ADAM10 deficient mice (ADAM10B-/-) resembles that of global TNFα knockouts prompting analysis of TNFα production in these mice. Our results indicated that ADAM10B-/- B cells stimulated with LPS/IL4 or anti-CD40/IL4 showed increased levels of soluble and membrane bound TNFα compared to WT, which was confirmed by qPCR and immunohistochemistry of naïve lymph nodes. In addition, ADAM17 expression, assessed by qPCR, was increased in stimulated ADAM10B-/- B cells, suggesting ADAM17 overcompensation. In contrast, stimulated ADAM10B-/- B cells exhibited impaired proliferation and activation as indicated by decreased 3H-thymidine incorporation and less B220 upregulation respectively. We reason that ADAM17 overcompensation generates aberrant TNFα signaling and enhanced TNFα expression in stimulated ADAM10B-/- B cells; however, other ADAM17 substrates may be involved and are currently under investigation. In conclusion, this work institutes a key interplay between ADAM10, ADAM17, and B cell-TNFα with critical implications in autoimmune pathogenesis and therapeutics where deliberate inhibition of one ADAM may be accompanied by upregulation of another, resulting in unintended consequences.
Human B cell development in a novel xenograft model that provides selective TSLP and IL-7 stimulation (P4390)
Authors: Milford Terry-Ann , Su Rui-jun , Baez Ineavely , Francis Olivia , Zhang Xiaobing , Payne Kimberly
Published: January 2023

IL-7 is critical for mouse B cell development and available data suggest that TSLP has overlapping functions. Our previous in vitro studies show that IL-7 expands human B cell progenitors by ~60 fold (JI 2009 82:4255). Our recent experiments provide evidence that TSLP similarly expands human B cell progenitors in vitro and that primary human bone marrow stroma provides an in vivo source of both cytokines. To study interplay between IL-7 and TSLP in early stages of in vivo human B cell lymphopoiesis, we developed a novel xenograft model system that selectively provides IL-7 and/or TSLP stimulation. While mouse IL-7 can act on human cells, TSLP is species-specific. To overcome this obstacle we coupled a xenograft model system engineered to provide human TSLP (hTSLP) with IL-7 neutralizing antibodies. Preliminary data obtained using this model system transplanted with cord blood CD34+ cells shows that the production of B cell precursors is reduce by ~90% in mice that lack both IL-7 and hTSLP stimulation as compared to mice where both are present. The absence of hTSLP stimulation, alone, reduced B cell production by about 50%, while the effects due IL-7 neutralization, alone, were about half this. These data suggest that IL-7 and TSLP play an essential and at least partially overlapping role in human B cell development. Current studies are aimed at defining the differential stage-specific effects of TSLP and IL-7 on survival and proliferation of human B cell progenitors in vivo.
Cellular characterization of V cell populations (novel antigen specific immunoglobulin expressing cell lineage) from immunized mice (P4402)
Authors: Elia Jeanne , Chronopoulou Efthalia , Uribe-Benninghoff Alejandro , Rabenstein Jacob , Nguyen Andrea , Fisher Janean , Berry Jody
Published: January 2023

In our efforts to improvemonoclonal antibody production, we identified novel populations of antigen-specific cells which can be classified as neither a B cell, nor an Eosinophil using classical surface markers. One novel antigen specific population is identified by the phenotype CD49b+/IgG+/IgE+/B220- (IgG/E V-cell). Immune animals have identifiable IgG/E V cell populations in multiple tissue sites including bone marrow, spleen, and blood. No IgG/E V cells were detected in the lymph nodes, thymus, or peritoneal cavity. The percentages of IgG/E V cells differ in each of the positive tissue compartments tested. Higher levels of IgG/E V cells are routinely seen in blood compared to bone marrow or spleen of immune animals. These cells have been sorted and successfully cultured on several attempts. Supernatants from sorted V cell populations were assayed for cytokine and immunoglobulin production via CBA sandwich immunoassay. No soluble IgG was detected, but low levels of IL-4 and TNF were.
A pre-BCR/NFκB pathway suppresses STAT5-dependent pre-B cell leukemia (P4408)
Authors: Katerndahl Casey , Heltemes-Harris Lynn , Willette Mark , Ramsey Laura , Koeuth Thearith , Baechler Emily , Farrar Michael
Published: January 2023

