bluuebunny/crossref_metadata_2025 huggingface dataset:
Toxoplasma gondii elicits a phagocytosis dependent pro-inflammatory cytokine response in human primary myeloid cells (INC4P.327)
Authors: Tosh Kevin , Singer Steven , Sher Alan , Jankovic Dragana
Published: January 2023

Toxoplasma gondii is an intracellular protozoan parasite and the etiological agent of toxoplasmosis, a disease seen in immunocompromised individuals such as HIV patients. Studies in murine models have shown that the innate immune response depends on the MyD88-dependent pattern-recognition receptors, TLR11 and TLR12. Thus, mice deficient in TLR11/12 rapidly succumb to acute infection due to uncontrolled parasitemia. While these findings highlighted a critical role for the innate recognition of toxoplasma, they also reveled a major gap in translation to clinical disease since humans do not express TLR11 or TLR12. In order to address how human innate immune cells detect and respond to T. gondii, we infected primary human monocytes, dendritic cells and monocyte-derived macrophages with T. gondii. We found that while all three cell-types become infected and support parasite replication, only monocytes and DC produce significant levels of TNFα and p40 IL-12. Although infection with live parasites was required for an optimal response, we demonstrated that parasite invasion was dispensable while phagocytosis of parasites by monocytes and dendritic cells was necessary for cytokine production. Upon sort-purification, we found that the minor CD16+ monocyte subsets produced the majority of the cytokines in response to infection. Taken together, these results suggest that human monocytes utilize a distinct recognition system from mice that requires phagocytosis of live T. gondii.
Limiting cholesterol biosynthesis acts as a danger signal that engages STING-dependent inflammation (INM2P.349)
Authors: York Autumn , Tong Ann-Jay , Argus Joseph , Gray Elizabeth , Wu Nicholas , Zhen Anjie , Kitchen Scott , Smale Stephen , Stetson Daniel , Bensinger Steven
Published: January 2023

Pathogen invasion alters host lipid metabolism, however the mechanisms underlying this remain poorly understood. Here, we show that transcriptional repression of cholesterol and fatty acid biosynthesis is driven by TRIF-dependent danger signals. Unexpectedly, decreasing cholesterol biosynthesis induces type I interferon (IFN)-mediated inflammation in the absence of conventional danger signals, resulting in resistance to pathogens. Spontaneous induction of inflammation was traced to a reduction in cellular cholesterol levels, and normalizing cholesterol attenuated IFNb and interferon stimulated gene expression. Epistasis studies indicate that the Stimulator of Interferon Genes (STING) links cellular cholesterol balance to type I IFN-mediated inflammation. These findings reveal that perturbing cholesterol homeostasis acts as an independent “danger” signal sensed by host defense machinery, and provides mechanistic insights as to how lipid metabolism influences inflammation.
Vibrio cholerae ghosts elicit the production of soluble immunostimulatory factors and prevent development of Chlamydia infection (INM3P.408)
Authors: Stevens Mumbi , McKeithen D. , Martinez O. , Igietseme J , Eko Francis , Black C , He Q , Ananaba G
Published: January 2023

Infections by the obligate intracellular pathogen Chlamydia trachomatis cause infertility in women of childbearing age. Currently infections caused by the pathogen are treated with antimicrobial therapy. Bacterial ghosts offer a cost-effective, long-term, protective therapy as vaccine delivery vectors. In the present study we tested the hypothesis that factors contained in culture supernatant exhibit anti-chlamydial activity. THP-1 monocytes were differentiated into macrophages (Mdm) by incubating with phorbol myristate acetate (PMA). Activated Mdm were pulsed with Vibrio cholerae ghosts (VCG) for 24 h, followed by the addition of murine splenocytes for an additional 48 h. After 72 h the supernatant was collected and assayed for cytokine concentration. Samples presented observable levels of immunostimulatory factors necessary for containing or clearing infections with chlamydia. Following these assay results, Chlamydia pneumonia (MoPn) elementary bodies (EBs) were suspended in VCG-pulsed THP-1 Mdm supernatant (conditioned media) and used to culture HeLa cells. As a positive control, MoPn was suspended in Earle’s MEM - a medium used for HeLa cell culture. Immunostimulatory factors contained in the conditioned media prevented the development of Chlamydia infection forming units (5.87%) vs. our positive control (94.1%). These results indicate that immunostimulatory factors induced by VCG confer protective immunity against the development cycle of Chlamydia.
The role of the novel ion channel TRPM7 in B cell activation (IRM12P.655)
Authors: Treanor Bebhinn , Krishnamoorthy Mithunah , Schmitz Carsten , Perraud Anne-Laure
Published: January 2023

The process of B cell spreading and the formation of signalling BCR microclusters are critical to the effective activation of B cells. These processes are dependent on the actin cytoskeleton; however the molecular pathways leading to reorganization of the actin cytoskeleton upon B cell encounter with membrane-bound antigen are poorly understood. Recent work has highlighted a potential role for the novel ion channel protein Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) in actomyosin reorganization. Intriguingly, this protein also contains a kinase domain, and thus, TRPM7 may play an important role in receptor-mediated signal transduction by phosphorylating downstream effector molecules. Interestingly, studies in B cells have implicated TRPM7 kinase in the regulation of phospholipase C (PLC), a key BCR signalling molecule and critical regulator of B cell spreading and the formation of BCR microclusters. Here, we show that TRPM7 plays a critical role in the early events of B cell activation through both its ion channel and kinase functions. B cells deficient in TRPM7 or expressing a point mutation which abolishes phosphotransferase activity show increased antigen accumulation and prolonged BCR signalling. Our data suggests this is likely due to altered actomyosin organization and defective antigen internalization.
B cell-intrinsic expression of the RNA-binding protein HuR is essential for humoral immunity by facilitating B-T communication in the lymphoid follicle. (IRM12P.645)
Authors: DeMicco Amy , Naradikian Martin , Cancro Michael , Bassing Craig
Published: January 2023

