bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptidase inhibitor 16 identifies a unique subset of memory T helper cells with hyperproliferative and proinflammatory properties (IRC8P.477)
Authors: Mohandas Arunesh , Zola Heddy , Barry Simon , Krumbiegel Doreen
Published: January 2023

T helper (Th) cells play a major role in protecting the body against pathogens. Any imbalance in Th cell subsets could lead to autoimmune and inflammatory diseases. Peptidase inhibitor 16 (PI16), also known as prostate secretory protein of 94 amino acid - binding protein, was recently discovered to be expressed on memory regulatory T cells. This study investigates the expression and function of PI16 on Th cells. In healthy adults, 5-25% of CD4+ Th cells express PI16 with over 90% showing a memory phenotype. PI16+ Th cells have an increased expression of chemokine receptors CCR4 and CCR6 compared with PI16- Th cells. Transwell migration assays showed that more PI16+ Th cells migrated towards the CCR4 and CCR6 ligands (CCL17 and CCL20) compared with PI16- Th cells. After 7 day stimulation using CD3 / CD28 beads, PI16+ Th cells produce more IL-17A and less IFN-g compared with PI16- Th cells. PI16+ Th cells also have a higher expression of ROR-gt compared to PI16- Th cells. Furthermore, in comparison to PI16- Th cells, PI16+ Th cells have an increased proliferative potential and are less responsive to suppression by Treg. The memory phenotype of PI16+ Th cells, the high expression of Th17-like chemokine receptors, increased ROR-gt expression and high production of IL-17A suggest an active role of PI16+ Th cells at the site of infection or inflammation. Further studies are ongoing to understand the functional role of PI16 on Th cells in autoimmune and inflammatory diseases.
Two distinct subsets of skin-infiltrating memory T cells, TRM and TEM, define cutaneous immune responses against systemically-administered soluble antigens (IRC9P.702)
Authors: Egawa Gyohei , Miyachi Yoshiki , Kabashima Kenji
Published: January 2023

Cutaneous immune reactions to soluble antigens, such as drugs, are common, but their precise pathomechanism remains unclarified. To this end, we newly developed a murine model for skin eruptions against soluble antigens using ovalbumin (OVA)-specific TCR transgenic line, OT1. OT1 T cells were adoptively transferred to naïve mice and were immunized with OVA/CFA. Five days later, a skin inflammation was induced to the right ear with topical PMA treatment, which induced skin-resident memory T cells (TRM). Two weeks after PMA-painting, a large number of CD103+CD69+ OT1-TRM were found in the epidermis of PMA-painted site, whereas only a marginal number of OT1 T cells were detected in the untreated left ear. Then, OVA peptide was injected intravenously, which exhibited immediate ear swelling response only in the right ear where OT1-TRM exist in the epidermis. Five days after the systemic administration of OVA peptide, CD44+CD62L- effector memory OT1 T cells (TEM) were significantly increased in the blood and, intriguingly, were detected in the dermis of the untreated left ear. In addition, a repeated intravenous administration of OVA peptide developed chronic necrotic skin lesion even without extrinsic stimuli. These observations implicate respective roles of TRM and TEM in cutaneous immune responses: antigen-specific TRM in the epidermis cause immediate and localized skin eruptions, whereas TEM infiltrate from the blood into the dermis and induce chronic disseminated dermatoses.
Utilizing dual-costimulation through CD134 and CD137 with varying levels of antigen presentation to elicit heterogeneous CD4+ T cell responses (IRC9P.703)
Authors: Morales Crystal , Maxwell Joseph , Adler Adam , Vella Anthony
Published: January 2023

Many cancer vaccines target CD8 T cells, but cytotoxic CD4+ T cells are emerging as important players. Our lab utilized a dual-costimulation immunotherapy approach through CD134 (OX40) and CD127 (4-1BB) agonist antibodies together with T cells specific to a widespread antigen which induced cytotoxic CD4+ T cell differentiation. Drawing upon a novel transgenic mouse model where CD11c+ cells present a specific antigen, we now demonstrate that antigen-specific dual-costimulated CD4+ T cells can be heterogeneous with differential expression of Eomes, KLRG1, GzmB, and the IL-33R (ST2). Specifically, in one transgenic mouse line with lower levels of CD11c antigen presentation, CD4+ T cells developed into Eomes+KLRG1+GzmB- cells. Despite comparable costimulation however, CD4+ T cells developed into Eomes-KLRG1-GzmB+ cells in a mouse line with greater levels of CD11c antigen presentation. Analysis of known helper T cell transcription factors and cytokines also showed expression of other helper T cell subset markers in these cells. Further, the capacity of these cells to become activated and kill target cells is currently being investigated with an in vitro assay. These findings are novel, demonstrating that cytotoxic CD4+ T cells may in fact be a heterogeneous population influenced by antigen presentation levels. Thus, this data should greatly impact immunotherapeutic targeting of cytotoxic CD4+ T cells.
Human thymopoiesis and naïve T cell maintenance throughout life (LYM7P.724)
Authors: Thome Joseph , Ventevogel Melissa , Ohmura Yoshiaki , Kato Tomoaki , Sempowski Gregory , Farber Donna
Published: January 2023