Mice expressing a constitutively active form of STAT5b (called Stat5b-CA mice) develop pre-B leukemia with low frequency (~1-2%). Microarray analysis of these leukemias shows decreased expression of genes involved in pre-BCR signaling. Stat5b-CA mice crossed to mice with defects in a coherent signaling pathway downstream of the pre-BCR involving BLNK, BTK, or PKCβ developed pre-B leukemia in > 75% of progeny. These leukemias exhibited reduced expression of a subset of NFκB target genes, which was important for transformation as Stat5b-CA x Nfkb1-/- mice also developed pre-B leukemia. Our results point to an unexpected role for NFκB1 as a tumor suppressor in pre-B cells. We found that STAT5 cooperates with defects in this pre-BCR-dependent pathway to repress expression of the potential tumor suppressor genes Foxo1, Foxo3a, Ikzf1, and Ikzf3. Our findings demonstrate that STAT5 activation antagonizes a pre-BCR-dependent pathway that limits pre-B cell proliferation, promotes pre-B cell differentiation, and inhibits pre-B cell transformation.
B cell transcription factors define a novel tumor suppressor gene network in acute lymphoblastic leukemia (P4410)
Authors: Farrar Michael , Heltemes Harris Lynn , Kornblau Steven , Larson Jon , Starr Tim , Largaespada David
Published: January 2023

We recently found that STAT5 activation correlates with poor response to therapy in B-ALL. To identify genetic defects that cooperate with STAT5 activation to initiate transformation we carried out a forward genetic screen involving the mutagenic transposon Sleeping Beauty. This screen identified a number of cooperating partners including gain-of-function mutations in Sos1 and loss-of-function mutations in Ebf1 and Ikzf1 (i.e., Ikaros). Haploinsufficiency of Ebf1 or the related transcription factor Pax5 synergized with STAT5 to rapidly induce progenitor B-ALL in 100% of mice. The leukemic cells displayed reduced expression of Ebf1 and Pax5, which affected a small subset of EBF1 or PAX5 target genes, including tumor suppressor genes and oncogenes. To test whether compound haploinsufficiency of Ebf1 and Pax5 alone is sufficient to cause transformation, we generated Ebf1+/- x Pax5+/- mice. Over 80% of these mice developed progenitor B-ALL. We observed similar results in Ebf1+/- x Ikzf1+/-, Pax5+/- x Ikzf1+/-, and Pax5+/- x Ebf1+/- x Ikzf1+/- mice; interestingly, although most leukemias were B-ALL we also observed T-ALL suggesting that T-All may arise from a B cell progenitor. Our findings suggest a model in which STAT5 activation cooperates with small perturbations in a self-reinforcing network of transcription factors critical for B cell development to initiate ALL and that loss of any two alleles in the network results in a loss of network tumor suppressor function.
Ikaros directly upregulates transcription of B lineage-specific genes in human B cell leukemia (P4412)
Authors: Dovat Sinisa , Song Chunhua , Payne Kimberly
Published: January 2023

Ikaros encodes a DNA-binding protein that functions as a master regulator of hematopoiesis and a tumor suppressor in pre-B acute lymphoblastic leukemia (B-ALL). Loss of Ikaros function is associated with impaired hematopoiesis and high-risk B-ALL in humans. The mechanism of Ikaros tumor suppressor activity is unknown. Using quantitative chromatin immunoprecipitation (qChIP) we demonstrate that Ikaros binds to the promoters of genes required for B cell differentiation in primary leukemia cells. Luciferase reporter assays demonstrated that Ikaros activates transcription of these B lineage-specific genes. Increased expression of Ikaros via retroviral transduction in B-ALL cells resulted in increased transcription of these critical B cell differentiation genes, as well as increased binding of Ikaros to their promoters. The inhibition of Casein Kinase II (CK2) resulted in dephosphorylation of Ikaros and enhanced Ikaros-mediated transcriptional activation of the B lineage-specific genes in B-ALL. Treatment of the pre-B ALL cell line, Nalm6, with a CK2 inhibitor, resulted in increased Ikaros binding to the promoters of B lineage genes, increased transcription of B cell differentiation genes, and cell cycle arrest. Results suggest that Ikaros functions as a positive regulator of B cell differentiation by directly upregulating the transcription of B lineage-specific genes in B-ALL. The presented data suggest that CK2 has a critical role in B cell differentiation and in leukemogenesis.
The thymic hypoplasia common to 22q11.2 deletion syndrome patients is linked to a deficiency of several novel RNA species (P4448)
Authors: Hoover Ashley , Eitson Jennifer , Kahn Shaheen , Ratliff Lyndsay , Cleaver Ondine , de la Morena M. Teresa , van Oers Nicolai
Published: January 2023