Post-transcriptional regulation of gene expression is critical for the survival, differentiation, and function of B lymphocytes. To investigate roles of the RNA-binding protein HuR in B cells, we generated mice with B-cell specific deletion of HuR using Cre recombinase driven by the Mb1 promoter. These mice have modestly reduced numbers of naïve B cells, but show profound loss of serum antibodies of all isotypes and low numbers of germinal center B cells, plasma cells, and switched memory cells. Further, these mice fail to form germinal centers or undergo affinity maturation upon immunization with NP-OVA. In contrast, HuR-deficient B cells survive, proliferate, class switch, and undergo typical gene expression changes upon in vitro stimulation. We find that HuR regulates expression of CD81, CD70, and CD86, which promote the ability of B cells to physically interact with T cells. Biochemically, HuR binds to mRNAs of genes involved in cytoskeletal remodeling upon B cell activation. Because cytoskeletal reorganization is critical for antigen internalization, these data suggest that HuR-deficient B cells may have an antigen presentation defect. Consistent with the notion that B cell-intrinsic functions of HuR promote B-T communication in vivo, mice with HuR-deficient B cells have reduced numbers of T follicular helper cells, whose differentiation and maintenance are dependent on interactions with B cells. In sum, we find that HuR has critical roles in B cell function.
The genetic regulation of the longevity of CD4 memory T cells. (LYM5P.660)
Authors: Song Nianbin , Khoruzhenko Stanislav , Sadegh-Nasseri Scheherazade
Published: January 2023

We have previously reported that CD4 memory T cells enter a stage of quiescence upon resolution of infection and the consequent reduction in antigenic levels. The hyporesponsive memory T cells become responsive again upon challenge with a secondary infection, or antigen and Toll-like receptor agonists. To better understand the genetic mechanisms of the longevity of CD4 memory T cells, we performed cDNA microarray analysis of CD4 memory T cells developed after immunization with vaccinia-OVA virus using DO11.10 transgenic cells in an adoptive transfer experiment. Gene expression levels of CD4 T cells were evaluated at 9 days, 5 weeks, 6 months and 10.5 months post immunization. We repeated the experiment in DR1 transgenic mice immunized with H5N1 influenza vaccine because they have diverse polyclonal TCR repertoire and are better representative of the natural conditions. We used HLA-DR1/H5N1-HA(259-274) tetramers for staining of the antigen specific CD4 memory T cells. We found up-regulation of genes mediating DNA repair and inhibiting apoptosis, and down-regulation of glucose metabolism and up-regulation of lipid metabolism are important in longevity of CD4 memory T cells. Using FACS staining we verified several genes that were detected as significantly up-regulated by microarray experiments. These observations verified our microarray data as well as confirming our findings that long-term CD4 memory T cells are quiescent and switch from glucose to lipid metabolism.
Dysregulated THEMIS results in the escape of pathogenic auto-reactive T cells. (LYM6P.719)
Authors: Baudewijn Magdalena , Spiering Rachel , Lambolez Florence , Cheroutre Hilde
Published: January 2023

THEMIS is required for setting the threshold for conventional selection of thymocytes by dampening TCR signals, resulting in the accumulation of “naïve” T cells in the periphery. In contrast, agonist selection, which is an alternative way to induce self tolerance, requires strong TCR signals and does not involve regulation by THEMIS. Using the OVA-specific monoclonal MHC class II restricted DO11.10 TCR transgenic system, we found that many DO11.10 thymocytes are selected along the agonist selection path and appear as co-receptor double negative (DN) TCRab cells in the intestine. Interesting, some DO11.10 cells also appear as conventional-like CD4 T cells in the spleen, suggesting they escaped agonist selection. Compared to the DN DO11.10 T cells or normal WT conventional selected T cells, these escaped DO11.10 CD4 T cells expressed much higher levels of THEMIS, suggesting that excessive levels of THEMIS might have diverted DO11.10 thymocytes to be selected along the conventional path, resulting in the accumulation of T cells with high affinity for self in the naïve pool. Although these aberrant selected THEMIShigh CD4 T cells remain quiescent at steady state, they caused severe intestinal pathology in RAG-/- recipient mice that were fed an OVA-diet. These data indicate that dysregulated THEMIS can lead to the accumulation of auto-reactive T cells with high potential to induce immune pathology when exposed to their cognate antigen in the periphery.
The molecular signature of T cells undergoing recurrent homeostatic proliferation. (LYM6P.720)
Authors: Fortner Karen , Bond Jeffrey , Austin James , Boss Jeremy , Budd Ralph
Published: January 2023

T lymphocyte homeostatic proliferation, driven by the engagement of TCR with self-peptide/MHC complexes and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by Fas. However, the functional consequences of sequential rounds of homeostatic expansion are not well defined. We examined the gene expression profiles of wild-type and Fas-deficient lpr CD8+ T cell subsets that have undergone different amounts of homeostatic proliferation as defined by the level of CD44 expression, and the CD4-CD8-TCRαβ+ T cell subset that results from extensive homeostatic expansion of CD8+ T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as PD-1 and Lag3. These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed paradox in clinical immunology of simultaneous autoinflammatory syndromes and immunodeficient states and provide further insight into the regulatory programs that control chronically stimulated T cells.
Regulation of granuloma formation via Myd88 and Nox2 in a model of pulmonary sarcoidosis (MPF5P.741)
Authors: Werner Jessica , Escolero Sylvia , Hewitt Jeff , Mak Tim , Eishi Yoshinobu , Núñez Gabriel
Published: January 2023

