bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the nitric oxide-mitochondrial hyperpolarization-mTOR-Rab4 signaling pathway precedes disease development in lupus T cells (93.36)
Authors: Perl Andras , Telarico Tiffany , Singh Ram , Fernandez David
Published: January 2023

Nitric oxide (NO) dependent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation in Systemic Lupus Erythematosus. Activity of mTOR, which senses the mitochondrial membrane potential, is increased in lupus T cells, which promotes HRES-1/Rab4 expression. Rab4 overexpression enhances recycling and lysosomal degradation of immune synapse components, including CD4 and TCRζ. We investigated activity of the NO-MHP-mTOR-Rab4 signaling pathway in lupus-prone mice. Overexpression of Rab4 and loss of TCRζ were detected prior to disease onset of NZB/NZW (F1) mice at 5 months. Increased NO production, mitochondrial transmembrane potential, and mitochondrial mass were present after disease onset at 11 months. MRL/lpr mice exhibited increased expression of the mitochondrial voltage-dependent anion channel 1(VDAC1) protein, transaldolase (TAL), Rab4, mTOR activation, and enhanced CD3 and CD4 recycling at 2-3 months, prior to disease development. In conclusion, lupus-prone mice exhibit mitochondrial dysfunction, with increased NO production, MHP, elevated expression of VDAC1 and TAL, and mTOR activation. Increased Rab4 expression was associated with enhanced endocytic recycling of CD4 and CD3. The results suggest that activation of the NO-MHP-mTOR-Rab4 signaling pathway is detectable and precedes disease onset in lupus-prone mice.
Generation of a proinflammatory response by macrophage following infection with an O3:K6 serotype of Vibrio parahaemolyticus. (94.3)
Authors: Parent Michelle , Waters Stephanie , Blumerman Seth , Whitaker Brian , Joerger Torsten , Boyd E.
Published: January 2023

Vibrio parahaemolyticus, a Gram-negative bacterium, is the leading cause of seafood-related bacterial gastroenteritis in the United States. This emerging pathogen can causes self-limiting gastrointestinal infection, wound infection, and, in the immunocompromised host, significant systemic disease, leading to death. The CDC has recently reported a 47% increase in the number of culture confirmed V. parahaemolyticus infections since 1996-1998. There is a dearth of literature regarding the host response to, and subsequent elimination of, infection with this pathogen. In order to elucidate the immune response to infection, we have developed an in vitro model using the virulent clinical isolate V. parahaemolyticus RIMD2210633, an O3:K6 serotype. First, we determined that this isolate could infect RAW264.7 cells, a murine macrophage cell line. Additionally, infection resulted in the simultaneous detection of 7-amino-actinomycin D (7-AAD) and annexin-V positive cells, suggesting cell death with a loss of membrane integrity. Lastly, infection resulted in the production of the early proinflammatory cytokines interleukin-1 alpha (IL-1α) and interleukin 6 (IL-6). We a currently investigating the cellular pathways involved in this proinflammatory response and macrophage cell death. Here, we have begun to investigate those aspects of the immune response required to eliminate this gastrointestinal pathogen from infected host tissues.
Adoptive transfer of Tc17 CD8 T cells leads to tumor inhibition and anti-tumor response (95.15)
Authors: Yen Hung-Rong , Harris Timothy , Radojcic Vedran , Huang Dih-Dih , Grosso Joseph , Getnet Derese , Wada Satoshi , Goldberg Monica , Bruno Tullia , Durham Nicholas , Khazaie Khashayarsha , Luznik Leo , Lin Tzou-Yien , Pardoll Drew , Huang Ching-Tai , Drake Charles
Published: January 2023

Although CD4+ T cells that secrete IL-17 (TH17) have been fairly well-studied, we and others have shown that CD8 T cells can be cultured under similar skewing conditions, resulting in CD8 cells with a robust ability to secrete IL-17. The physiological role such CD8 T cells (Tc17) is not well understood. In our previous data, we showed that Tc17 can functionally convert to IFN-g producing cells, expand, and mediate autoimmunity in a self-antigen murine model. Because autoimmunity and antitumor immunity both involve the breaking of peripheral T cell tolerance, we performed experiments to test whether Tc17 could mediate anti-tumor immunity in implanted tumor models. In a preventive model, small numbers (0.1M) of sorted IFN-g-producing Tc1, IL-17-producing or IFN-g-producing Tc17 TCR-transgenic HA-recognizing CD8 T cells were adoptively transferred to immunocompetent recipient (Balb/C) mice on day -1, and 0.5M CT-26 tumor cells expressing HA as antigen were implanted into the footpad on day 0. Adoptive transfer of Tc17 was significantly superior to Tc1 in mediating an antitumor response and a better survival rate. Additional mechanisms are being explored.
Identification and characterization of putative proline directed serine/threonine kinase(s) directed against a functional PSP motif in STAT5 (95.6)
Authors: Mitra Abhisek
Published: January 2023

