bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT3 regulates iNKT cell development and function but is not required for NKT17 cell development (LYM6P.715)
Authors: Mi Qing-Sheng , Zhang Xilin , Li Guihua , Koralov Sergei , Zhou Li
Published: January 2023

Signal transducer and activator of transcription 3 (STAT3) controls the expansion of T help 17 cell (Th17) lineage through induction of transcription factor orphan nuclear receptor (RORγt). CD1d-restricted invariant natural killer T (iNKT) cells are potent regulators of diverse immune responses and have three sublineages, NKT1, NKT2, and NKT17 cells. However, it still remains unclear how to regulate NKT17 cell development. Using lymphocyte-specific STAT3 hyperactivation mice, we found that overactivation of STAT3 universally suppressed iNKT cell cytokine secretion, including IL-17, while it enhanced conventional Th17 cell development. Consistently, STAT3 hyperactivation reduces the RORγt expression of iNKT cells. Surprisingly, bone marrow chimera experiments revealed that STAT3 overactivation disturbed iNKT cell maturation and increased IFNγ- and IL-4-secreting iNKT cells while it did not affect IL-17 secreting iNKT cells and did not alter RORγt expression of iNKT cells. Our results demonstrate that STAT3 regulates the development and function of iNKT cells, but unlike conventional Th17 cells, STAT3 is not required for IL-17 production and RORγt expression in iNKT cells.
A role for dectin-1 in the CNS during chronic Toxoplasma gondii infection (MPF5P.739)
Authors: Worth Danielle , Nance John Philip , Jang Jessica , David Clement , Nair Meera , Wilson Emma
Published: January 2023

The latent stage of T.gondii infection is characterized by the formation of tissue cysts within the CNS. These tissue cysts are formed by manipulation of the parasitophorous vacuole, and chitin is a structural component of the cyst wall. Our lab has shown that Toxoplasma cysts induce an M2 phenotype in macrophages in a contact-dependent manner. Upon contact, these macrophages actively secrete the mammalian chitinase, AMCase, to break down the cyst wall. We have previously demonstrated that AMCase production by macrophages is essential for control of cyst burden in the brain during T. gondii infection. The molecular interactions involved in macrophage-cyst recognition, and the signaling pathway for AMCase production are as yet unknown. Although chitin recognition has been poorly defined, studies suggest dectin-1 may be a receptor. Here, we present data on a role for dectin-1 during T. gondii infection. Dectin-1 is upregulated in the brain in response to infection and is expressed on arginase-1+, M2 macrophages. Consistent with dectin-1 expression on M2 macrophages, dectin-1 is not required to control parasite burden or induce an inflammatory immune response in vivo. However, dectin-1 deficient mice do exhibit trends towards decreased cyst numbers in the brain and a concomitant increase in AMCase transcripts and AMCase activity ex vivo. Thus, this data suggests that dectin-1 is modulating the immune response and chitinase production during chronic Toxoplasma infection.
Intravital imaging of the colonic mucosa: analysis of iNKT physiology during health and disease (MUC5P.752)
Authors: Zemp Franz , Waterhouse Christopher , Kubes Paul
Published: January 2023

Invariant natural killer T-cells (iNKTs) are specialized innate-like T cells that respond to glycolipid antigens with the rapid secretion of polarizing cytokines. Recent literature has shown that iNKTs are important in mouse models of IBD, but have failed to demonstrate the role of colonic iNKTs specifically. Further, the behavior of these cells has never been observed in the gut. Using intravital spinning disk confocal microscopy and transgenic mice that label iNKT cells, we found that iNKTs actively survey the colonic lamina propria. These cells arrested upon administration of a glycolipid antigen, which was largely presented by colonic epithelial cells. Activation of iNKTs with this ligand produced a rapid inflammatory response typified by proximal colon edema and neutrophil recruitment in the submucosal venules. We extended these studies to the DSS model of colitis, and found iNKT deficiency exacerbates disease. This is even more apparent in CD1d deficient animals, which lack iNKT cells and glycolipid antigen presentation. Current work is evaluating the activation of colonic iNKT cells in this model of colitis. Our data suggest that iNKT activation in the colon results in an acute inflammatory response. However, during colitis, iNKTs and possibly another CD1d-utilizing cell type, play a protective role. Current studies are examining the mechanism(s) behind iNKT activation in the colon and how this activation orchestrates colonic inflammation.
Vitamin A deficiency creates persistent shedding of Citrobacter rodentium and potential reservoirs for the spread of enteric pathogens (MUC5P.755)
Authors: McDaniel Kaitlin , Restori Katherine , Dodds Jeffery , Kennett Mary , Ross A. , Cantorna Margherita
Published: January 2023

Vitamin A deficiency remains a public health concern in developing countries and is associated with increased susceptibility to infectious disease and childhood mortality. We utilized Citrobacter rodentium to model human enteric infections. 40% of vitamin A deficient (A-) mice developed a severe and lethal infection. All of the vitamin A sufficient (A+) mice survived and cleared the infection by day 25. Retinoic acid (RA, active form of vitamin A) treatment of A- mice before and at the time of infection resulted in clearance of C. rodentium with the same kinetics as the A+ mice. Additionally, RA treatment of A- mice at the peak of the infection eliminated C. rodentium within 16 days of treatment. Although the A- mice remained infected at day 37, colon inflammation had resolved to that found in A+ mice. All of the A- and A+ mice were able to clear systemic challenge with C. rodentium indicating that A- mice are able to control systemic bacterial infection. Furthermore, the increased mortality of A- mice during infection was not due to systemic cytokine production or increased C. rodentium pathogenicity. Instead the A- mice seem to have developed a severe gut infection but 60% of the A- mice were able to survive and resolve the inflammation. Our work suggests that improvements in vitamin A status and/or RA treatments may decrease susceptibility to enteric pathogens and eliminate potential carriers from spreading infection to susceptible populations.
Intestine lamina propria CD4+T cells promote the bactericidal activity of macrophages via galectin-9 and Tim-3 interaction during Salmonella typhimurium infection (MUC5P.760)
Authors: Zhang Cai , Zhang Hongru , Zuo Zengyan , Li Zhiyuan , Han Qiuju , Zhang Jian
Published: January 2023