The extent to which thymopoieis is diminished over the human lifespan and how this affects the naïve T cell repertoire in peripheral tissue sites is not well characterized. In collaboration with the New York Organ Donor Network, we have attained novel access to thymus and secondary tissues from deceased organ donors. From this, it is possible to correlate naïve T cell development to distribution in tissue sites from the same individual effectively identifying when functional thymopoiesis is halted and how this affects the naïve T cell pool over time. We have found marked cessation of functional thymopoiesis in donors over 40 years of age but strikingly naïve T cells are maintained in lymph node tissue sites for decades undergoing low levels of proliferation and turnover comprising over 20% of total T cells in donors aged 40-73. When profiling double negative (DN) stage thymocytes using CD25 and CD44, DN1 thymocytes accumulate in donors with negligible thymic output indicating a lack of progenitor cells to progress to maturation while identified recent thymic emigrants (RTE) exhibit differential distribution and regulation in tissues during periods of active thympopoiesis but are evenly distributed in the spleen and lymph nodes when the export of new naïve T cells is halted. Together this indicates that naïve T cell populations are uniquely maintained as a function of thymic activity in order to preserve the ability to respond to new pathogen throughout the human lifetime.
Lck is essential in the non-canonical T cell receptor CR signaling pathway regulated pressed by Cbl during T cell development (LYM7P.727)
Authors: Chiang Yungping , Hodes Richard
Published: January 2023

A canonical pre-TCR/TCR signaling pathway has been extensively characterized that involves sequential phosphorylation and signaling through Lck, ZAP-70, LAT, and SLP-76, and that plays a critical role in thymic T cell development as well as TCR-mediated activation of mature T cells. However, recent studies have reported that genomic deletion of Cbl substantially reverses the defect in thymic development that is seen in ZAP-70-deficient mice. To understand this ZAP-70 independent non-canonical pre-TCR/TCR signaling pathway, we have characterized the effect of Cbl inactivation on the physiologic and signaling defects in mice deficient in the proximal signaling molecules Lck and ZAP-70. We demonstrated that inactivation of Cbl reversed the defects in phosphorylation of multiple signaling molecules, including ERK, PLC-γ1 and AKT, in TCR-stimulated ZAP-70-/- thymocytes, and partially reversed the defective phenotype of T cell development in ZAP-70-/- mice. In contrast, inactivation of Cbl was unable to reverse either defective phosphorylation of TCR signaling molecules or the defective T cell developmental phenotype of Lck-/- mice. Lck thus plays a critical role in both the canonical TCR signaling pathway and in a ZAP-70-independent non-canonical pathway that is normally inhibited by Cbl but is capable of mediating productive thymocyte development.
iNKT cell development is modulated by TCR avidity (LYM7P.728)
Authors: Mallevaey Thierry
Published: January 2023

Invariant Natural Killer T (iNKT) cells are versatile innate T cells that influence immune responses associated with infection, cancer, inflammation and autoimmunity, notably through the production of a wide array of cytokines and chemokines. iNKT cells respond to lipid antigens presented by the MHC Class Ib molecule CD1d. iNKT T cell receptors (TCRs) have limited diversity, due to exclusive usage of a canonical Vα14-Jα18 TCRα chain and a limited set of TCR Vβ gene segments, but with extensive CDR3β diversity. Recent structural and mutational studies revealed that CD1d-lipid-TCR interactions differ markedly from classical pMHC-TCR interactions. Remarkably, docking onto CD1d-lipid complexes is unaffected by the nature of the lipid involved, nor TCRβ composition (Vβ gene segment usage or CDR3β diversity). Rather, we and others have demonstrated that TCRβ composition modulates TCR avidity for CD1d-lipids. Here, we have generated TCR retrogenic mice using TCRs with various avidities, and found a strong correlation between the frequency and numbers of iNKT cells that develop in vivo and TCR avidity for CD1d-lipids. In addition, TCR strength impacts on iNKT cell maturation as well as cytokine production upon immunization with the prototypical lipid αGalCer. In summary, our results highlight a crucial role for iNKT TCR strength on the selection and functional programing of iNKT cells.
Immune perturbation in patients with TGFbeta pathway defects (LYM7P.729)
Authors: Tran Dat , Regalado Ellen , Milewicz Dianna
Published: January 2023

Intro: Mutations in TGFbR1, TGFbR2 and SMAD3 are associated with familial thoracic aortic aneurysms and aortic dissections (TAAD). These patients offer an opportunity to study their immune development. Methods: PBMC from TAAD (n=9) and controls (CT, n=8) were analyzed by FACS. Th1 (IFNg) and Th17 (IL17A) were determined with intracellular cytokine staining. Foxp3+ Tregs were detected with anti-Foxp3. CD19+ were analyzed for naive (IgD+CD27-), unswitched (IgD+CD27+) and switched memory (IgD-CD27+). Plasmacytoid (CD303+pDC) and myeloid (CD1c+mDC) were defined within lineage-1 negative population. Results: %CD3-CD16+NK, CD3+CD16+NKT, CD4+, CD8+ and CD4+CD45RA+ in TAAD were similar to HC. Average %CD19+ (20.8vs7.3, p=0.006) and naive B cells (81.3vs66.6, p=0.004) were higher in TAADvsCT. The unswitched were similar but the switched B cells were lower (8.6vs15.5, p=0.01). While the %Tregs were similar, there was a remarkable reduction (1/2-3 folds, p<0.05) in Foxp3 concentration based on median fluorescence intensity of Foxp3 in TAAD. There was a significant reduction of %Th17 (0.14vs0.61, p=0.01), while the Th1 were similar. %pDC (9.4vs24.1, p=0.009) and %mDC (11.4vs17.9, p=0.01) were also lower in TAAD. Conclusions: These results demonstrate the involvement of TGFbeta signaling in B cells, DCs, Th17 and Treg development. Further studies and monitoring of the clinical effects of these immunological perturbations are needed to appreciate the impact of their underlying disease.
The absence of thymus involution in K5.Cyclin D1 transgenic mice sustains the output of recent thymic emigrants during aging. (LYM7P.717)
Authors: Bolner Michelle , Fink Pamela , Richie Ellen
Published: January 2023