22q11.2 deletion syndrome (22qDS) is the most frequent chromosomal microdeletion syndrome in humans (1 /4000 births). It arises from erroneous interchromosomal exchanges during meiosis, resulting in a hemizygous microdeletion of ~60 genes (3Mb) and 4 microRNAs on chromosome 22. Sixty percent of 22qDS patients have a thymic hypoplasia, resulting in a peripheral T cell lymphopenia. The thymic hypoplasia, hypoparathyroidism, and cardiac anomalies originate from defective pharyngeal pouch development during embryogenesis. The molecular causes remain unresolved. We profiled the RNA species in thymic tissues from normal and 22qDS patients. The miR-200 family and miR-205 were deficient in the 22qDS samples. MiR-205 is localized within a long intergenic non-coding RNA (lncRNA). This lncRNA and three transcription factors, Pax1, Foxn1, and Foxg1, were severely under-expressed in those 22qDS patients with a thymic hypoplasia. The functional role of these RNAs is being addressed with viral knockdown approaches in fetal thymic organ and pharyngeal pouch explant cultures. Preliminary findings suggest a role for several of these RNAs in thymic and parathyroid tissue specification. Results from our study could lead to better therapies for reconstituting T cell development in patients undergoing stem cell transplants and chemoablative therapies and for the elderly, who have a loss of thymic epithelial tissue with diminished T cell output.
Generation of functional natural killer T cell subsets from human bone marrow derived adult hematopoietic stem progenitor cells (P4456)
Authors: Sun Wenji , Wang Yi , East James , Kimball Amy , Webb Tonya
Published: January 2023

Natural killer T (NKT) cells constitute an important subset of T cells that can both directly and indirectly mediate anti-tumor immunity. However, cancer patients have a reduction in both NKT cell number and function, and these deficits limit the potential clinical application of NKT cells for cancer therapy. To overcome the problem of limited NKT cell numbers, we have investigated whether NKT cells can be generated in vitro from bone marrow-derived adult hematopoietic stem-progenitor cells (HSPC).Our data demonstrate that co-culture of human bone marrow HSPC with OP9 stromal cells transduced with Notch ligand delta-like 1 (OP9-DL1), generates a CD3+ T cell population. These CD3+ precursors can be further differentiated into CD8- NKT cells by secondary culture with CD1d-Ig based artificial antigen presenting cells (aAPC), expressing anti-CD28mAb and loaded with α-GalCer. Importantly, these in vitro-generated NKT cells are functional as demonstrated by their ability to proliferate and secrete IFN-γ following stimulation. These findings demonstrated for the first time that functional human NKT cells can be effectively generated from HSPC in the absence of genetic manipulation or viral transduction. These studies will serve to help understand and design NKT cell-based approaches to enhance current immunotherapeutic strategies for cancer.
Tanshinones increase natural killer cell maturation via activating p38 phosphorylation (P4464)
Authors: Choi Inpyo , Kim Won Sam , Yoon Suk Ran
Published: January 2023

The maturation of natural killer cells is regulated by various growth and nuclear transcription factors. Tanshinones, such as cryptotanshinone (CTS) and tanshinone IIA (TS), are the major lipophilic constituents extracted from the rhizome of Salvia miltiorrhiza Bunge. Here, we demonstrated that tanshinones are positive regulators for NK cell maturation. The tanshinones augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells. Tanshinones also increased IFN-gamma production in NK cells in the presence of IL-15. They also induced the expression of several transcription factors, including Id2, GATA3, T-bet, and Ets-1. Additionally, tanshinones increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, the p38 inhibitor SB203580 blocked the developmental effects of the tanshinones and suppressed Id2, T-bet, and Ets-1 expression during NK cell differentiation. These results suggest that tanshinones significantly increased IL-15-induced NK cell differentiation via enhancing the p38 phosphorylation and the expression of transcription factors.
Cell density during differentiation can alter the phenotype of bone marrow-derived macrophages (P4474)
Authors: Lee Chan Mi , Hu Jim
Published: January 2023