The intestinal immune system is crucial for protection the host from pathogenic infection and maintenance of mucosal homeostasis. The intestine lamina propria macrophages are main effector cells in innate resistance to intracellular microbial pathogens. In the present study, we found that Salmonella typhimurium infection augmented the Tim-3 expression on intestinal lamina propria CD4+T cells and enhanced galectin-9 expression on F4/80+CD11c+macrophages. Moreover, CD4+T cells promote the activation and bactericidal activity of F4/80+CD11c+macrophages via galectin-9 and Tim-3 interaction during Salmonella typhimurium infection. Blockade the interaction of Tim-3 and galectin-9 with a-Lactose significantly attenuated the killing of intracellular bacteria by macrophages. Furthermore, the interaction of Tim-3 and galectin-9 also promoted the formation and activation of inflammasome, which leads to the cleavage of caspase-1 and IL-1β. The secretion of active IL-1β further improved bactericidal activity of macrophages and expression of galectin-9 on macrophages. These results demonstrated the critical role of the cross-talk between CD4+T cell and macrophage, in particularly the interaction of Tim-3 and galectin-9, in antimicrobial immunity and control intestinal pathogen infection.
Effector CD4+CD25- T cells from Stearoyl CoA Desaturase deficient mice drive chronic colitis in RAG-1KO mice (MUC5P.761)
Authors: Yeoh Beng San , Singh Vishal , Vijay-Kumar Matam
Published: January 2023

Fatty acids in lipid bilayer substantially influence membrane fluidity and thus dictate cellular functions. Stearoyl CoA Desaturase (SCD-1) is a lipogenic enzyme that assists in the de novo biosynthesis of oleate (C18:1, n9), a major fatty acid in the phospholipids of lipid bilayer. Accordingly SCD-1KO mice display substantially reduced oleate in cell membranes. We investigated the colitogenic capacity of effector T-cells from SCD-1KO mice and their WT littermates in adoptive T-cell transfer colitis model. Splenic effector T-cells (CD4+CD25-) from age and gender matched WT and SCD-1KO mice were isolated by cell sorting using magnetic beads and flow cytometry. RAG-1KO (n=5) were administered 5.0x105 CD4+CD25- cells/mouse intraperitoneally and monitored for body weights and colitis development. RAG-1KO mice which received CD4+CD25- cells from SCD-1KO mice displayed colomegaly, splenomegaly, elevated colonic, systemic lipocalin-2, a general inflammatory marker and hallmarks of colitis at histologic level. Interestingly, colonic TNFα and IL-10 gene transcripts were significantly reduced in SCD-1KO CD4+CD25- recipients whereas the opposite is true for iNOS and lipocalin-2. Further, systemic sIL-1Ra was significantly reduced in SCD-1KO CD4+CD25- recipients when compared to WT T cell recipients. Our results demonstrate that effector T-cells from oleate poor environment attain colitogenic properties and may be associated with increased immunoreactivity to gut microbiotal antigens.
Supplementation of the aryl hydrocarbon receptor ligand indole-3-carbinol protects mice from Clostridium difficult associated disease. (MUC5P.762)
Authors: Julliard Walker , Fechner John , O'Driscoll Chelsea , Safdar Nasia , Mezrich Joshua
Published: January 2023

We have previously presented the dramatic benefit in survival that the dietary aryl hydrocarbon receptor (AHR) ligand indole-3-carbinol (I3C) confers in C. difficile disease in mice. Other groups have demonstrated that γδ T cells and Tregs play an important role in preventing other models of colitis. The current study sought to determine whether dietary I3C could protect mice from C. difficile by increasing the amounts of protective γδ T cells and Tregs. Furthermore, we examined the role of I3C in preventing antibiotic mediated dysbiosis. Mice were fed an experimental AHR ligand free diet (“base”) or the same diet supplemented with I3C and then infected with C. difficile. Pre-infection, base mice had less γδ T cells and Tregs in their cecum compared to I3C mice. Furthermore, I3C mice had a more robust and less pathologic microbiome pre-infection. Post-infection, I3C mice continued to have higher levels of γδ T cells and Tregs. These data suggest that AHR ligand supplementation can protect the host from C. difficile by reducing antibiotic associated dysbiosis as well as augmenting the local immune system to be more protective from C. difficile associated inflammation. Given the poor treatment options currently available as well as the high morbidity of infection, this represents an exciting new therapeutic possibility.
Differential metabolic features and pathogenic potential of effector CD4 T cells associated with IFNγ production during intestinal inflammation (MUC5P.749)
Authors: Shin Boyoung , Kramer Philip , Darley-Usmar Victor , Harrington Laurie
Published: January 2023