Naïve T cells are continually exported from the thymus and maintain peripheral TCR repertoire diversity. As the thymus involutes with age, thymopoiesis and T cell output wane, reducing the number of recent thymic emigrants (RTEs). Although homeostatic proliferation of memory T cells sustains peripheral T cell numbers, the decrease in naïve T cells restricts immune responsiveness to new antigens. It has been proposed that RTE output would be sustained if thymus involution were prevented. To test this hypothesis, we analyzed RTEs in mice that do not undergo thymus involution due to a keratin 5 promoter-driven Cyclin D1 transgene (K5.D1) expressed in thymic epithelial cells. This transgene maintains functional thymic microenvironmental niches that support thymocyte progenitor maturation. We introduced a RAG2p-GFP transgene to discriminate RTEs from mature naïve (MN) T cells in K5.D1 mice. As previously reported, RTEs decline with age in RAG2p-GFP controls. In contrast, the number of RTEs increases with age in K5.D1;RAG2p-GFP mice. Despite a lower frequency of MN T cells in K5.D1 mice, the number of MN T cells is increased by ~4 fold in 5-6 month old K5.D1;RAG2p-GFP mice compared to RAG2p-GFP littermates. The increase in naïve T cells does not displace existing memory T cells, supporting the notion that naïve and memory T cells occupy distinct homeostatic niches. These data demonstrate that preventing thymus involution sustains the naïve T cell compartment during aging.
Type II NKT cells play a role in the regulation of CD4 T cell subsets (LYM8P.730)
Authors: Robertson Faith , Lu Xiuju , Terabe Masaki , Berzofsky Jay
Published: January 2023

The immunological response to cancer and other diseases depends on the interplay between the innate and adaptive immune systems. CD4 T helper cells are key modulators of immune responses. As an intermediate between the innate and adaptive systems, CD1d-restricted NKT cells may impact the activity of these CD4 T cells. It has been shown that type II NKT cells are immunosuppressive in many disease settings. However, their interaction with CD4 T cells is not well understood. In this study, we investigated the role of type II NKT cells on the activation of various CD4 T cell subsets by stimulating them with sulfatide, an endogenous glycolipid antigen and known type II NKT cell activator. Naïve ovalbumin-specific CD4 T cells isolated from DO11.10 mice were either tested immediately, or polarized in vitro with cytokines and neutralizing antibodies. CFSE dilution was used to assess CD4 proliferative responses upon antigenic stimulation in the presence or absence of sulfatide-activated type II NKT cells. We found that activated type II NKT cells substantially inhibited proliferation of naïve CD4 T cells, and preliminary data show that the polarized populations have different sensitivities to type II NKT cell mediated suppression. Ongoing elucidation of these interactions may not only enhance our understanding of CD4 T cell regulation, but also improve our capability to utilize type II NKT cells for clinical applications.
Comparison of the protective roles of Trypanosoma cruzi-specific Th1 and Th17 cells. (MPF6P.746)
Authors: Blase Jennifer , Eickhoff Christopher , Hoft Daniel
Published: January 2023

Trypanosoma cruzi is the protozoan etiology of Chagas disease. T. cruzi-specific Th1 cells are known to be important for protective immunity, while the role of T. cruzi-specific Th17 cells has yet to be explored in detail. TCR-transgenic, T. cruzi-specific CD4+ T cells were differentiated in vitro into Th1 or Th17 cells and co-cultured with T. cruzi-infected peritoneal exudate macrophages. Protection was measured as inhibition of intracellular amastigote replication. In vivo, T. cruzi-specific Th1 or Th17 cells were adoptively transferred into Rag-/- mice along with naïve polyclonal CD8+ T cells. Mice were challenged with T. cruzi, persistent immune responses were measured, and protection was assessed by parasitemia and survival. In vitro, both Th1 and Th17 cells led to protection against intracellular T. cruzi growth in macrophages. Th1-mediated protection involved IFN-γ signaling and nitric oxide production by macrophages. In contrast, Th17-mediated protection was dependent on IL-17A but independent of iNOS induction. The role of reactive oxygen species, autophagy, apoptosis, and mechanisms of lysosomal killing are being studied. More importantly, Th17 cells were significantly more protective than Th1 cells in vivo, involving enhanced expansion of pathogen-specific CD8+ T cells. Thus, Th17 cells have both direct and indirect effects for T. cruzi protective immunity. Furthermore, we report the novel finding that Th17 cells provide important helper effects for CD8+ T cells.
A new model to study mucosal immunity at the ocular surface (MUC7P.758)
Authors: St. Leger Anthony , Karauzum Hatice , Datta Sandip , Caspi Rachel
Published: January 2023

The ocular surface represents a mucosal interface between the environment and the host. Similarly to the lymphoid tissue of the gut, cells of the conjunctiva-associated lymphoid tissue (CALT) are organized into follicles. Humans exhibit CALT in healthy eyes, and the number and size of follicles increase with inflammation caused by infections, allergies, dry eye, and conjunctivitis, suggesting a functional role for CALT in immunity at the ocular surface. Due to the limited amount of data from animal models, functional studies of CALT are lacking. We aimed to develop a mouse model to study the dynamics and functional parameters of CALT in normal and immunologically perturbed hosts. Towards that end, we used mice deficient in IL-10 and/or IL-22. Upon ocular infection with Staphylococcus aureus (Strain USA300 LAC), we observed an expansion of immune cells in the CALT. This included elements of the adaptive and innate immune systems, with prominent expansion of group 3 innate-like lymphocytes (ILC3), which have hitherto only been shown to localize to the gut and lungs. The increase in cellular infiltrate correlated with an increase in the numbers of TNFα and IL-17 producing cells. Our studies lay the basis for a working mouse model to analyze the functional characteristics of CALT, an understudied component of the mucosal immune system. Also, this model may permit identification of factors that induce ocular inflammation leading to various ocular pathologies.
IL-33/ST2 regulate innate IL-17 production and viral replication in the cornea following a herpes infection (MUC7P.760)
Authors: Rowe Alexander , Harvey Stephen , Hendricks Robert
Published: January 2023