Sarcoidosis is characterized by non-caseating granulomas with an unknown etiology that present primarily in the lung. Propionibacterium acnes, an immunogenic commensal skin bacterium involved in acne vulgaris, has been implicated as a causative agent of sarcoidosis because it has been both detected and cultured from granuloma lesions and lymph nodes of sarcoidosis patients. Here we demonstrate that a strain of P.acnes isolated from a sarcoidosis patient and instilled intratracheally into wild type mice can generate pulmonary granulomas. When mice deficient in the innate immunity adapter protein Myd88 are instilled with P.acnes the number and size of granulomas are increased compared to wild type. Additionally there was an increase in granuloma number and size after instillation with P.acnes in mice deficient in Nox2, a critical component of the phagocyte NAPDH that generates reactive oxygen species for microbial killing within the phagosome. Furthermore mice without Myd88 or Nox2 displayed defective killing of P.acnes in the lung, and wild type mice that were given heat-killed P.acnes produce significantly less granulomas then when given viable P.acnes, suggesting that bacterial killing and/or clearance plays a critical role in the development of pulmonary granulomas. In conclusion viable P.acnes can be used to generate a mouse model of pulmonary sarcoidosis, and Myd88 and Nox2 appear to play a key role in the development of these granulomas.
Escherichia coli colonization restores mucosal immune tone during antibiotic treatment (MUC5P.759)
Authors: Caballero Silvia , Samilo Dane , Carter Rebecca , Pamer Eric
Published: January 2023

Molecules derived from gut-colonizing microbes contribute to intestinal homeostasis by stimulating innate immune receptors and maintaining a basal level of activation of host antimicrobial defenses. Depletion of intestinal microbes by treatment with antibiotics severely dampens these responses, increasing the risk for colonization and infection with pathogenic bacteria. A major consequence of antibiotic treatment is downregulation of the antimicrobial protein RegIII-γ. RegIII-γ expression is markedly decreased during antibiotic treatment but can be induced by administration of Toll-like receptor (TLR) ligands derived from Gram-negative bacteria. This observation suggests a role for Gram-negative organisms in stimulating RegIII-γ intestinal levels. In this study, we investigated the ability of several E. coli and K. pneumoniae strains to induce RegIII-γ in the mouse intestine during antibiotic stress. We find that although both bacterial species colonize the gastrointestinal tract of antibiotic-treated mice, only E. coli restores RegIII-γ expression in the ileum. E. coli, but not K. pneumoniae, induces IL-22 expression, a requisite step for RegIII-γ, induction, in the small intestinal lamina propria. Flagellin and TLR5 signaling are not required for E.coli-mediated RegIII-γ induction. Our findings suggest that Gram-negative bacteria colonizing the intestine differ in their ability to mediate IL-22-dependent RegIII-γ.
Campylobacter jejuni induces autoimmune peripheral neuropathy via Siglec-1 and IL-4 axes (MUC5P.746)
Authors: Malik Ankit , Gadsden Barbie , Ethridge Alexander , Dybas Leslie , Mansfield Linda
Published: January 2023

Campylobacter jejuni is the most common bacterial cause of gastroenteritis worldwide. C. jejuni infection has also been causally linked with development of the peripheral neuropathy called Guillain Barré Syndrome (GBS). We have previously shown that C. jejuni isolates from human enteritis patients induce a Type1/17 cytokine dependent colitis response in IL-10-/- mice. In contrast, isolates from human GBS patients colonize the IL-10-/- mice without inducing colitis but instead induce autoantibody elicitation targeted against peripheral nerve antigens. We show here that the autoantibody response is dependent upon blunted Type1/17 but enhanced Type2 cytokine production by T helper cells. Autoantibody elicitation also correlated with enhanced macrophage infiltration in the sciatic nerve and its dorsal root ganglia. Autoantibodies and these histological changes were significantly decreased in mice depleted of IL-4, without leading to colitis induction. Histological damage in the sciatic nerve was associated with abnormal gait and hind limb movements in the IL-10-/- mice, consistent with this syndrome’s manifestation in humans. Furthermore, we show here that Siglec1 is a central antigen presenting cell receptor that mediates GBS but not colitogenic isolate uptake, T cell differentiation and autoantibody elicitation. Therefore, this is the first mouse model of an autoimmune disease induced directly by a bacterium and it is dependent upon Siglec1 and IL-4 axes.
Relapse of lupus after B cell depletion therapy is associated with loss of apoptotic debris clearing marginal zone macrophages in BXD2 mice (THER5P.911)
Authors: Hsu Hui-Chen , Yang Zhaoqi , Li Hao , Yang PingAr , Wu Qi , Li Jun , Luo Bao , Harrington Laurie , Mountz John
Published: January 2023