Interferon γ (IFNγ) production is a hallmark feature of mucosal CD4 T cells in inflammatory bowel disease (IBD) patients and multiple mouse models of colitis. However, the contribution of the IFNγ producing cell population to chronic intestinal inflammation remains controversial and requires better understanding. Recent studies have linked cellular metabolism to effector functions of immune cells, but it is unknown if discrete effector T cell subsets present during IBD possess divergent metabolic properties and differential T cell pathogenicity. Using IFNγ reporter mice, viable IFNγ secreting and non-secreting CD4 T cells were isolated from mice with active colitis and examined for metabolic characteristics. While both IFNγ producing and non-producing cells exhibit a high rate of glycolysis, interestingly, the mitochondrial function differs; IFNγ producing cells have lower oxidative phosphorylation and blunted spare respiratory capacity, indicating defective mitochondrial function compared to IFNγ non-producing cells. Consistent with decreased mitochondrial function, IFNγ producing cells have lower cell recovery upon secondary transfer and result in more attenuated intestinal inflammation. Therefore, these data suggest that CD4 T cells that upregulate IFNγ mark a unique effector cell subset with distinctive metabolic features, impaired survival, and reduced pathogenicity, which is paradoxical to the proinflammatory nature of IFNγ during chronic intestinal inflammation.
Non-human primate model of collagen II-induced arthritis: a comprehensive system simulating human rheumatoid arthritis for evaluation of novel therapeutic candidates (THER5P.901)
Authors: Zeng Jia , Dai Feihong , Cheng Yu , Yang Nan , Yang Ping , Zheng Hellen , Fu Yunfeng , Duan Jeff
Published: January 2023

Non-human primate (NHP) model provides the advantage of closer phylogenetic relationship to human and high cross-reactivity to the human target specific biological molecules that do not react with rodent counterpart. PharmaLegacy has been dedicated in developing NHP inflammation models since 2009. Here, we report a NHP model of Collagen II induced Arthritis in Cynomolgus monkey, which developed similar symptoms as human RA in terms of body weight loss, clinical score lifting, joint swelling enlargement and rising PIP incidence. Terminal analysis by radiographic imaging and the multi-dimensional scaling for histopathology and histomorphometry on joints demonstrated similar characteristics of human RA joint deformity, including the significant cell infiltration, loss of proteoglycans (PG) in cartilage, Pannus development, osteophyte formation, loss of articular structure and bone erosion, well supporting for the arthritic disease. Therapeutic treatment with mAb HSF082D significantly reversed the above disease index. In a study of biomarkers profile in Serum or RA joint, CRP/IL-1β/IL-6/IL-17A/RANKL/GM-CSF were found to correlate with the arthritic severity at mRNA or protein levels. Th17 cell activities in synovial fluid were also detected.
Transcriptome analysis of circulating lymphoid progenitors after allogeneic stem cell transplantation in humans reveals profound alterations of cellular metabolism (TRAN1P.930)
Authors: Toubert Antoine , Glauzy Salome , André-Schmutz Isabelle , Lachuer Joel , Peffault de Latour Regis , Servais Sophie , Socié Gerard , Clave Emmanuel
Published: January 2023

T-cell reconstitution is delayed by acute Graft-Versus-Host Disease (aGVHD) during allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we analyzed transcriptional programs of cell-sorted circulating lymphoid progenitors and CD34+Lin-CD10- non lymphoid progenitors in 12 allo-HSCT patients having (n=4) or not developed (n=8) grade 2 aGVHD and in 4 age-matched healthy donors. Gene expression was quantified on the GeneChip HG u133 plus 2.0. Pathway analyses were performed with KEGG, GSEA and DAVID. Major deregulated pathways included protein synthesis, energy production, cell cycle regulation and response to stress. Notably, Genes from ribosome protein biogenesis, translation machinery (EEF1D, EEF1G, EIF3K) and cell cycle (CCND1, CDK6) were over-expressed in progenitors from patients without aGVHD compared with those from patients affected by aGVHD and from healthy donors. Expressions of genes from the oxidative phosphorylation metabolic pathway (NDUFS2, SDHA, ATP5A1) and genes involved in stress resistance (BTG2, MGST3, HPX) were specifically increased in CD34+Lin-CD10+CD24- lymphoid progenitors from patients without aGVHD. In all, we show that circulating lymphoid T-cell progenitors undergo profound changes in metabolism favoring energy production and response to stress after allo-HSCT in humans. These mechanisms are abolished in case of aGVHD, indicating a persistent cell-intrinsic defect in addition to the impact of aGVHD on the bone marrow environment.
Donor CD103+ dendritic cells in the colon engender lethal graft-versus-host disease (TRAN1P.938)
Authors: Koyama Motoko , Cheong Melody , Markey Kate , Gartlan Kate , Kuns Rachel , Locke Kelly , Lineburg Katie , Teal Bianca , Bunting Mark , Vuckovic Slavica , Zhang Ping , Teng Michele , Varelias Antiopi , Tey Siok-Keen , Wockner Leesa , Engwerda Christian , Smyth Mark , Belz Gabrielle , McColl Shaun , MacDonald Kelli , Hill Geoffrey
Published: January 2023

The spatial and temporal nature of donor antigen presentation during graft-versus-host disease (GVHD) and its relevance to disease severity are poorly understood. Here we use luciferase expressing TCR transgenic CD4+ T cells specific for indirect alloantigen presentation within MHC class II to demonstrate that GVHD markedly enhances indirect presentation within the mesenteric lymph nodes (mLN). By using antibody specific for alloantigen complexed with donor MHC and IRF4 and CCR7 deficient donors we demonstrate that this presentation is entirely mediated by the donor CD103+CD11bneg dendritic cell (DC) subset that migrates from the colon under the influence of CCR7. We used IL-12, IL-6, MyD88/TRIF and receptor for advanced glycation end products (RAGE) deficient grafts to demonstrate that this differentiation of donor T cells in the mLN during GVHD is driven by high levels of alloantigen and local IL-12 and IL-6 secretion elicited by Toll-like receptor and RAGE signals. Notably, alloantigen presentation by CD103+ DC migrating from the colon imprints gut-homing integrin signatures on donor T cells in the mLN, leading to their emigration into the gastrointestinal (GI) tract where they mediate fulminant GVHD. These data highlight multiple therapeutic targets and the ability of GVHD, once initiated by recipient APC, to generate a profound and localized feed-forward cascade of indirect antigen presentation and cytokine secretion within the GI tract, culminating in lethality.
Jak/STAT signaling inhibition enhances costimulation blockade and promotes transplant acceptance (TRAN1P.943)
Authors: Raimondi Giorgio , Khalifian Saami , Miller Devin , Oh Byoungchol , Lee WP , Brandacher Gerald
Published: January 2023