Poorly controlled infections and inflammation can both be very deleterious to tissues such as the cornea whose function requires clarity, so understanding the mechanisms controlling the balance between protective immunity and immunopathology is critical. Since the anti-inflammatory cytokine IL-33 and its receptor ST2 are constitutively expressed in the cornea, we investigated their role in regulating early stages of the immune response to herpes simplex virus type 1 (HSV-1) in the cornea. HSV-1 replicates in the corneal epithelium and is simultaneously axonally transported to the cornea innervating neuronal cell bodies in the trigeminal ganglion (TG). Compared to wild type controls, corneas of ST2 KO mice exhibited elevated IL-17 production from 4 hrs to at least 48 hrs after infection, which correlated with enhanced viral clearance from the cornea at 48 hrs (infectious virus in eye swabs, and viral genome copies in the cornea). Other anti-viral mediators such as IL-6 and type I interferons were not elevated in corneas of ST2 KO mice. Infected corneas of ST2 KO mice had increased numbers of neutrophils, but not macrophages or dendritic cells 2 days after infection. Surprisingly, viral titers in TG of infected ST2 KO mice were not significantly changed relative to wild type controls. We conclude that IL-33 selectively regulates an innate immune response to HSV-1 in the cornea at the expense of impaired viral clearance.
Group 2 innate lymphoid cells are present in mouse uterine tissue and may play roles in healthy pregnancy (MUC7P.763)
Authors: Bartemes Kathleen , Kita Hirohito
Published: January 2023

Innate lymphoid cells (ILCs) are emerging as important effector cells in innate immunity and tissue homeostasis. Group 2 ILCs (ILC2s) have been identified in organs and tissues, such as adipose tissue, small intestine, lungs and skin, where they are involved in helminth immunity, type 2 immune responses, and tissue pathology and recovery. Using flow cytometry, we identified ILC2s in mouse uteri and confirmed IL-5, but not IL-13, production in response to IL-33 both in vivo and in vitro. Furthermore, intraperitoneal administration of IL-33 induced recruitment of activated ILC2s to uteri as well as to lungs. To investigate the possibility that uterine ILC2s might play a role in maintaining a healthy pregnancy, we mated ST2-/- females (on a Balb/c background) with ST2-/-, MHC-matched Balb/c and MHC-mismatched C57B6 males (ST2 is the IL-33 receptor). Balb/c, C57B6 and Balb/c x C57B6 pairs were used as controls. Litter sizes were not significantly different among the pairings. We defined viable pups as those still alive 24 hours after parturition. Total numbers of non-viable pups, percent of litters with at least one non-viable pup and percent of non-viable pups per litter were significantly increased in ST2-/- x C57B6 pairs when compared to control pairs. Thus, IL-33-responsive ILC2s are present in murine uterine tissue and may play roles in successful reproduction.
Exosomes in human semen impair antigen-presenting cell function and decrease antigen-specific T cell responses (MUC7P.767)
Authors: Vojtech Lucia , Hughes Sean , Levy Claire , Tewari Muneesh , Hladik Florian
Published: January 2023

Exposure to semen is the primary route of transmission for many sexually transmitted infections. Mounting evidence suggests that components in semen directly influence leukocytes, which could help to explain the difficulty of inducing vaccination-based protection in the genital mucosa. Exosomes, small microvesicles with demonstrated immunomodulatory functions, are present at an average concentration of 2.2 x 1013 particles per ejaculate (n = 18 healthy semen donors). These seminal exosomes (SE) efficiently and rapidly entered peripheral and vaginal dendritic cells (DCs), whereas T cell uptake of SE was poor. SE impaired the immune responses of DCs stimulated by bacterial lipopolysaccharide, where cytokine production was reduced at both the mRNA level in the cells and the protein level in supernatants. Preliminary experiments also indicated that SE down-regulate CD80 and CD83 expression on DCs. In PBMC cultures, SE impaired memory T cell function, reducing the production of TNF-alpha and/or IFN-gamma in response to virus-derived peptides an average of 73% for CD4+ T cells and 55% for CD8+ T cells, in a dose-responsive manner (n = 4 PBMC donors). Thus, SE likely inhibit antigen-specific T cell responses by affecting the co-stimulatory capacity of antigen-presenting cells. Understanding how programmed immune responses are altered by the presence of semen is important to developing the next generation of vaccine and preventative treatments against sexually transmitted disease.
Topical electrophilic nitro-fatty acids potentiate cutaneous immune responses. (MUC7P.775)
Authors: Mathers Alicia , Carey Cara , Killeen Meaghan , Salvatore Sonia , Schopfer Francisco , Freeman Bruce , Falo Louis
Published: January 2023