Bone marrow-derived macrophages (BMDMs) are widely used primary cells for studying macrophage function. However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories. In this study, we addressed the effect of cell density on the resulting macrophage population. With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4 x 10^5 cells or 5 x 10^6 cells per 10cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals. Reproducibly, cells plated at lower density gave a pure population of CD11b+F4/80+ macrophages (~98%) with majority (~80%) being Ly-6C-Ly-6G- and c-Fms+, while those plated at higher density produced less CD11b+F4/80+ cells and a considerably higher proportion of CD11c+ (~60%) and Ly-6C-Ly-6G+ (~80%) cells. BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation. Overall, our findings indicate that altering cell density during BMDM differentiation can give rise to distinct macrophage populations that could vary the outcome of a functional study.
Cross-talk between HDAC4 and STAT6 in the transcriptional regulation of Arginase-1 during dendritic cell differentiation in mouse (P4486)
Authors: Zhou Jie , Yang Quan , Zhong Limei , Shi Maohua , Zhu Mingjiang , Qin Aiping , Yin Huiyong
Published: January 2023

L-arginine and L-arginine-metabolizing enzymes play important roles in the immune system. Arg1 is one of the key enzymes in L-arginine metabolism and plays critical roles in the biological function of myeloid cells, including macrophage polarization and suppressive function of myeloid-derived suppressor cells. In this study we provide evidence for a novel role of Arg1 in the regulation of myeloid dendritic cells (DCs) differentiation. Expression of Arg1 and thus the L-arginine usage was dramatically induced during the course of mouse DC differentiation. Modulation of Arg1 expression in mouse bone marrow affects DC differentiation. Mechanistic studies showed that the induction of Arg1 in differentiating DCs was caused by an increased recruitment of HDAC4 to Arg1 promoter, which led to decreased acetylation of both Histone 3 and STAT6 proteins and subsequent transcriptional activation of Arg1. Further ChIP analysis identified a novel STAT6 binding site on Arg1 promoter that was essential for HDAC4-mediated Arg1 regulation. In addition, over-expression of Arg1 abrogated the suppressive effect of HDAC inhibitor on DC differentiation. In summary, this study reveals that Arg1 is a novel regulator of DC differentiation and that cross-talk between HDAC4 and STAT6 is the pathway that regulates Arg1 transcription during DC differentiation.
Metabolic control of dendritic cell development and function (P4490)
Authors: Wang Yanyan , Huang Gonghua , Zeng Hu , Chi Hongbo
Published: January 2023

Cell metabolism refers to the intracellular chemical reactions that convert nutrients and endogenous molecules into energy and biomass. Emerging evidence highlights an intimate interaction between metabolism and immunity. Whereas most of the current studies of immunometabolism have focused on T cells in adaptive immunity, we are just beginning to appreciate that activation of innate immune cells is also metabolically demanding. The evolutionarily conserved mechanistic target of rapamycin (mTOR) pathway integrates various environmental signals to regulate fundamental physiological functions, such as cell growth and proliferation, autophagy, and nutrient sensing and uptake. Although much less investigated, emerging evidence also suggests a role of mTOR signaling in the development and function of dendritic cells (DCs). Here we found that the differentiation and activation of DCs is associated with dynamic regulation of metabolic programs and the mTOR activity. Combining genetic and pharmacological modulation of mTOR activity, we found that mTOR is a crucial regulator of DC development and function. Mechanistically, mTOR signaling impinges upon distinct cell metabolic programs that in turn connect with cell cycle and apoptosis machineries. Our results demonstrate an important role of mTOR in linking cell metabolism and DC development and function.
How functional are human immune cells developed in mice? (P4376)
Authors: Lange Franziska , Scholbach Johanna , Rodewohl Anja , Koeberle Margarethe
Published: January 2023