Transplant tolerance induction through costimulation blockade (CoB) remains an elusive goal. Recent evidence suggests that inflammatory cytokines (IC) contribute to the activation of alloreactive T cells in a CD28- and CD40-independent manner. We aimed to delineate the possible synergism between CD28 blockade and inhibition of IC signaling via Jak/STAT inhibition. First, we measured the impact on mouse T cell activation via quantification of IL-2 secreting cells at 24h post in vitro stimulation. Although CTLA4-Ig diminished the proportion of IL-2+ cells, the concomitant addition of the supernatant (MATSup) from maturing dendritic cells completely abolished this effect. This suggested an early impact of IC on T cell activation. MATSup counteracted the anti-proliferative effect of CTLA4-Ig on both CD4 and CD8 T cells. However, addition of the Jak3/1 inhibitor Tofacitinib (Tofa) completely restored the effect of CTLA4-Ig in presence of MATSup. In a BALB/c to B6 heart transplant model a short course of Tofa (d0-6) synergized with CTLA4-Ig promoting long term graft survival (MST untreated: 11d, CTLA4-Ig only: 36d, Tofa+CTLA4: 114d). Survival was associated to lower Th1 cell production and, unexpectedly, an increment in graft infiltrating Treg. Overall, our results indicate that inflammatory cytokines counteracts the efficacy of CoB. However, this effect can be neutralized via transient inhibition of Jak signaling - a promising new immunoregulatory strategy we define Enhanced CoB.
Human mesenchymal stem cell-educated macrophages: a potential therapy for GVHD and radiation injury (TRAN1P.944)
Authors: Bouchlaka Myriam , Kim Jaehyup , Bloom Debra , Hematti Peiman , Capitini Christian
Published: January 2023

Total body irradiation is typically used to condition patients for allogeneic hematopoietic stem cell transplant, leading to release of inflammatory cytokines from damaged tissues. After transplant, many patients develop graft-versus-host disease (GVHD) resulting from donor T cells attacking host tissues leading to further inflammation. Mesenchymal stem cells (MSCs) have immunosuppressive and tissue repair properties, but clinical trials using MSCs to treat GVHD have shown mixed results. Macrophages (MQs) are important regulators of immunity and can promote tissue remodeling. We have previously shown that MSCs can educate MQs toward a unique anti-inflammatory phenotype, however the implications for in vivo models of inflammation have not been studied. Using flow cytometry, we found that in comparison to MQs, mesenchymal stem cell-educated macrophages (MEMs) have increased expression of the inhibitory molecules PD-L1 (p=0.039), PD-L2 (p=0.0002), and markers of alternatively activated macrophages: CD206 (p=0.04) and CD163 (p= 0.0004). MEMs also show increased expression of TGF-β (p<0.0001), Arginase-1 (p<0.0001) and IL-6 (p=0.0006), and decreased expression of IL-12 (p=0.004) and TNF-α (p<0.0001) by RT-PCR. In two xenogeneic mouse models, we show that MEMs significantly enhance survival from lethal GVHD (p=0.028 vs MSC) and from lethal radiation (p=0.0044 vs MSC). MEMs could be a novel cellular therapy for the management of GVHD and protection from radiation-induced injury.
Delayed CTLA-4Ig treatment of established acute allograft rejection successfully abrogates ongoing B cell responses (TRAN1P.950)
Authors: Young James , Chen Jianjun , Yin Dengping , Sciammas Roger , Chong Anita
Published: January 2023

Once rejection is initiated, halting the immune response becomes much more difficult and effective therapies are necessary. Despite evidence that CTLA4Ig is ineffective at controlling memory T cell responses, we here report that CTLA4Ig is unexpectedly effective at controlling established B cell responses and treating established acute rejection. When C57BL/6 mice were sensitized with BALB/c splenocytes and treated with CTLA4Ig from 7d post-sensitization, B cell germinal center responses rapidly collapsed and alloantibody production was halted. In contrast, the effector CD4 and CD8 T cells response, which was monitored with seeded TCR-tg or endogenous allospecific cells, was not inhibited, and the extent of CD44 differentiation, IFNg production, and Tfh differentiation was similar to untreated controls. We also tested the effect of delayed CTLA-4Ig in established acute allograft rejection. In C57BL/6 recipients of BALB/c hearts, delayed treatment of CTLA4Ig till 6d post-transplant (just 2d before rejection) inhibited alloAb production and prevented acute rejection. 10 of 15 grafts survived to ≥ 30d post-transplant, despite significant T cell infiltration in the graft. Thus significant control of B cell responses and rejection can be achieved through delayed CTLA-4Ig treatment. This study raises the exciting possibility of using CLTA-4Ig therapeutically, not just prophylactically, in the clinic to control established B cell responses and established acute rejection.
The adaptor protein ASC controls transplantation outcomes independently of the inflammasome (TRAN1P.951)
Authors: Cheong Melody , Gartlan Kate , Tey Siok-Keen , Kuns Rachel , Lor Mary , Lineburg Katie , Teal Bianca , Shi Wei , Raju Jyothy , Zhang Ping , Varelias Antiopi , Leveque-El Mouttie Lucie , Olver Stuart , Bunting Mark , Lane Steven , Boyle Glen , Ting Jenny , Schroder Kate , Engwerda Christian , Khanna Kum Kum , Smyth Mark , MacDonald Kelli , Koyama Motoko , Hill Geoffrey
Published: January 2023