Endogenous electrophilic fatty acids mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction reactions. In this regard, there is considerable interest in employing nitro-fatty acids and other electrophilic fatty acids for the prevention and treatment of inflammatory disorders. We evaluated nitro-oleic acid (OA-NO2) in a preclinical murine model of allergic contact dermatitis (ACD) as a potential topical therapy for the treatment of ACD. In marked contrast to previous findings, topical OA-NO2 potentiated hapten-dependent inflammation inducing a sustained psoriasis-like inflammatory response characterized by psoriasiform histological features, increased angiogenesis, and an inflammatory infiltrate that included neutrophils, inflammatory monocytes, and γδ T cells. Interestingly, analysis by HPLC-MS-MS of skin from psoriasis patients revealed a significant increase in nitro-fatty acids in lesional skin compared to non-lesional skin. Overall, when applied topically to the cutaneous microenvironment nitro-fatty acids can potentiate inflammatory responses. Thus, it is paramount that a better understanding be obtained of how electrophilic fatty acid derivatives regulate cutaneous immunity due to the widespread use of fatty acids in topical creams and the abundant formation of electrophilic nitro-fatty acids during inflammation.
The role of gap junction protein connexin 43 in Chlamydial pathogenesis (MUC7P.752)
Authors: Murthy Ashlesh , La Salle Sophie , Nicholson Bruce , Manam Srikanth , Holtzman Michael , Zhang Yong
Published: January 2023

We have shown that Chlamydia-specific CD8 T cells do not significantly affect bacterial clearance but cause oviduct pathology, suggesting that they do not target infected cells efficaciously but cause pathology presumably by targeting uninfected cells. This suggests the possibility that chlamydial peptides are presented to CD8 T cells by uninfected cells, but begs the question: “How do uninfected cells acquire chlamydial peptides”? We hypothesized that gap junction mediated antigen transport (GMAT) via channels formed by connexin (Cx) proteins, predominantly Cx43 expressed on oviduct epithelium, contributes to this effect. We have found that HeLa cells engineered to express Cx43, but not those without, can efficaciously transfer Ova257-264 to co-cultured mouse APC, which subsequently activate CD8+ T cells from Ova primed OT-1 mice. Additionally, using Cre-Lox technology, we generated mice (Foxj1Cre-Cx43flox mice) with a conditional deficiency of Cx43 only in ciliated columnar epithelia, including oviducts. The Foxj1Cre-Cx43flox mice displayed comparable kinetics of vaginal chlamydial clearance, comparable splenic antigen-specific cytokine and serum antibody response, but significantly reduced oviduct pathology compared to WT mice on day 80 after intravaginal chlamydial infection. We have thus demonstrated the role of GMAT in immunopathogenesis of microbial infections.
Analysis of CNS tissue-specific Tregs and dendritic cells in T cell responses and autoimmunity (MUC7P.757)
Authors: McPherson Scott , Heuss Neal , Pierson Mark , Gregerson Dale
Published: January 2023

We assessed locally generated, locally acting peripheral regulatory T cells (pTregs) in retinal immune homeostasis and ability of retinal dendritic cells (DC) to support T cell activation/expansion within retina. We used transgenic (Tg) mice expressing beta-galactosidase (bgal mice) as retinal neo-self antigen, TCR Tg mice (BG2) specific for bgal, and mice with labeled, depletable Tregs or DC, based on expression of diphtheria toxin (DTx) receptor and/or GFP controlled by FoxP3 or CD11c promoter (FDG or CDG mice). Treg and DC depletion from retina was done by DTx injection into the eye. T cells and DC were assayed by FACS of retina or blood. Retinal autoimmunity was assessed by histological analysis. After bgal (FDG/bgal mice) or IRBP (FDG mice) immunization, Treg depleted retinas showed increased incidence and severity of autoimmunity. We found local depletion of Tregs from retina sufficient to induce spontaneous autoimmunity in FDG/bgal/BG2 mice but not in single or double Tg mice. Dose of DTx used to eliminate retinal Tregs did not induce retinal autoimmunity if given systemically. DC depletion from the retina prevented Treg and Teff generation within retina after bgal injection. Retinal microglia remaining after DC depletion did not make up for the loss of DC-dependent antigen presentation. We conclude local Tregs within the retina protect against spontaneous organ-specific autoimmunity and local DC must be present within the retina for antigen presentation to T cells.
DC-expressing TRAF6 is important mediator of gut homeostasis in the context of dietary antigen responses (MUC8P.812)
Authors: Han Daehee , Surh Charles
Published: January 2023

The intracellular signaling mediator TRAF6 is critical for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). Recently we have reported that TRAF6 in DCs is important to maintenance of intestinal immune tolerance through balanced induction of regulatory T cells versus Th2 immunity. In addition, we found a clue that loss of TRAF6ΔDC mucosal tolerance requires the presence of commensal microbiota in a manner that appears independent of the Myd88 pathway. However, which environmental factors induce abnormal Th2 immunity and disease phenotype in TRAF6ΔDC mice remains unclear. Here we suggest that spontaneous gut inflammation occurred in TRAF6ΔDC mice is strongly associated to dietary antigens as well as microbiota.
Optimal CXCL8 secretion by IL-1 stimulated Caco-2 epithelial cells requires an effect of the Rho associated kinase, ROCK, on the JIP-3 scaffold signaling to JNK (MUC9P.815)
Authors: Banerjee Sayantan , McGee Dennis
Published: January 2023