After transplantation with human umbilical cord blood cells NOD-SCID IL2Rγ(null) rodents develop an human immune system which is characterized by the presence of B cells, T cells, monocytes, granulocytes and dendritic cells. The extent of the several subpopulations differs during the time after transplantation. A clear development of the immune system can be seen from immature immune system which contains mainly B cells, a few T cells in peripheral blood, during a transitional state with an additional existence of monocytes, granulocytes and denritic cells in bone marrow and spleen of the mice to a state which reminds of Graft-versus-Host-Disease. This development can be observed during a period of 10 to 20 weeks after transplantation. The B cells are able to produce high amounts of IgM in the early state and few IgG. The antigen presenting cells in the transitional state are able to express activation molecules like HLA-DR and CD86 after the injection of LPS as an immune modifying stimulus. Moreover the cells are able to produce human TNFα, IL-10, IL-1b, IFNγ and high amounts of IL-6 after stimulation with LPS in every state. Release of murine TNFα, IL10, IL-6, MCP and IL-12 was detected as well. The presence of human and murine PTX3 was also measureable after in vivo stimulation with LPS. Depending on the time point after transplantation the human immune cells are able to produce human cytokines and to express several activation markers after immune modifying stimulus.
Role of TGF-β in the creation of an immunosuppressive microenvironment during leukemia (P2025)
Authors: Carli Cedric , Caron Louis-Philippe , Jean Catherine , Dahmani Amina , Giroux Martin , Delisle Jean-Sebastien
Published: January 2023

Tumors develop a milieu that fosters their development and counteract the total efficacy of immune system through different mechanisms. TGF-β, a potent growth suppressor and immunosuppressive cytokine has the capacity to inhibit the anti-tumor immune response in several models. However, all the mechanisms underlying the effects of TGF-β are not well known. Specifically, the role of TGF-β in shaping the cancer microenvironment is ill-defined. Our objective is to characterize the role of TGF-β in shaping the neoplastic microenvironment using a leukemia model. Using the TGF-β producing EL4 cell line, we characterized the immune cell infiltration and the levels of several cytokines in the context of systemic TGF-β neutralization. When compared to tumor extracted from untreated animals, leukemic masses that had evolved in the context of TGF-β inhibition had increased levels of inflammatory cytokines and a tendency to accumulate more activated T cells and surprisingly more myeloid cells, unveiling a potential role for these cells in leukemia specific immunity. More precisely, secretion of cytokines involved in migration and activation of leucocytes, namely IL-2, GM-CSF, KC and MIP-1α was significantly induced in treated animals. Our data suggest that TGF-β blockade alters the leukemic microenvironment. Further studies will confirm the impact of TGF-β on the leukemic milieu including immune cells, stromal cells and angiogenesis and how this cytokine impedes anti-cancer immunity.
Myeloid-derived suppressor cells are associated with disease progression and decreased overall survival in advanced-stage melanoma patients (P2027)
Authors: Jordan Kimberly , Amaria Rodabe , Ramirez Oscar , Callihan Eryn , Borakove Michelle , Manthey Elizabeth , Borges Virginia , McCarter Martin
Published: January 2023

Myeloid-derived suppressor cells are increased in the peripheral blood of advanced-stage cancer patients and prevent effective antitumor immune responses. Although these cells have been well characterized in mouse studies, few studies have shown a correlation of these immunosuppressive cells with functional and clinical outcomes in melanoma patients. We characterized the frequency and suppressive function of two subsets of myeloid-derived suppressor cells in the peripheral blood of 34 patients with Stage IV melanoma. The frequency of CD14+ MDSCs (Lin- CD11b+ HLA-DR- CD14+) and CD33+ MDSCs (Lin- CD11b+ HLA-DR- CD14- CD33+) were increased in the peripheral blood of Stage IV melanoma patients relative to healthy donors. These frequencies correlated with increased expression of PD-1 on peripheral T cells and an increased frequency of regulatory T cells. CD33+ MDSCs isolated from the peripheral blood of Stage IV melanoma patients, but not healthy donors, suppressed T cell activation and the frequency of these cells correlated with disease progression and decreased overall survival. Our study provides evidence that the frequency of CD33+ MDSCs negatively correlates with clinical outcomes in advanced-stage melanoma patients. These data indicate that suppressive MDSCs should be considered as targets for future immunotherapies.
ABCG1 as a novel link between cholesterol homeostasis and tumor immunity (P2031)
Authors: Sag Duygu , Cekic Caglar , Wu Runpei , Linden Joel , Hedrick Catherine
Published: January 2023