The adaptor protein ASC is known to facilitate caspase-1 activation essential for innate host immunity via the formation of the inflammasome complex - a multi-protein structure responsible for processing IL-1b and IL-18 to their active moieties. Here we report for the first time, a unique inflammasome-independent role for ASC in the control of transplant outcome following allogeneic bone marrow transplantation (BMT). We demonstrate that ASC-deficient donor CD8+ T cells fail to induce GVHD lethality due to an inability to differentiate into fully cytolytic effectors after BMT, with a developmental bias instead towards CD127+KLRG1- memory CD8+ T cells. Despite this, graft-versus-leukaemia effects against BCR-ABL NUP98/HOXA9 primary leukemia remained largely intact. This phenomenon was inflammasome independent, since GVHD lethality and T cell differentiation were not altered in recipients of caspase-1-deficient T cells. We also confirmed a reduced capacity for human T cells in which ASC was knocked down by shRNA to induce xenogeneic GVHD. In a model of bone marrow rejection, ASC expression in recipient CD8+ T cells profoundly impaired graft rejection and was permissive of robust engraftment across MHC barriers and long term survival. Taken together, these findings demonstrate an inflammasome-independent role for ASC in controlling GVHD and graft rejection. Thus, the inhibition of ASC in the clinic represents an important new therapeutic target to manipulate transplant outcomes.
Hyperglycemia is indispensable for delayed islet graft rejection in STZ-induced diabetes mouse model. (TRAN1P.952)
Authors: Bogacz Marek , Drutel Robert , Leanhart Silvia , Kern Joanna , Pacholczyk Rafal
Published: January 2023

Transplantation of allogeneic pancreatic islets or pancreas is one of the methods to control euglycemia in patients with type 1 diabetes. However despite emergence of new tolerance induction protocols these patients require lifelong immunosuppression which is associated with many adverse effects including toxicity towards transplanted pancreatic β-cells. It has been previously shown that streptozotocin (STZ)-induced diabetes can delay or abrogate rejection of allografts in CBA/J or C57BL/6 mice. To test this effect on autoimmune prone background we performed a series of allogeneic-islet transplantation experiments using NOD mice as recipients. Our results show that if transplanted during specific time window after STZ treatment (2-3 days) graft rejection will be delayed but not completely abrogated. Using insulin pump to control blood glucose level we demonstrate that STZ administration without episode of acute hyperglycemia is not sufficient to delay graft rejection. It is despite the fact that STZ injection alone results in increased frequency of regulatory CD4Foxp3+ T cells. Comparison of gene expression in CD4 T cells between STZ-treated and untreated mice shows selective increase of apoptosis in effector but not regulatory T cells.
Generation, cryopreservation, function and in vivo persistence of ex vivo expanded cynomolgus monkey Treg (TRAN1P.927)
Authors: Guo Hao , Zhang Hong , Lu Lien , Ezzelarab Mohamed , Thomson Angus
Published: January 2023

Foxp3+ regulatory T cells (Treg) function to promote immune tolerance and prevent immunopathogenesis. Preclinical studies have demonstrated that adoptive transfer of Treg prevents rejection of organ allografts in mice and humanized mouse models. In this study, we optimized a protocol to expand flow-sorted cynomolgus Treg >1000-fold after three rounds of stimulation with anti-CD3 mAb-pulsed artificial APCs, rapamycin and IL-2. The expanded Treg from each round maintained high level expression of Treg signature markers, -CD25, CD27, CD39, Foxp3, Helios, CTLA-4, as well as CXCR3, a homing receptor of Th1-like cells. In contrast to expanded Teff, Treg produced minimal IFN-γ and IL-17 and no IL-2. In addition, the expanded Treg from each round showed high suppressive capacity in vitro. Compared to freshly-expanded Treg, Treg immediately thawed from cryopreserved stocks maintained Treg signature markers and suppressive capacity, but were less viable. However, additional rounds of stimulation/expansion restored maximal viability. Adoptively-transferred autologous Treg labeled with CFSE (approximately 2x107/kg, i.v.) could be detected in vivo 2-3 months post infusion in peripheral blood, inguinal LN, mesenteric LN and spleen. Moreover, these cells maintained CD25, CCR7, CXCR3 expression and acquired CD62L after infusion in vivo.
c-Met identifies a population of matrix metalloproteinase 9-producing monocytes in peritumoral stroma of hepatocellular carcinoma (TUM6P.966)
Authors: Wu Yan , Zhao Lan , Zheng Limin
Published: January 2023

Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. Previously, we found that tumors could alter the normal developmental process of Mφ to trigger transient activation of monocytes in the peritumoral stroma of human hepatocellular carcinoma (HCC). In the present study, we showed that a fraction of monocytes in the peritumoral stroma, but not in HCC cancer nests, expressed surface c-Met molecules. Monocytes exposed to tumors strongly expressed c-Met proteins with kinetics similar to their activation status, and significant correlations were found between c-Met levels and HLA-DR expression on tumor-infiltrating monocytes. NF-κB-mediated autocrine TNF-α stimulated the expression of c-Met on activated monocytes, and by interacting with its ligand HGF, c-Met regulated the retention, accumulation, and matrix metalloproteinase (MMP) 9-producing capacity of monocytes in the peritumoral stroma of HCC. The intensity of c-Met expression on tumor-infiltrating monocytes was associated with high mortality and reduced survival of patients with HCC. Therefore, the expression of c-Met on activated monocytes/Mφ may represent a novel mechanism by which a tumor actively and precisely regulates the distribution and functions of these cells to facilitate disease progression.
Suppression of tumor growth by a M2b macrophage reverter in humanized murine chimeras created with peripheral blood monocytes of patients with advanced stage hepatocellular carcinoma (TUM6P.967)
Authors: Tsuchimoto Yusuke , Asai Akira , Ohama Hideko , Yokohama Keisuke , Nakamura Ken , Fukunishi Shinya , Tsuda Yasuhiro , Higuchi Kazuhide , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Previously, we have reported that humanized murine chimeras created with peripheral blood monocytes from patients with early stage hepatocellular carcinoma (E-hccM) are resistant against tumor inoculation, while tumors grow in the same chimeras created with advanced stage patient monocytes (A-hccM). A majority of E-hccM are shown to be M1 monocytes, and a predominance of M2b monocytes is shown in A-hccM. In this study, we tried to suppress tumor growth in humanized chimeras created with A-hccM by CCL1 antisense ODN treatment, which reverts Mf from M2b to resident. Chimeras, created in X-irradiated NSG mice after inoculation of A-hccM (i.v., 1 x 106 cells/mouse, every 2 weeks), were inoculated with HepG2 cells (2 x 106 cells/mouse, s.c.). CCL1 antisense ODN (6 mg/mouse) were administered 4 times within 36 hrs of each A-hccM inoculation. In the results, 843 ± 227 mm3 of tumors were detected in the chimeras inoculated with A-hccM 8 weeks after the tumor inoculation, while tumors were not demonstrated in A-hccM chimeras treated with CCL1 antisense ODN, and 753 ± 165 mm3 of the tumors were detected in A-hccM chimeras treated with scrambled ODN. A-hccM were characterized as IL-12-IL-10+CCL1+iNOS-CD14+ cells, while IL-12+IL-10-CCL1-iNOS+CD14+ cells with tumoricidal activities were detected in A-hccM treated with the antisense ODN. These results indicate that M2b monocytes present in A-hccM play a key role in the tumor growth seen in advanced stage hepatocellular carcinoma patients.
Emodin inhibits breast cancer growth by modulating the phenotype of tumor-associated macrophages (TUM6P.1004)
Authors: Iwanowycz Stephen , Wang Junfeng , Jia Xuemei , Fan Daping
Published: January 2023

Infiltration of macrophages into breast tumors is correlated with increased metastasis and decreased patient survival. Activated macrophages display a spectrum of phenotypes, at either end of which are pro-inflammatory/anti-tumor M1 or anti-inflammatory/pro-tumor M2 macrophages. Tumors recruit macrophages to the primary site as well as to pre-metastatic niches, educate them to adopt an M2-like phenotype, and thereby create an immunosuppressive microenvironment. We previously showed that Chinese herb derived compound emodin has the potential to inhibit tumor metastasis by targeting tumor cell-macrophage interactions. Here we tested the ability of emodin to inhibit breast cancer growth in mice and examined the underlying mechanisms. Emodin was used to treat mice bearing EO771 or 4T1 breast tumors. We found that emodin treatment reduced M2-like macrophages in the primary tumors and the lungs, blunted the growth of primary tumors, and attenuated lung metastasis. Further in vitro studies showed that emodin modulates macrophage polarization and function through affecting multiple intracellular signaling pathways as well as through possible regulation of the epigenetic landscape. These results suggest that emodin can be developed as an anti-breast cancer therapy by its virtue of modulating the phenotype of tumor-associated macrophages.
Targeting dysfunctional myeloid cells delays disease development and improves immune function in a CLL mouse model (TUM6P.1009)
Authors: Hanna Bola , McClanahan Fabienne , Zaborsky Nadja , Dürr Claudia , Kalter Verena , Egle Alexander , Lichter Peter , Gribben John , Seiffert Martina
Published: January 2023

Previous in vitro studies have shown that myeloid cells play a key role in apoptosis-resistance in chronic lymphocytic leukemia (CLL). However, their composition and function have not been thoroughly investigated in vivo. Using the Eµ-TCL1 mouse model of CLL, we observed severe skewing of myeloid cell populations along with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes expressing high levels of inflammatory factors like IL-10, TNFα and CXCL9 accumulated also in the spleen, the main site of disease, and were severely skewed towards Ly6Clow phenotype. Along with that, the percentage of MHC-IIhi dendritic cells (DCs) and macrophages significantly dropped in the spleen, suggesting monocyte differentiation arrest. Myeloid cell depletion using liposomal clodronate resulted in a significant control of CLL development, repair of innate immune cell phenotypes and partial resolution of systemic inflammation. Also, CLL-induced loss of naive T cells was resolved. CLL-associated monocytes and DCs aberrantly express high PD-L1 levels suggesting their contribution in T cell defects. Importantly, PD-L1 blockade very effectively prevented CLL development and was accompanied with a re-activation of immune effector cells. This included restoration of mature macrophages and MHC-IIhi DCs. Our preclinical data suggests that targeting myeloid cell survival and immunosuppression can serve as a novel strategy for CLL immunotherapy.
Induction of cell death following restimulation of the T cell receptor results in activation of the DNA damage response pathway in a JNK and ROS-dependent manner (TUM7P.1026)
Authors: Scheffel Matthew , Scurti Gina , Simms Pat , Garrett-Mayer Elizabeth , Nishimura Michael , Mehrotra Shikhar , Voelkel-Johnson Christina
Published: January 2023