Elevated levels of Interleukin-1 (IL-1) accompany inflammatory bowel disease. IL-1-stimulated intestinal epithelial cells (IECs) secrete potent chemokines like CXCL-8 (IL-8), which can attract neutrophils and exacerbate inflammation. Previously, we found that inhibiting the Rho-Associated Kinase, ROCK, using the compound Y27632 could inhibit IL-1-induced CXCL8 secretion by the Caco-2 colonic epithelial cell line. We also reported that ROCK inhibition during IL-1 signaling did not affect signaling through IκBα, but suppressed the phosphorylation of JNK. Therefore, ROCK inhibition-mediated suppression of CXCL8 secretion may be due to an inhibition of JNK and not the NF-κB pathway. We now confirm that ROCK inhibition had no effect on the formation of the upstream IRAK1-TRAF6 signaling complex in IL-1-stimulated cells. Also, inhibiting ROCK had no effect on p38 MAPK phosphorylation or the stability of CXCL8 mRNA. Yet, inhibiting ROCK suppressed the IL-1-induced phosphorylation of MKK4, a known activator of JNK, but not MKK7. Immunoprecipitation of the JIP3 scaffold protein from IL-1-stimulated cells showed that inhibiting ROCK suppresed both the association of JNK with JIP3 and the phosphorylation of JNK. Hence, a ROCK-JIP3-JNK interaction may be a crucial regulatory sequence during IL-1-induced chemokine responses by IEC. These studies indicate a novel signaling event, which could provide a target for suppressing IEC chemokine responses involved in mucosal inflammation.
GPR15 mediated homing controls immune homeostasis in the large intestine mucosa (MUC9P.817)
Authors: Kim Sangwon , Xiang Wenkai , Kwak Changsoo , Yang Yi , Lin Xiyao , Ota Mitsuhiko , Sarpel Umut , Rifkin Daniel , Xu Ruliang , Littman Dan
Published: January 2023

The large intestine is the site most commonly affected in inflammatory bowel diseases. However, the mechanism of T cell homing to the large intestine, which contributes to inflammation, had remained unclear. We show here that an orphan G-protein coupled receptor GPR15 controls the specific homing of T cells, particularly FOXP3+ regulatory T cells (Tregs), to the large intestine lamina propria (LILP). GPR15 expression is promoted by gut microbiota and TGF-β1, but not by retinoic acid. GPR15-deficient mice had fewer Tregs in LILP and were prone to develop more severe inflammation in the large intestine, which was rescued by the transfer of GPR15-sufficient Tregs. Our findings thus indicate that GPR15 is a T cell homing receptor for LILP and that GPR15 plays a key role in maintaining gut immune homeostasis, largely by regulating the influx of Tregs. Our study also demonstrates that adaptive immune responses in the gut are functionally compartmentalized through the differential requirements for T cell homing to the small and large bowel.
FcγRIIB regulates T cell autoreactivity, ANCA production and neutrophil activation to suppress anti-myeloperoxidase glomerulonephritis (THER5P.834)
Authors: Ooi Joshua , Gan Poh-Yi , Chen Tong , Eggenhuizen Peter , Odobasic Dragana , Holdsworth Stephen , Kitching A Richard
Published: January 2023

Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis involves innate and adaptive immune cells in the induction of autoimmunity and in autoimmune effector responses. Most Fcγ receptors (FcγRs) activate immune cells, but FcγRIIB, found in humans and mice on B cells and innate cells, is an inhibitory receptor. These studies tested the hypothesis that endogenous FcγRIIB negatively regulates autoreactivity and effector responses in experimental anti-myeloperoxidase (MPO) glomerulonephritis, using wild type and FcγRIIB-/- mice. After MPO immunization, FcγRIIB-/- mice developed higher MPO-ANCA titers and increased anti-MPO T cell responses. Transfer of FcγRIIB deficient dendritic cells loaded with a nephritogenic MPO peptide (MPO409-428) into wild type mice induced stronger autoimmunity than dendritic cells derived from wild type mice. Transferring anti-MPO antibodies into LPS primed mice resulted in increased glomerular neutrophil accumulation and injury in FcγRIIB-/- mice, showing a role for FcγRIIB in suppressing neutrophil activation. Inducing active autoimmunity to MPO followed by triggering T cell mediated glomerular injury by transfer of subnephritogenic doses of LPS and anti-MPO antibodies resulted in more disease in FcγRIIB-/- mice. Therefore endogenous FcγRIIB negatively regulates anti-MPO autoimmunity and glomerulonephritis via dendritic cells, B cells and neutrophils to limit MPO-ANCA production, T cell responses and neutrophil activation.
Marked induction of long-term islet graft survival in gastric submucosa engrafted rats (THER5P.841)
Authors: Wang Zhiliang , Li JIaxin , Chen Xiang , Wang Ximo
Published: January 2023

Since the first report of successful islet transplantation to treat diabetes mellitus in rodents, selecting the optimal site for transplantation has become a key step for successful treatment. To date, the portal vein (PV) has been the primary site for clinical islet transplantation. Despite some limited success, complications, particularly instant blood-mediated inflammatory reaction (IBMIR), due to the graft site location are not conducive to long-term survival of islets and improvement of diabetes. Therefore finding a more suitable site for islet transplantation has become urgent. The gastric submucosa (GS) and kidney capsule (KC) space are fast becoming the two most common sites for islet transplantation in pre-clinical studies. Thus, these sites were compared for induction of long-term islet graft survival in rats. 24 Sprague Dawley recipients had diabetes induced by streptozocine (STZ); the STZ-induced rats were then distributed into 3 groups based on the different sites for transplantation. In our study, rats that received islets in the GS obtained better control of diabetes compared to KC and PV engrafted animals. IBMIR and pro-inflammatory cytokines also indicated an early loss of the islets in PV transplanted animals that was not evident in GS and KC animals.
A specific tolerance inducing vector for therapeutic treatment of autoimmune diseases (THER5P.842)
Authors: Hansson Charlotta , Schön Karin , Hasselberg Annemarie , Lycke Nils
Published: January 2023