ATP-binding Cassette Transporter G1 (ABCG1) promotes cholesterol efflux from cells for intracellular cholesterol homeostasis. ABCG1 has been shown to regulate macrophage and lymphocyte immune responses. In this study, we demonstrate a role of ABCG1 as a novel modulator of tumor immunity. MB49 bladder carcinoma or B16 melanoma cells were injected into Abcg1-/- or B6 mice subcutaneously. Both groups of mice were fed with either high-fat diet or chow diet starting a week before injection of tumor cells. Abcg1-/- mice on high-fat diet had dramatically reduced (80 %) tumor growth compared to B6 mice. Abcg1-/- mice on high-fat diet also showed diminished tumor metastasis and prolonged survival. Furthermore, we showed that reduced tumor size in Abcg1-/- mice on high-fat diet was associated with an increase in the frequency of NK cells and T cells and decrease in the frequency of macrophages and Tregs in tumor. Selective inhibition of ABCG1 in myeloid cells, but not in T cells, reduced tumor growth in vivo, demonstrating that impaired tumor growth in the absence of ABCG1 is mediated through myeloid cell-intrinsic factors. Moreover, ABCG1 deficiency caused an increase in apoptosis of macrophages and a shift of macrophages to an M1 phenotype in the tumor under high-fat diet conditions. Overall, our study shows that absence of ABCG1 inhibits tumor growth through regulation of macrophage function within the tumor and provides a link between diet, cholesterol homeostasis, and cancer.
TGF-β dependent suppression of effector T cell function within the tumor microenvironment (P2033)
Authors: Budhu Sadna , Schaer David , Li Yongbiao , Houghton Alan , Silverstein Samuel , Merghoub Taha , Wolchok Jedd
Published: January 2023

Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microcopy, we found that Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and Teff hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific T cells. In vivo depletion of Tregs in foxp3-DTR mice prior to tumor excision reversed the suppression. In vivo modulation of Tregs by GITR ligation had a similar effect, reducing the number of intra-tumor Tregs leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-β reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 T cells recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-β dependent manner shedding light into novel ways to inhibit intratumoral Tregs.
FOXO3 regulates tumor-associated DC tolerogenicity by binding and sequestering NF-kB. (P2037)
Authors: Watkins Stephanie , Hurwitz Arthur
Published: January 2023

Tumor-associated dendritic cells (TADC) are a major contributor of immune tolerance in the tumor microenvironment. We previously reported that the transcription factor FOXO3 is a critical mediator of TADC tolerogenicity and that silencing Foxo3 expression promoted anti-tumor immunity. In the current study, we sought to identify the mechanism by which FOXO3 regulates TADC function. It was previously reported that FOXO3 to inhibits NF-kB activity in T cells through the transcriptional regulation of IkB proteins. In contrast, our data demonstrate that unlike in non-DC populations, FOXO3 was principally detected in the cytoplasm of TADC. Further analysis revealed that despite extensive phosphorylation, cytoplasmic FOXO3 was stable. These findings suggested that FOXO3 may not solely function as a transcriptional regulator. Instead, our findings show for the first time that FOXO3 binds both the RelA and p50 NF-kB subunits and sequesters them to the cytoplasm. The formation of this protein complex prevents nuclear translocation and downstream pro-inflammatory signaling by NF-kB. In the absence of FOXO3, NF-kB re-localizes to the nucleus and turns on expression of pro-inflammatory genes. We further demonstrate that NF-kB enhances cytoplasmic FOXO3 stability. These new insights into the regulation of DC tolerogenicity provide new potential targets for the enhancement of immune-based therapies for cancer, where FOXO3 may reduce pro-inflammatory activities of TADC.
Soluble CD80 restores T cell activation and overcomes tumor cell Programmed Death Ligand-1-mediated suppression (P2041)
Authors: Haile Samuel , Dalal Sonia , Clements Virginia , Tamada Koji , Ostrand-Rosenberg Suzanne
Published: January 2023