The p53 protein is activated by various forms of cellular stress and its function is modulated though a multiplicity of posttranslational modifications to dictate signaling outcomes. A recent study demonstrated that inhibition of p53 by pifithrin-m effectively blocked cell death caused by restimulation of the T cell receptor (TCR). Using human T cells, we investigated the phosphorylation status of p53 following TCR restimulation and detected a time-dependent increase in phosphorylation on Ser15, which correlated with onset of cell death. Phospho-p53 (Ser15) was limited to the nucleus and coincided with phosphorylation of ATM. Blocking ATM function through KU55933 or caffeine prevented cell death. Phosphorylation of ATM suggested activation of the DNA damage response pathway and we confirmed coordinate increases in both yH2AX and SMC-1. Activation of the ATM-p53-dependent DNA damage response pathway following restimulation of the TCR was reduced by inhibition of JNK by SP600125 as well as the anti-oxidant N-acetyl cysteine (NAC). Activation of the DNA damage pathway occurred in an antigen-specific manner and was also observed in T cells derived from melanoma patients undergoing adoptive T cell therapy. In vitro treatment with NAC improved survival of T cells while increasing melanoma cell killing. Thus our data highlight a role for activation of the DNA damage response pathway following TCR restimulation and its potential translational significance in adoptive T cell therapy.
PD-1+CD28H+ selectively identifies for superior tumor-reactive T lymphocytes in pancreatic cancer (TUM7P.1013)
Authors: Zhu Yuwen , Koenig Michelle , Byers Joshua , Paniccia Alessandro , schulick alex , schulick richard , Chen Lieping , edil barish
Published: January 2023

Tumor-infiltrating lymphocytes (TILs) are enriched in tumor-specific T cells and have been used as a source for adaptive cell therapy (ACT) of cancer. TILs are a mixture of different T cell subsets with both anti-tumor and pro-tumor capacities. It is valuable to isolate potent tumor-reactive T cells while eliminating suppressor T cells from TILs to maximize anti-tumor immune response. CD28H is a newly discovered co-receptor of the B7 family that interacts with its ligand B7-H5 to costimulate human T cells. CD28H is constitutively expressed on native T cells. However, repetitive antigenic exposure induces the loss of CD28H expression in T cells. T cells that have lost CD28H have the phenotypic characteristics of terminally differentiated and replicative senescent cells. Here we characterized PBMCs and TILs from patients with pancreatic cancer based on the expression of CD28H and PD-1. We found that a subset of T cells, PD-1+CD28H+ (DP) cells, are exclusively enriched in TILs. Based on phenotypic and functional analysis, our preliminary results suggest that PD-1+CD28H+ cells represent a subset of superior tumor-reactive cells in TILs. By targeting these DP cells by CD28H agonistic mAb and/or with a PD-1 blocking antibody, we are expecting to reprogram TIL functions in human pancreatic cancer.
Effector lymphocytes induce lymph node-like vasculature in tumors, which enables naïve T cell infiltration and enhanced anti-tumor immunity (TUM7P.1020)
Authors: Peske J , Thompson Elizabeth , Gemta Lelisa , Baylis Richard , Fu Yang , Engelhard Victor
Published: January 2023

Intratumoral lymph node (LN)-like vasculature, characterized by the expression of peripheral node addressin (PNAd) and chemokine CCL21, has been correlated with T cell infiltration and a positive prognosis in breast cancer and melanoma patients. However, the mechanisms controlling its development, and how it contributes to a beneficial outcome for cancer patients, are unknown. Here we demonstrate that LN-like PNAd+ CCL21+ vasculature develops in murine models of melanoma and lung carcinoma growing in multiple anatomic locations. PNAd expression on tumor-associated endothelium and CCL21 expression by tumor-associated endothelial cells and gp38+ fibroblasts was controlled by a novel mechanism dependent on tumor infiltrating effector lymphocytes that secreted LTα3. In intraperitoneal (IP) tumors, CCL21 was also controlled by IFNγ. While effector CD8 T cells induced LN-like vasculature in both subcutaneous (SC) and IP tumors, NK cells only promoted its development in SC tumors. In IP but not SC tumors, LN-like vasculature was associated with organized aggregates of B-lymphocytes and gp38+ fibroblasts that resembled tertiary lymphoid organs. Importantly, this LN-like vasculature enabled the infiltration of adoptively transferred naïve T cells. These T cells underwent activation and effector cell differentiation in the tumor, and significantly delayed tumor outgrowth. These results establish LN-like vasculature as both a consequence of and key contributor to anti-tumor immunity.
Anti-CEA chimeric antigen receptor T cells in combination with suppressor cell targeting for the treatment of colorectal cancer peritoneal metastases (VAC7P.1043)
Authors: Point Gary , Thorn Mitchell , Cunetta Marissa , Bais Anthony , Boutros Cherif , Hanna Nader , Espat N , Junghans Richard , Katz Steven
Published: January 2023