We have developed a novel platform for intranasal treatment of autoimmune diseases in which ADP-ribosylation determines whether immunity or tolerance is induced. Hence, cholera toxin A1-subunit based immunomodulation through the CTA1-peptide-DD fusion protein promotes enhancement, while inactive mutants induce suppression. Whereas the target population after intranasal administration of both constructs was CD103+CD11blow DC’s, both active and inactive mutants induced strong CD4 T cell priming, but they differentially affected CD4 T cell differentiation. This way inactive mutant constructs generated regulatory CD4 T cells producing IL-10 and effectively preventing experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA), when carrying relevant peptides. Targeted DCs expressed low levels of CD80, CD86 and CD40, while, by contrast, ADP-ribosylating CTA1-peptide-DD constructs gave significantly enhanced co-stimulation. Suppression was global in that regulatory T cells following treatment were able to suppress adoptively transferred naïve CD4 T cells and could subsequently maintain tolerance. The use of specifically tolerance-inducing fusion proteins may be a way forward in the search of effective treatments against autoimmune diseases. The immunomodulating effect on the CD103+CD11blow DCs will be described and the requirement for CTA1-binding to Gsa evaluated in detail, using Cre-lox Gsa-deficient mice.
Conjugated linoleic acid reduces inflammation via PPARγ in murine autoimmune glomerulonephritis (THER6P.861)
Authors: Hammond Sarah , Regna Nicole , Chafin Cristen , Reilly Christopher
Published: January 2023

Conjugated linoleic acid (CLA) has been shown to reduce inflammation via Peroxisome Proliferator-Activated Receptor (PPAR)-γ in inflammatory disorders. We sought to determine whether CLA isomers would reduce inflammation via PPARγ in cultured mesangial cells, and in murine autoimmune glomerulonephritis. Mesangial cells were cultured with pure CLA isomers (c9,t11 or t10,c12) or a 50:50 mixture prior to immune stimulation. Next, cultured mesangial cells were transfected with siRNA targeting PPARγ and treated with CLA isomers prior to immune stimulation. ELISA and RT-qPCR were performed to measure cytokine production and mRNA expression. Treatment with c9,t11 CLA reduced IL-6 production in cultured mesangial cells, but not in siRNA-treated mesangial cells, suggesting c9,t11 CLA acts through PPARγ. IL-1RA mRNA expression was significantly higher in c9,t11-treated siRNA-transfected cells than in t10,c12-treated cells, suggesting alternate pathways in addition to PPARγ. For in vivo studies, wild-type and PPARγ -/- mice were fed a control diet or a CLA-supplemented diet (50:50 mixture of c9,t11 and t10,c12) prior to induction of glomerulonephritis by anti-glomerular basement membrane antisera administration. CLA treatment reduced renal TGFβ in the CLA-treated wildtype mice, but not in the CLA-treated PPARγ-knockout mice. Taken together, these studies suggest that CLA acts in part through PPARγ to decrease inflammatory mediator production in autoimmune glomerulonephritis.
A pharmacological protein kinase D inhibitor ameliorates experimental arthritis. (THER6P.850)
Authors: Yi Ae-Kyung , Yoon Tae Won , Brand David , Kim Young-In , Park Jeoung-Eun , Stuart John
Published: January 2023

While the factors that initiate and perpetuate inflammation in rheumatoid arthritis (RA) are not well understood, it is thought that RA may be initiated in part by signaling through Toll-like receptors (TLRs). Disruption of the TLR signaling pathway may provide an opportunity to halt the RA process in the earliest stages of development. We previously found that protein kinase D1 (PKD1) is essential for proinflammatory responses mediated by MyD88-dependent TLRs. Here, we investigated whether PKD1 is involved in exaggerated proinflammatory responses seen in RA patients, and in the development of experimental arthritis. PKD1 was constitutively hyperactivated in synoviocytes of RA patients, and was rapidly activated in synoviocytes of normal donors and RA patients in response to TLR/IL-1R ligands. Silencing PKD1 expression in RA synoviocytes resulted in substantial inhibition of both spontaneous and TLR/IL-1R-mediated expression of cytokines/chemokines. Suppression of PKD1 expression also resulted in inhibition of TLR/IL-1R-mediated cytokine production in IL-1R antagonist-deficient (IL-1rn-/-) macrophages. In addition, daily treatment with a PKD inhibitor substantially reduced the incidence and severity of spontaneously occurring inflammatory arthritis in IL-1rn-/- mice, and collagen-induced arthritis in humanized HLA-DR1 mice. This implies that PKD1 might be one of the key regulatory factors that modulate proinflammatory responses in RA, and can be a therapeutic target for RA.
IL-12/Th1 and IL-23/Th17 biliary microenvironment in primary biliary cirrhosis (THER6P.852)
Authors: Yang Chen-Yen , Ma Xiong , Tsuneyama Koichi , Huang Shanshan , Takahashi Toru , Chalasani Naga , Bowlus Christopher , Yang Guo-Xiang , Leung Patrick , Ansari Aftab , Wu Linda , Coppel Ross , Gershwin Merrill
Published: January 2023