B-cell depletion therapy (BCDT) is used to treat autoimmune diseases but development of autoreactive B cells can occur during repopulation. Marginal zone macrophages (MZMs) are essential for tolerogenic clearance of apoptotic debris. Maintenance of MZMs requires MZ B cells. To determine if BCDT using anti-CD20 depletes MZ B cells and promote the loss of MZMs, lupus prone BXD2 mice were administered α-CD20 or isotype control (250 µg/single dose i.v.). This resulted in depletion of >80% B cells in the spleen at week 2 in BCDT group. This was associated with increased uncleared apoptotic debris in the spleen MZ and a dramatic decline in MZMs. By 5 weeks of BCDT, the B-cell repopulation rate was >50%. However, there was a significantly elevated expression of type I IFN signature genes Usp18 and Oasl in repopulated MZ precursor B cells in anti-CD20 group. There was a 2.8-fold increase in PNA+Fas+ CD19+ germinal center B cells in the spleens of anti-CD20-treated BXD2 mice, compared to isotype-treated mice. At week 9, the serum titers of IgG autoantibodies against DNA, histone, and malondialdehyde, a lipid peroxidation product associated with apoptotic blebs, were markedly increased in the α-CD20-treated BXD2 mice than control group. These results indicate that BCDT in BXD2 mice promotes an environment with excessive uncleared apoptotic debris which enhanced the effects of type I IFNs on repopulating B cells, leading to the return of autoreactive B cells.
Male microbiota protects female BWF1 mice from lupus and death by restoring CD103DC function possibly via metabolite(s) production (THER5P.914)
Authors: Kosiewicz Michele , Alard Pascale , Zhang Xiang , Fausnaught Thomas , Chhabra Anita
Published: January 2023

Alterations in microbiota composition are associated with autoimmune diseases. Little is known about the effect of microbial metabolite production on disease development. CD103 dendritic cells (CD103DC) induce regulatory T cells (iTregs) in the periphery. We have found that lupus-resistant male BWF1 mice exhibit increased disease incidence/mortality when treated with anti-CD103 antibody. Female CD103DC exhibit lower tolerogenic function than male cells in vitro and in vivo (i.e., oral tolerance) possibly due to a defect in the retinoic acid synthesis pathway. We also found that microbiota composition differed significantly between female and male BWF1 mice, and transfer of male microbiota decreased autoantibody production in female BWF1 mice. Here we report that transfer of male microbiota prevents kidney disease and dramatically increases survival of female BWF1 mice which correlates with enhanced female CD103DC function in both in vitro and in vivo assays, and increased iTregs. Furthermore, our preliminary study suggests that treatment with anti-CD103 antibody prevents male microbiota-mediated protection. Finally, metabolomic analysis of feces from female and male BWF1 mice revealed significant quantitative differences in 17 metabolites, including one that exhibits retinoid X receptor agonist activity. Taken together, these data suggest that male microbiota may protect against lupus through the production of metabolite(s) that enhance the retinoic acid synthesis pathway.
Repository corticotropin injection (H.P. Acthar® Gel) attenuates established collagen-induced arthritis when used alone or as adjuvant therapy with etanercept (Enbrel®) (THER5P.902)
Authors: Decker Dima , Higgins Paul , Bendele Alison , Becker Patrice
Published: January 2023

Melanocortins have anti-inflammatory and immunomodulatory properties mediated by receptors expressed on cells relevant to the pathophysiology of immune-mediated arthritis. Repository corticotropin, a melanocortin preparation, is approved as adjunctive therapy for rheumatoid arthritis (RA), but its mechanism of action in RA has not been determined. We therefore explored the efficacy of corticotropin when used alone or as adjunct therapy with etanercept in a rat collagen-induced arthritis (CIA) model. Corticotropin (160 or 400 U/kg) administered after disease onset significantly attenuated CIA-related changes in ankle diameter and paw weight compared to vehicle treatment (p ≤ 0.05). Corticotropin (400 U/kg) also significantly reduced clinical (4.15 ± 0.38 vs 5.65 ± 0.21, p ≤ 0.01) and summed ankle histopathology (9.90 ± 0.88 vs 15.20 ± 0.85, p ≤ 0.0001) scores below those in vehicle treated rats. Overall efficacy of corticotropin and etanercept used alone in established CIA were comparable, however corticotropin in combination with etanercept synergistically attenuated anti-collagen antibodies and both clinical and histopathologic measures of CIA vs etanercept alone (p ≤ 0.05). In addition, corticotropin administered with etanercept significantly attenuated CIA-induced decreased average bone density, whereas neither drug alone was efficacious (p ≤ 0.05). These data support the potential clinical efficacy of repository corticotropin as an adjunctive therapy for patients with RA.
Efficacy in collagen induced arthritis models with a selective, reversible covalent Bruton’s tyrosine kinase inhibitor PRN473 is driven by durable target occupancy rather than extended plasma exposure (THER5P.904)
Authors: Bisconte Angelina , Hill Ronald , Bradshaw Michael , Verner Erik , Finkle David , Brameld Ken , Funk Jens , Goldstein David , Nunn Phil
Published: January 2023

Bruton’s Tyrosine Kinase (BTK) is an essential signaling element downstream of the B-cell receptor (BCR). Inhibition of BTK activity in B cells produces phenotypic changes consistent with blockade of the BCR, including inhibition of cell proliferation, differentiation, maturation, and survival. A selective BTK inhibitor has the potential to treat diseases involving inflammation and autoimmunity. Using Principia Biopharma’s proprietary Tailored Covalency™ technology, we discovered PRN473, a reversible covalent BTK inhibitor that selectively binds BTK with a slow off-rate as assessed in biochemical and cell based assays. A slow off-rate molecule with rapid systemic clearance may lead to a long duration of action and high efficacy, while reducing the potential for off target toxicities. The duration of binding of PRN473 was measured in vivo, with prolonged BTK occupancy confirmed after drug was cleared from plasma. In mouse and rat collagen-induced arthritis (CIA) models, PRN473 when dosed therapeutically demonstrated dose-dependent inhibition, reversal of arthritis, with almost complete abrogation of clinical scores and preservation of joint integrity and histology. BTK occupancy in splenocytes was found to be a robust measure of BTK inhibition and correlated with efficacy in the CIA models. These data in combination with GLP toxicology studies, where no organ specific toxicities were observed, validate using a reversible covalent BTK inhibitor to treat autoimmune diseases.
Control of lupus by novel tryptophan metabolite 5-methoxytryptophan. (THER5P.907)
Authors: Wu Hsu-Feng , Wang YiFu , Kuo Cheng-Chin
Published: January 2023