Many tumor cells escape anti-tumor immunity through their expression of Programmed Death Ligand 1 (PDL1 or B7-H1), which interacts with T cell-expressed PD1 and results in T cell apoptosis. We previously reported that transfection of human tumor cells with a membrane-bound form of the costimulatory molecule CD80 prevented PD1 binding and restored T cell activation. We now report that membrane-bound CD80 similarly reduces PDL1-PD1-mediated suppression by mouse tumor cells, and that a fusion protein consisting of the extracellular domains of CD80 fused to an Fc domain of IgG1 overcomes PDL1-mediated suppression by human tumor cells. T cell activation experiments assessing costimulation indicate that the soluble CD80 fusion protein mediates its effects by binding to PDL1 and inhibiting PDL1-PD1 interactions. Comparison of the CD80 fusion protein to antibodies specific for PD1 or PDL1 demonstrate that soluble CD80 treatment is more effective in restoring T cell activation than treatment with mAb to either PD1 or PDL1. These studies identify soluble CD80 as an alternative and potentially more efficacious therapeutic agent for overcoming PDL1-induced immune suppression and facilitating tumor-specific immunity.
Deregulation of the Fas-mediated apoptosis pathway confers MDSCs ability to evade apoptosis to persist in tumor-bearing hosts (P2055)
Authors: Paschall Amy , Hu Xiaolin , Bardhan Kankana , Yang Dafeng , Waller Jennifer , Park Mary , Nayak-Kapoor Asha , Samuel Thomas , Abrams Scott , Liu Kebin
Published: January 2023

Myeloid-derived suppressive cells (MDSCs) are potent immune suppressive cells that accumulate with tumor progression. Compelling data from mouse models and human cancer patients have shown that tumor-induced inflammation induces MDSC differentiation. However, the mechanisms underlying MDSC persistence is unknown. Here, we made use of orthotopic mammary and colon carcinoma mouse tumor models and demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis in vivo, as well as decreased sensitivity to FasL-induced apoptosis in vitro, as compared to myeloid cells with the same phenotype from tumor-free mice. Furthermore, tumor-induced MDSCs accumulated in a greater rate in Faslgld mice than in wild type mice. Analysis of gene expression profiles revealed that IRF8 is dramatically down-regulated in MDSCs from tumor-bearing mice and from human cancer patients as compared to tumor-free mice and normal human donors, respectively. Further analysis of IRF8 target gene expression indicated that Fas and Bax are down-regulated, whereas Bcl-xL is up-regulated in MDSCs from tumor-bearing mice. ABT-737 therapy increased MDSCs spontaneous apoptosis and decreased MDSC accumulation in vivo. Our data determine that MDSCs use down-regulation of IRF8 to deregulate the Fas-mediated apoptosis pathway to evade elimination by apoptosis in tumor-bearing hosts, and targeting Bcl-xL is potentially an effective approach to suppress MDSC persistence in cancer therapy.
Neuropilin-1 mediates infiltration of regulatory T cells into tumors favoring immune escape (P2063)
Authors: Buer Jan , Hutzler Marina , Helfrich Iris , Schadendorf Dirk , Hansen Wiebke
Published: January 2023

Infiltration of tumors by Foxp3+ regulatory T cells contributes to tumor development and progression by dampening an effective anti-tumor immune response. We show that the surface receptor Neuropilin-1, highly expressed by Foxp3+ regulatory T cells, controls the immunological anti-tumor response by directing regulatory T cells into the tumor in a Vascular Endothelial Growth Factor dependent mode of action. Deletion of Neuropilin-1 in CD4+ T cells or abrogation of Vascular Endothelial Growth Factor production by the tumor cells itself resulted in a major impairment of tumor development and progression accompanied by an increased CD8+ T cell response. Most importantly, the frequency of tumor-infiltrating Foxp3+ regulatory T cells was clearly reduced within tumors from both T cell-specific Neuropilin-1-deficient mice and wild type mice transplanted with Vascular Endothelial Growth Factor-deficient tumor cells. From this, we conclude that Neuropilin-1 represents a key mediator of Foxp3+ regulatory T cell infiltration into Vascular Endothelial Growth Factor producing tumor sites.
Immune checkpoint protein VISTA regulate autoimmunity and anti-tumor immunity (P2069)
Authors: Wang Li , LeMercier Isabelle , Lines Janet , Sergent Petra , Li Jiannan , Noelle Randolph
Published: January 2023