Colorectal peritoneal metastases (PM) is a grave condition in which tumor cells disseminate throughout the abdomen. Chimeric antigen receptor T cell (CAR-T) immunotherapy is a promising therapy for PM. We developed a novel regional delivery approach to maximize CAR-T efficacy against PM while limiting systemic toxicity. Murine tumor cells expressing CEA and luciferase were injected intraperitoneally (IP) in mice to induce PM (day 0). We injected murine anti-CEA CAR-T IP on days 3 and 6, alone or in combination with antibodies against Ly6G/Ly6C, PDL1 or GITR to target myeloid suppressor cells (MSC) or regulatory T cells (Treg), which we have shown to be present in PM. Tumor burden was measured by bioluminescence. In comparison to unmodified T cells, CAR-T alone mediated a 22-fold reduction of tumor burden (p=0.028). The combination of CAR-T with anti-PDL1, anti-GITR or anti-Ly6G/Ly6C treated groups showed 25-fold, 104-fold and 153-fold reductions, respectively. The CAR-T + anti-GITR and CAR-T + anti-Ly6G/Ly6C groups had a significantly better treatment response than CAR-T alone (p=0.005 and p=0.017). On days 8 and 10, CAR-T + anti-Ly6G/Ly6C tumor burdens dropped below detectable levels. Upon gross inspection, CAR-T treated mice did not have detectable tumor. Our findings demonstrate that targeting MSC and Treg in addition to regional IP CAR-T delivery represents a viable immunotherapeutic strategy that resulted in complete responses in mice with established PM.
Effects of trans-sialidase enzyme activity, secretion and antigen persistence on the induction of protective immunity against Trypanosoma cruzi infection (VAC8P.1054)
Authors: Cai Catherine , Guo Hongjie , O'Shea Anne , Eickhoff Christopher , Beverley Stephen , Hoft Daniel
Published: January 2023

Chagas Disease is a life-threatening illness caused by chronic infection with the Trypanosoma cruzi parasite. It affects up to 11 million people in Latin America, where it is a leading cause of heart disease, making vaccine development critical and worthwhile. Because the T. cruzi trans-sialidase (TS) enzyme is highly conserved and necessary for parasite virulence, it has strong potential as a vaccine target. We have shown that mice can be protected by vaccination with dendritic cells pulsed with a single pair of immunodominant TS epitopes, validating further exploration of TS as a target. We recently have generated live vaccines by transfecting Leishmania major with four TS variations: active secreted, active non-secreted, inactive secreted and inactive non-secreted. These also were transfected into an avirulent, dihydrofolate reductase knock-out strain of Leishmania to investigate the role of antigen persistence versus clearance on immune response. Mice will be infected with the recombinant Leishmania and later challenged with T. cruzi to analyze protective immunity. In preliminary experiments, recombinants were found to express the TS protein and immunizations protected BALB/c mice from infection. This project is the first examining the ability of recombinant Leishmania to serve as vaccine vehicles for Chagas Disease. These experiments will also elucidate the relative importance of inducing central and peripheral memory T cell populations for protective T. cruzi immunity.
Enhancing pulmonary function in women with lymphangioleiomyomatosis through exercise training (VAC9P.1109)
Authors: Lowder Thomas
Published: January 2023

Lymphangioleiomyomatosis (LAM) is a rare disease affecting women almost exclusively. There is no cure for LAM and few treatment options other than immunosuppressive drugs (e.g. sirolimus, everolimus) exist. Rather than attempting to cure LAM, our lab is focused on reversing declines in pulmonary function through aerobic exercise training. A total of 11 patients are enrolled (age from 27 to 59). Following initial assessments of pulmonary function testing, exercise capacity, and bone mineral density, patients underwent individualized, supervised exercise training (2d/wk). Every 3mo patients were reassessed for changes in the above measures. All patients improved in exercise tolerance (time to fatigue, increased work capacity) and in oxygen consumption (VO2max) following training. Most patients showed an improvement in pulmonary function measures (FEV1, FEV1/FVC) and in bone mineral density, both whole body and lumbar vertebrae. FVC increased in several patients. Anecdotally patients reported improvements in daily living tasks (energy levels, decreased dyspnea upon exertion) and quality of life measures (lower levels of depression, greater outlook on life, improved overall mood). This is the first non-drug intervention to show improvements in pulmonary function in this patient population. Although this study is not yet complete, preliminary results suggest that exercise training only two days per week can result in substantial improvements in patients with LAM.
Characterization of class II β chain major histocompatibility complex genes in a family of Hawaiian honeycreepers: ‘Amakihi (Hemignathus virens) (VET1P.1124)
Authors: Jarvi Susan , Farias Margaret , Bianchi Kiara , Txakeeyang Ann , MacFarland Thomas
Published: January 2023

The objective of this study was to estimate the number and degree of variation of Mhc class II β chain genes and alleles in a family of Hawaiian honeycreepers (`Amakihi, Hemignathus virens). The parents were bred in captivity and produced eight progeny. Partial antigen binding region (173 bp) fragments were produced via PCR+1 cloning and sequenced on a Beckman CEQ system, and two Roche 454 platform pyrosequencing runs. A total of 302 sequences were obtained via cloning. After quality filtering and removal of chimeras, a total of 2762 sequences were retained from 454 run 1 and 3958 sequences from 454 run 2. Unique sequences detected <3 times within an individual were discarded. All sequences were used to generate a neighbor-joining tree in MEGA v. 5.2 to evaluate sequences across family members. Singlet sequences were discarded. The remaining sequences were used to evaluate segregation and relationships of loci. A total of 19 putative loci were detected in this family. Allelic segregation was not observed among any of these loci thus they appear to be independent, but linkage was observed among four loci suggesting segregation of a haplotype among 5 progeny. The finding of 37 sequences in one individual also suggests a minimum of 19 Mhc class II loci in `Amakihi. Of these, 17 loci are classical Mhc loci thus designated as DAB, but mean diversity and evolutionary divergence values among groups suggest that two loci are non-classical and thus should be designated as DBB.