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the presence of pathogenic autoantibodies which potentially drive immune-complex related inflammation in various tissues and organs. We previously identified that 5-methoxytryptophan (5-MTP), a soluble factor released by fibroblasts, provides endogenous control of proinflammatory cytokine-induced cyclooxgenase-2 expression. However, its effect on controlling systemic inflammation and systemic inflammatory disorders such as SLE remains unclear. Clinical studies by measuring serum 5-MTP concentrations found that a considerable amount of 5-MTP was detected in healthy subjects and was significantly reduced in SLE patients. Mouse models of experimental lupus-like disease induced by pristine shown that exogenous supplementation of 5-MTP not only prevented serum anti-dsDNA IgG production but also rescued the loss of body weight. Notably, 5-MTP administrations rescued kidney by reducing the accumulation of IgG. The beneficial effect of 5-MTP was correlated with inhibition of IL-6 production in bone-marrow derived dendritic cells treated with TLR7 ligand, which is a stimulator of lupus progression. Additionally, 5-MTP also suppressed Th1 cell while increased regulatory T cells which play an essential role in preventing autoimmunity. These results that 5-MTP is a novel circulating anti-inflammatory molecule which protects against excessive systemic inflammatory responses in animal models.
Recipient IL-33 stimulates donor T cells to promote Type 1 alloimmunity and lethal acute GVHD (TRAN1P.931)
Authors: Matta Benjamin , Reichenbach Dawn , Schwarze Vincent , Tkachev Victor , Mathews Lisa , Liu Quan , Warncke Max , Ferrara James , Zeiser Robert , Blazar Bruce , Turnquist Heth
Published: January 2023

Graft-versus-host disease (GVHD) is a severe and often fatal complication of allogeneic (allo) hematopoietic cell transplantation (HCT). Total body irradiation (TBI) conditioning of the recipient and subsequent GVHD-associated alloimmune responses cause significant gut epithelial cell damage. As IL-33 is an epithelial cell-derived cytokine with pleiotropic functions, we investigated the impact of IL-33 on GVHD pathogenesis. Mice received TBI and alloHCT (allogeneic bone marrow (BM) +/- T cells). Where indicated, recipient mice, donor BM or T cells were deficient for IL-33 or its receptor, ST2. Survival, weight, and clinical score were monitored. Tissues and serum were harvested for IL-33 quantitation and analysis of alloimmune responses. We find that IL-33 expression is increased rapidly in CD45- cells of the gut after TBI, and sustained at least through day 14 in alloHCT recipients. Recipient, but not donor, IL-33 acting on ST2-expressing donor T cells fueled Type 1 responses and GVHD. The IL-33 antagonist ST2-Fc prevented GVHD and reduced Type 1 alloimmune responses, whereas IL-33 administration post-alloHCT (d+3 to d+7) accelerated GVHD lethality and Type 1 alloimmunity. These potent responses overcome regulatory functions of IL-33 to promote GVHD. In summary, our findings are the first to reveal a detrimental role for IL-33 underlying GVHD pathogenesis and establish the IL-33/ST2 axis as a targetable pathway to lessen the risk of GVHD following alloHCT.
An increase in the level of anti-Gal IgM is associated with an early graft failure in intraportal porcine islet xenotransplantation (TRAN1P.935)
Authors: Kang Hee Jung
Published: January 2023

In order to develop laboratory tests to predict graft outcome in porcine islet transplantation (ITx), we investigated the association between anti-Gal antibody responses and the graft failure in NHP recipients after porcine ITx. Total 35 naïve diabetic NHP recipients were intraportally administered with adult porcine islets and then graft function was monitored. Early graft failure (EGF) was defined as returning to diabetic state within a month after ITx. Among the recipients, 7 and 28 received induction immunosuppression (IS) without and with CD40 pathway blockade, respectively: 19, anti-CD154 and 9, anti-CD40. The levels of anti-Gal IgG and IgM in the recipients on the day 0 (0 d) and on the day 7 (7 d) of ITx were quantitatively measured by in-house ELISA. Whereas an increase in the level of anti-Gal IgG after ITx was suppressed by the use of either anti-CD154 or anti-CD40, a response of anti-Gal IgM was suppressed by anti-CD154, not by anti-CD40. The anti-Gal antibody ratio of 7 d/0 d was significantly higher in the recipients with EGF than in those without EGF (IgG ratio, 1.54 vs 0.75, P=0.0025; and IgM ratio, 1.48 vs 0.87 P=0.0035). An increase in the level of anti-Gal IgM on the day 7 of ITx is significantly associated with EGF in the recipients with porcine ITx regardless of use of CD40 pathway blockade, suggesting that an early response of anti-Gal IgM may reflect the innate immune responses which account for killing of porcine islets in early period of porcine ITx.
Potential of 131I-anti-TLR5 mAb for noninvasive assessment of monitoring rapamycin treated allograft efficiency (TRAN1P.936)
Authors: hou guihua , sun hukui
Published: January 2023