V-domain Ig Suppressor of T cell Activation (VISTA) is a newly discovered immune checkpoint ligand that suppresses T cell activation. Our study demonstrates the critical role of VISTA in controlling autoimmunity and anti-tumor immunity. Within the hematopoietic compartment, VISTA is highly expressed on myeloid antigen-presenting cells, naïve T cells and Foxp3+ Tregs. Aged VISTA ko mice developed inflammatory phenotypes. When bred onto a TCR transgenic background that is specific for myelin oligodendrocyte glycoprotein (MOG), VISTA ko mice developed severe autoimmune disease at 6-7 weeks of age. VISTA also regulates the suppressive function of Foxp3+CD4+ regulatory T cells (Tregs), as well as the induction of adaptive Tregs in the periphery. During tumor development, VISTA expression is upregulated within the tumor microenvironment. A VISTA-specific monoclonal antibody (mab)-mediated blockade enhanced tumor-specific T cell priming, as well as altered the suppressive character of the tumor microenvironment by reducing tumor-infiltration of myeloid-derived suppressor cells while enhancing the infiltration and effector function of tumor-specific effector T cells. Consequently, VISTA mab-mediated blockade as a single reagent significantly suppressed the growth of both transplantable and inducible melanoma tumors, and when combined with a cancer vaccine, effectively eradicated established tumors. VISTA thus represents a novel and promising target for cancer immunotherapy.
Role of NPRA in MDSC mediated suppression in cancer (P2071)
Authors: Nagaraj Srinivas , Nelson Allison , Mohapatra Subhra , Mohapatra Shyam
Published: January 2023

It is now evident that inadequate function of the host immune system is due to suppressive factors such as myeloid derived suppressor cells (MDSC). In our study we found that MDSCs isolated from tumor bearing mice expressed high levels of natriuretic peptide receptor A (NPRA) a receptor for atrial natriuretic peptide (ANP). Since MDSCs play a pivotal role in tumorigenesis, we addressed whether NPRA signaling may be linked with altered differentiation of myeloid cells and their suppressive function. We examined the wild type and NPRA deficient mice for MDSC differentiation, and the results showed that NPRA deficient mice exhibited restricted tumor growth and altered differentiation of MDSCs. Next we investigated the link between ANP levels and accumulation of MDSCs. Increase in ANP levels triggered expansion of MDSC. MDSCs suppressed T cells via reactive oxygen species (ROS), and increase in NPRA expression was associated with decreased miR150 as well as increased expression of Nox. NPRA KO mice showed increased expression of miR150 compared to WT mice.Our experiments suggested that activation of NPRA and was regulated by a key micro RNA, miR-150. Taken together, these results suggest a key role to NPRA signaling in myeloid cell differentiation and cancer progression.
Escape of myeloma cells from CD4 T-cell-driven immunoprotection (P2015)
Authors: Tveita Anders , Schjesvold Fredrik , Haabeth Ole Audun , Bogen Bjarne
Published: January 2023

Background: CD4+ T cells can mediate efficient immunosurveillance against MHC class II-negative cancer cells through indirect presentation of tumor-specific antigens on antigen-presenting cells (APCs). A CD4+ T cell response against an epitope on the immunoglobulin light chain of the Id peptide of MOPC315 myeloma cells can mediate an antitumor immune response that induces long term tumor dormancy, followed by eventual tumor escape. Interestingly, this escape occurs despite continued secretion of the tumor-specific antigen. Objective: To identify the immunoediting mechanisms underlying the loss of T-cell-mediated immunoprotection against MOPC315 myeloma cells Method: Mice injected with fluorescently labeled MOPC315 tumor cells were monitored using in vivo imaging. Parental and escape tumor cell clones were obtained, and gene- and protein expression characterized. Results: Escape tumor cells secrete an intact M315 Id peptide, but secretion of free light chains is selectively down-regulated. This limits exposure of the inciting antigen in a form that permits efficient antigen display by tumor-infiltrating APCs, despite preserved T cell reactivity within draining lymph nodes. Our study defines a novel mechanism of tumor immunoevasion by modified secretion of a natural tumor-specific antigen, which emphasizes the importance of antigen presentation within the tumor microenvironment, and has implications for the design of effective CD4+ T cell-based immunotherapy.