The interleukin (IL)-12/IL-23 mediated Th1/Th17 signaling pathway has been associated with the etiopathogenesis of primary biliary cirrhosis (PBC). To address the cytokine microenvironment specifically in the liver, we examined the localized expression of IL-12/IL-23 molecules and their corresponding receptors by immunohistochemistry with an extensive panel of antibodies with specificity for IL-12p70/p35, IFN-γ, IL-12RB2, IL-23p40/p19, IL-17 and IL-23R using liver from PBC (n=51) and non-PBC (n=80) control liver disease patients. Sections of portal tracts were blindly evaluated and individually scored. We report herein that although IL-12/Th1 and IL-23/Th17 staining were detected in all of the liver sections, they were primarily localized around the damaged interlobular bile ducts in PBC. Most importantly, the Th1/Th17 balance skewed toward Th17 prominently in liver tissues from advanced PBC patients with intensive secretion of IL-23p19 by inflamed hepatocytes around IL-23R, IL-12RB2, and IFN-γ expressing degenerated cholangiocytes. In conclusion, our novel finding on the direct association of Th17 skewing and disease severity illustrates the significance of the IL-23/Th17 pathway in the perpetuation of IL-12/Th1-mediated immunopathology in PBC and has important therapeutic implications. The imbalance toward Th17 at advanced stages of PBC suggests the necessity to consider blockade of both IL-12/Th1 and IL-23/Th17 signaling for therapeutic intervention.
Therapeutic treatment of multi-organ system, obstructive pulmonary and scleradermatous chronic graft-versus-host disease with the BTK and ITK inhibitor Ibrutinib (TRAN3P.873)
Authors: Flynn Ryan , Dubovsky Jason , Harrington Bonnie , Reichenbach Dawn , Yiming Zhong , Yang Carrie , Towns Will , Lehman Amy , Johnson Amy , Muthusamy Natarajan , Devine Steven , Jaglowski Samantha , Serody Jonathan , Murphy William , Munn David , Luznik Leo , Maillard Ivan , Koreth John , Cutler Corey , Soiffer Robert , Antin Joseph , Ritz Jerome , Panoskaltsis-Mortari Angela , Byrd John , Blazar Bruce
Published: January 2023

Chronic GVHD (cGVHD) is dependent on the ability of donor bone marrow (BM) B cells to produce pathogenic antibody (Ab) deposited in target organs, which is associated with increased germinal centers (GCs). BTK is necessary for B cells to enter GCs and ITK is necessary for T cell activation. We hypothesized that alloreactive GC B cells and T follicular helper cells require BTK or ITK and inhibition by ibrutinib (Ib) would prevent cGVHD. We evaluated the therapeutic effect of Ib in two models of cGVHD: a MHC-disparate (B6→B10.BR) multi-organ model complicated by bronchiolitis obliterans (BO) and a sclerodermatous (SCL) minor-mismatch (LP/J→B6) model. Mice treated with Ib had a decrease in pathogenic Ab, collagen deposition and GC B cells in target organs similar to healthy control mice. In contrast to cGVHD mice, pulmonary function in Ib treated mice was similar to control mice. To confirm that BTK and ITK are required for cGVHD we used donor mice with an ablation of BTK or ITK. Absence of BTK in B cells impeded GCs, Ab deposition and BO. Mice transplanted with T cells lacking ITK and WT BM did not develop BO. In the SCL model, mice treated with Ib did not develop clinical signs of cGVHD including less alopecia and SCL lesions. Furthermore, there was a decrease in lymphocyte infiltration surrounding the bronchioles and nephrons in treated mice. The inhibition of TEC-family kinases with Ib is a novel therapeutic for multi-organ system cGVHD with BO or SCL manifestations.
Endogenous memory CD8 T cells exhibit increased early proliferation in cardiac allografts subjected to prolonged ischemia (TRAN3P.880)
Authors: Su Charles , Iida Shoichi , Abe Toyofumi , Fairchild Robert
Published: January 2023

Memory T cells induced to microbial infections provide protection to subsequent exposures but can have cross-reactivity with allogeneic MHC molecules. High numbers of memory T cells with donor reactivity in human and non-human primate recipients prior to transplant are associated with poorer allograft survival and resistance to costimulatory blockade strategies given to induce long-term graft survival. Rodent transplant models have failed to replicate the detrimental impact of endogenous memory T cells on allograft survival and tolerance induction, raising questions about the sufficiency of these models to study this component of alloimmunity. We have recently shown a direct association between increasing duration of cold ischemic storage and numbers of early graft infiltrating memory CD8 T cells that markedly up-regulate effector functions and directly mediate costimulatory blockade resistant rejection of cardiac allografts in a mouse model. Here we show that endogenous memory CD8 T cells exhibit increased early proliferation in grafts subjected to prolonged ischemia that is supported by memory CD4 T cells. Recipient depletion of CD4 T cells or treatment with MR1 (anti-CD40L) at transplant significantly decreased early memory CD8 T cell proliferation and extended survival of grafts subjected to prolonged cold ischemia. These studies provide novel insights into endogenous memory CD8 T cell mediated graft injury and the poor outcomes associated with cadaver donor grafts.
Defining the alloreactive T cell repertoire using high-throughput sequencing of mixed lymphocyte culture (TRAN3P.884)
Authors: Emerson Ryan , Matthew James , Robins Harlan , Leventhal Joseph
Published: January 2023

The cellular immune response is a major barrier to patient tolerance of allografts. Positive followed by negative selection in the thymus ensures that T cell populations have some binding affinity for self-HLA, but culls T cells with high specificity for peptides presented on self-HLA. However, in the context of a patient with an allograft, these processes also allow for mature T cells with off-target specificity for self-peptides presented on allo-HLA. Currently, the breadth of the alloreactive T cell repertoire is unknown, as well as whether the alloreactive T cell repertoire is stable over time. We have used high-throughput sequencing to characterize the alloreactive T cell repertoire in three pairs of healthy adults using mixed lymphocyte culture, analyzing 4 reactions per pair: duplicate MLR reactions at baseline followed by duplicate MLR reactions 3 months later. Our results indicate that thousands of T cell clones proliferate in mixed lymphocyte culture, suggesting a very broad alloreactive T cell repertoire. This repertoire is consistent across biological replicates and across a span of three months, and is dominated by relatively high-abundance T cell clones. Our results suggest the existence of a broad alloreactive T cell repertoire consisting mainly of expanded memory T cells which remain stable over time. The presence of a large, stable population alloreactive T cells should allow for long-term tracking of alloreactive clones in patients receiving allografts.