Background Toll-like receptor 5 (TLR5) is a potentially target related with immunosuppressant rapamycin. Our aim was to evaluate 131I-anti-TLR5 mAb for non-invasive in vivo graft visualization and quantification in rapamycin treated allogeneic transplantation model compared with nonspecific tracer 18F-FDG. Methods Labeling, binding and stability studies were performed. Allografted BALB/c mice were divided into rapamycin-treated and non-treated group, respectively. In vivo dynamic whole-body phosphor-autoradiography and ex vivo biodistribution studies were conducted. Results Phosphor-autoradiography imaging showed clear graft localization from 12 h onward after 131I-anti-TLR5 mAb injection,significantly higher graft uptake in rapamycin-treated group. Pretreatment with anti-TLR5 mAb blocked graft uptake,which were consistent with ex vivo biodistribution studies. As the nonspecific radiotracer, high 18F-FDG uptake was only observed in non-treated allorejection group, not in treated group. The highest allograft skin-to-native skin ratio (A: N) of 131I-anti-TLR5 mAb uptake was significantly higher than the ratio for 18F-FDG (7.68 versus 1.16, respectively) in rapamycin-treated group. Immunohistochemistry analysis confirmed that 131I-anti-TLR5 mAb uptake significantly correlated with TLR5 expression in the allograft. Conclusion Radiolabelled anti-TLR5 mAb is capable of detecting allograft rejection with high target specificity after treatment with the immunosuppresant rapamycin.
The universal donor stem cell: removing the immune barrier to transplantation using CRISPR/Cas9 (TRAN1P.946)
Authors: Meissner Torsten , Strominger Jack , Cowan Chad
Published: January 2023

Recent advances in stem cell biology have made it possible to contemplate the use of a patient’s own cells as an unlimited source for transplantation. Unfortunately, the generation of iPSCs remains a costly, time consuming and highly variable process, with regards to pluripotency, epigenetic status, capacity for differentiation, and genomic stability. Moreover, changes occurring during reprogramming and prolonged culturing have been found to trigger an adaptive immune response, resulting in immune rejection of even autologous stem cell-derived transplants. To overcome this immune barrier, we propose to create a Universal Donor Stem Cell, a source for any transplantable cell type, which will not be rejected by the recipient’s immune system regardless of his/her genetic make-up. For this purpose we are using state-of-the-art genome editing technologies: We have successfully employed the CRISPR/Cas system to delete critical immune genes in human ES cells and iPSCs, rendering them potentially less prone to immune rejection. In a complementary approach, we are introducing well-established tolerance-inducing molecules, such as HLA-G, and PD-L1 into stem cells that can locally suppress the immune system and improve transplant engraftment, thus bypassing the detrimental systemic side effects caused by immunosuppressive drugs. Experiments assessing the immunogenicity of our modified stem cell lines and their derivatives in vivo in humanized mice are currently ongoing.
Antigen-specific killer polylactic-co-glycolic acid microspheres selectively deplete antigen-specific T cells in vitro and in vivo (TRAN1P.924)
Authors: Wang Wei , Li Miaochen , Fang Kun , Wu You , Gu Ning , Shen Chuanlai
Published: January 2023

The strategy of specifically depleting antigen-specific T cells can potentially be used to treat allograft rejection and autoimmunity because it does not suppress the overall immune systems. In this study, we generated killer polylactic-co-glycolic acid (PLGA) microspheres by covalently coupling H-2Kb-Ig and apoptosis-inducing anti-Fas monoclonal antibody (mAb) onto cell-sized PLGA microspheres. The Kb/OVA257-264-Killer PLGAs resulted in a dramatic reduction in the number of OVA257-264-specific CD8+ T cells in an antigen-specific manner in vitro and in vivo, by using a transgenic OT-1 mice model. The bioluminescence imaging revealed that Killer PLGAs dominantly distributed in lung, liver and gut, and was maintained for up to 48 hours in OT-1 mice and a longer time in OT-1 mice than in C57BL/6 mice. Moreover, the Kb-Killer PLGAs were administered intravenously into bm1 mice (H-2Kbm1) that had previously been grafted with ear skin from C57BL/6 mice (H-2Kb). The killer PLGAs selectively decreased the H-2Kb alloantigen-reactive CD8+ T cells infiltrating into the alloskin graft but not CD4+ T cells, and significantly prolonged allograft survival for 41.5 days without the impairment of host overall immune function. Thus, our data highlight the therapeutic potential of this biocompatible and biodegradable killer effectors for the treatment of allograft rejection and autoimmune disease. Keywords: Allograft rejection, PLGA, H-2Kb-Ig
Tumor-derived GM-CSF promotes JAK/STAT3-dependent expression of IDO and PD-L1 in myeloid suppressor cells (TUM6P.954)
Authors: Thorn Mitchell , Cunetta Marissa , Point Gary , Guha Prajna , Espat N , Junghans Richard , Katz Steven
Published: January 2023

Myeloid suppressor cells (MSC) are a tolerogenic population of immune cells that poses a major obstacle to cancer treatment. Intrahepatic MSC associated with liver metastases (LM) are especially detrimental by enhancing immunosuppression. We have recently found that MSC in a mouse model of LM suppress endogenous T cell function and anti-tumor cell-based immunotherapy. Mice with LM induced by a splenic injection of MC38 tumor cells, showed an expansion of MSC (5-fold, p=0.0005), while GM-CSF blockade reduced MSC frequency and increased the overall survival (6-fold, p=0.03). As MC38 and other tumors overexpress GM-CSF, we wanted to further investigate the importance of GM-CSF in MSC-driven immunosuppression. A significant proportion of MSC in LM co-expressed IDO and PD-L1 (34±5%), while maintaining high levels of pSTAT3, a transcription factor important to MSC function. Ex vivo, MSC in culture with MC38 cells or supplemented with GM-CSF maintained high IDO/PD-L1 expression. Conversely, when MSC were co-cultured with tumor cells supplemented with anti-GM-CSF, IDO/PD-L1 levels were significantly reduced (p=0.03), implicating GM-CSF in promoting IDO/PD-L1 expression in MSC. Small molecule STAT3 and JAK2 inhibitors abolished IDO/PD-L1 expression in MSC (p=0.003 and p=0.002), suggesting the importance of STAT3/JAK2 signaling pathway in IDO/PD-L1 expression. STAT3 and JAK2 pathways represent viable targets for blocking GM-CSF induced immunosuppressive pathways in liver MSC.
Interactions within the tumor microenvironment imprint metastatic traits in cancer cells (TUM6P.1001)
Authors: Sharma Rajesh , Chheda Zinal , Jala Venkatakrishna , Haribabu Bodduluri
Published: January 2023

Metastasis accounts for majority of deaths across cancer types. Tumor microenvironment plays an impotant role in enhancing metastatic propensity of cancer cells. To explore the mechanisms, we herein established a model by in vivo passaging the Lewis lung carcinoma (3LL) cells with 1-2 week of culture in between passages. When injected subcutaneously, 3LL cells have minimal metastasis, while in vivo passaged (p-)3LL cells showed robust metastasis. Sorted p-3LL cells from tumors of the whole body RFP transgenic mice were equally metastatic suggesting that cancer cell intrinsic changes are responsible for the metastatic imprinting. To identify the stromal cell population/s that crosstalk and imprint the metastatic traits in cancer cells, in vivo passages were performed in Rag2-/- mice and WT mice depleted of NK cells that resulted in similar metastatic imprinting excluding the role of adaptive immune and NK cells in this process. Depletion studies in WT mice and/or co-culture of 3LL cells with sorted myeloid cells indicated a critical role for CD11b+Gr1dim myeloid cells in metastatic imprinting. Comparison of gene expression profiles between 3LL and p-3LL cells by microarray showed enhanced expression of claudin-9 in p-3LL cells which was validated at protein levels in mouse and human lung cancers. These results indicate that interaction of CD11b+Gr1dim myeloid cells with cancer cell within tumor microenvironment imprints claudin-9 dependent metastatic traits in cancer cells.
C/EBP homologous protein expression regulates immunosuppressive activity in myeloid derived suppressor cells (TUM6P.1006)
Authors: Thevenot Paul , Sierra Rosa , Raber Patrick , Al Khami Amir , Ochoa Augusto , Rodriguez Paulo
Published: January 2023

Myeloid-derived suppressor cell (MDSC) suppression of anti-tumor T cell responses is a significant barrier in cancer immunotherapy. Although pathways controlling MDSC regulatory activity have been characterized, therapies to globally and specifically inhibit MDSC function are limited. Targeting central mediators of MDSC-regulatory activity may overcome T cell suppression and increase the efficacy of T cell-based immunotherapy. We investigated the role of the stress sensor C/EBP homologous protein (Chop) on MDSC function. Elevated Chop expression in tumor-bearing mice was restricted to malignant cells and tumor MDSCs. Chop elevation was also detected in tumor MDSCs from colon carcinoma patients. Interestingly, increased CD8+ T cell-mediated anti-tumor effects were found in both systemic Chop -/- and Chop -/- bone marrow chimeric mice bearing s.c. Chop-competent tumors, suggesting the importance of MDSC-Chop in tumor-induced T cell anergy. MDSCs from Chop -/- mice showed a decreased expression of major MDSC-inhibitory pathways, reduced inhibitory activity on T cells, and a surprising ability to prime T cell proliferation and induce anti-tumor effects. The reduced regulatory activity in Chop -/- MDSCs was caused by decreased IL-6 production, resulting from decreased C/EBPb activity. Altogether the results demonstrate a central role of Chop in MDSC inhibitory activity and suggest targeting Chop to restore protective immunity in cancer.
Tumor-derived osteopontin suppresses antitumor immunity by accumulating myeloid progenitor cells and myeloid-derived suppressor cells (TUM6P.1012)
Authors: Song Boyeong , Kim Eun Kyung , Jeon Insu , Seo Hyungseok , Park Youngjun , Chung Yeonseok , Kang Chang-Yuil
Published: January 2023

Extramedullary myelopoiesis occurs commonly in tumor-bearing animals and is known to lead to accumulation of peripheral myeloid-derived suppressor cells (MDSC), which play an important role in immune escape. However, the cellular and molecular mechanisms by which tumors induce extramedullary myelopoiesis are poorly understood. In this study, we found that osteopontin expressed by tumor cells enhances extramedullary myelopoiesis in a CD44-dependent manner through the Erk1/2-MAPK pathway. Osteopontin-mediated extramedullary myelopoiesis was directly associated with increased MDSCs in tumor-bearing hosts. More importantly, osteopontin silencing in tumor cells delayed both tumor growth and extramedullary myelopoiesis, while the same treatment did not affect tumor growth in vitro. Finally, treatment with an antibody against osteopontin inhibited tumor growth and synergized with cell-based immunotherapeutic vaccines in mediating antitumor immunity. Our findings unveil a novel immunosuppressive role for tumor-derived osteopontin and offer a rationale for its therapeutic targeting in cancer treatment.
Progressive loss of differentiated effector CD8 T cells infiltrating melanoma (TUM7P.1021)
Authors: Stevens Aaron , Bullock Timothy
Published: January 2023

CD8 tumor infiltrating lymphocytes (TIL) are critical for tumor control and their presence is a positive prognostic factor. Understanding CD8 TIL dysfunction is vital for developing cancer immunotherapies. Here, using a Granzyme B Cre ROSA YFP reporter system, we characterized the differentiation of CD8 T cells over the course of tumor growth using a murine melanoma model. At early stages of the CD8 T cell response (day 10 after tumor injection), the majority of the cells had differentiated to express Granzyme B (YFP+). However, over a period of four days, most of these effector cells were replaced by CD8+ T cells that had no evidence of effector differentiation. Concurrently, we found an increase in expression of the inhibitory receptor PD-1 in differentiated (YFP+) relative to non-differentiated (YFP-) CD8 TIL in late stage tumors. This suggests that there is both a loss of differentiated CD8 TIL and that the differentiated CD8 T cells that remain are more dysfunctional in late stage tumors. To provide insight into the cause and effect of this fluctuation in differentiation, we present data establishing the contribution of checkpoint inhibitory molecules and dendritic cell activation state in the loss of effector characteristics, and whether cytokine production and other effector activities correlate with the shift in effector differentiation status.
Chronic cold-stress suppresses chemokine production and CD8+ T cell infiltration in the tumor microenvironment (TUM7P.1024)
Authors: Alexander Matthew , Kokolus Kathleen , Costa Amanda , Clancy-Thompson Eleanor , Repasky Elizabeth , Mullins David
Published: January 2023

Recently it was reported that maintenance of mice at thermoneutral temperatures (TT, the temperature at which basal metabolism is sufficient to maintain body temperature, 30oC) enhanced antitumor immune responses and reduced tumor growth, relative to standard vivarium temperatures (ST, 22oC). We hypothesized that mice maintained at ST may have impaired production of T cell chemoattractant cytokines and subsequent T cell infiltration. In mice bearing breast tumors or solid or metastatic-like melanomas, intratumoral expression of CXCL9 and CXCL10 (CXCR3-cognate chemokines) was enhanced by maintenance of hosts in TT conditions. Further, tumors of TT-housed mice contained increased numbers of CD8+ T cells. We further hypothesized that the effect of temperature on T cell chemokine pathways may be a result of chronic cold stress, which may induce norepinephrine (NE) production and subsequent activation of β-adrenergic signaling pathways. We observed that pharmacologic blockade of β-adrenergic receptors in mice bearing solid or metastatic-like melanomas recapitulated the increased intratumoral expression of CXCL9 and CXCL10 and increased CD8+ T cell numbers. Because melanoma cells express β-adrenergic receptors, we suggest that chronic cold stress may induce systemic NE production, leading to tumor local activation of receptors and inhibition of tumor-derived chemokine production. These studies suggest potential interventions to improve existing T cell-based immunotherapies.
Novel kinetic live cell imaging assays for T cell killing of tumor cells (TUM7P.1031)
Authors: APPLEDORN DANIEL , Bevan Nicola , Szybut Clare , O'Callaghan Tim , Dale Tim , Trezise Del
Published: January 2023

Cancer immunotherapy offers the potential for specific eradication of tumor cells and the prevention of cancer recurrence. In this study we describe fully kinetic live cell imaging assays designed to quantify T cell mediated killing of tumor cells in co-culture. RFP-nuclear labelled tumor cells were seeded overnight in 96 or 384 well plates. Peripheral Blood Mononuclear Cells (PBMCs) or isolated CD8+ T cells were then added to the wells in combination with various activators and a non-perturbing green fluorescence caspase-3/7detection reagent. Phase and 2-colour fluorescence images were captured every 2h for 4-7 days using IncuCyte ZOOM™. Tumor cell numbers were quantified by direct nuclear object counting (RFP). The number of apoptotic tumor cells were measured using green object counting and size/brightness filtering to remove dying T cells. This method is illustrated and validated with anti CD3, IL-2 and CD28 mediated cell killing, and a study on the impact of target/effector cell ratios. These validation data demonstrate that live cell imaging can be used to discern the full time course and specificity of T cell killing, without the need to lift cells, use Ab labels or radioisotopes (e.g. 51Cr). HD images and time-lapse movies facilitate a clear understanding and compelling verification of the underlying biology.
Metabolic competition in the tumor microenvironment determines cancer progression (TUM7P.1016)
Authors: Chang Chih-Hao , Qiu Jing , Pearce Erika
Published: January 2023

T cells produce cytokines that are critical for tumor immunity. However, during cancer, T cells often experience a decline in function preventing tumor regression. Why T cell hyporesponsiveness develops is unclear, but it is thought to result from lack of dominant epitope-specificity, continuous antigen exposure, and suppression by other immune cells. Previously we have shown that T cells need to engage aerobic glycolysis to optimally produce IFN-γ. Using a well-characterized mouse sarcoma model we show here that tumor mediated glucose consumption metabolically restricts tumor-infiltrating T cells (TILs), which dampens their mTOR activity, decreases their glycolytic capacity and therefore, limits their IFN-γ production, leading to tumor progression. We show that anti-CTLA-4, anti-PD-1, or anti-PD-L1 blockade therapy, which induces regression of progressing sarcomas, restores glucose in the tumor microenvironment and rescues glycolysis in TILs. Moreover, we demonstrate that blocking PD-L1 directly on tumor cells dampens tumor glycolysis and glucose uptake. Finally, we show that an antigenic sarcoma that normally is rejected can turn into a progressing tumor when its glycolytic rate is enhanced. Our data show that metabolic competition in the tumor microenvironment dictates T cell function and that this influences cancer progression. Combining therapies that blunt tumor cell metabolism with those that promote glycolysis in TILs could provide new effective treatments for cancer.