Hematological malignancies, such as leukemia and lymphoma, can develop from aberrant changes in cell signaling molecules that drive uncontrolled cell proliferation. Although there are numerous effector molecules that promote activation and expansion of T cells, one key pathway is the JAK (Janus Kinase)-STAT (Signal transducer and activator of transcription) cascade. These signaling proteins are activated in response to an array of cytokines, however constitutively activated JAKs and STATs have been described in several T cell malignancies. Indeed, a growing body of evidence suggests that dysregulated STAT5 may be a major contributor to certain types of T cell cancers. In addition to tyrosine phosphorylation of STAT5, serine phosphorylation within a Proline-Serine-Proline (PSP) or PXSP motif, located in the transactivation domain may also be equally important to its activation status, however the STAT5 serine-threonine kinase is not immediately known. Therefore, using a siRNA directed “loss of function” approach, proline directed serine/threonine kinases were screened for their ability to phosphorylate STAT5 in an IL-2 dependent manner. The results from this assay provide interesting insight into this family of kinases and their role in STAT5 regulation. Additionally, the data offers new information into the functional role of STAT5 serine phosphorylation and provides possible new therapeutic strategies for controlling diseases dependent upon this key signaling pathway.
A leukemic plasmacytoid dendritic cell line (PMDC05) as potent antigen presenting cells for anti-tumor cellular immunothrerapy (95.8)
Authors: Yamahira Akie , Narita Miwako , Nakamura Takeshi , Watanabe Norihiro , Kaji Masami , Taniguchi Tomoyo , Hashimoto Shigeo , Takahashi Masuhiro
Published: January 2023

We established a leukemic plasmacytoid dendritic cell (pDC) line (PMDC05) with potent antigen presenting capacity from leukemia cells of a patient with pDC acute leukemia. PMDC05 was positive for CD4, CD56, CD33, HLA-DR, CD123 (IL-3Rα) and CD86 in the absence of lineage markers. mRNA of TLR1, TLR2, TLR4, TLR7 and TLR9 were clearly expressed and among these TLRs, TLR7 was prominent. By culturing PMDC05 with IL-3, CpG-B or LPS, the expression of CD1a, CD80, CD83, CD86 or HLA-DR was remarkably enhanced. Production of IFN-α and IL-12p70 was enhanced by the stimulation with CpG-A and LPS, respectively. PMDC05, which was stimulated with 0.1 μg/mL LPS and loaded with 10 μg/mL WT1 peptides (HLA-A*2402-restricted, modified-type 9-mer peptide; CYTWNQMNL) for 24 hours, was irradiated (60 Gy) and co-cultured with allogeneic CD8+ T cells purified from PB-MNCs of HLA-A*2402+ healthy donor (PMDC05:CD8+ Tcells = 1:10). Induction of WT1-specific CTLs was evaluated by flow cytometry analysis using HLA-A*2402 WT1 tetramer every week. Although Daudi pulsed with WT1 peptides could not induce WT1 tetramer+CD8+ T cells during the co-culture up to 7 weeks, PMDC05 began to induce WT1 tetramer+CD8+ T cells at week 4 and the percentage of WT1 tetramer+ T cells in CD8+ T cells rose to more than 75% at week 7. These data suggested that PMDC05 could be efficiently used for generating CTLs specific for antigens of tumors and could be applicable for cellular immunotherapy for tumors.
Single Dose Nasal Immunotherapy Rapidly Recruits NK Cell-Dependent Regulatory T (Treg) Cells into Central Nervous System (CNS) to Ameliorate Experimental Autoimmune Encephalomyelitis (EAE) (96.12)
Authors: Huarte Eduardo , Rynda* Agnieszka , Maddaloni Massimo , Riccardi Carol , Pascual David
Published: January 2023

Reovirus protein sigma 1 (pσ1), engineered to deliver OVA mucosally, induced tolerance in mice even with a single, low dose. To test the efficacy of pσ1-based therapy, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG29-146) was genetically fused to pσ1 (MOG-pσ1). Susceptible C57BL/6 mice were nasally dosed with PBS, MOG-pσ1, or recombinant (r)MOG and 3 wks later were challenged with MOG35-55-induced EAE. MOG-pσ1, not rMOG or PBS, abrogated EAE incidence evidenced by >8-fold reductions in IFN-γ, IL-17, and IL-21, but greatly elevated IL-4 and IL-10. To test its therapeutic potential at the peak of MOG35-55-induced EAE (around day 15), mice were nasally treated with 50 μg of MOG-pσ1 or PBS. Strikingly, within 24 hrs of treatment with MOG-pσ1, >2-fold reduction in clinical symptoms were obtained. This improved clinical response was accompanied by a significant increase of at least 4-fold in FoxP3+ Treg cells in the spinal cords of treated mice; however, such increases were not evident in head and neck lymph nodes or the spleen. This increase in Treg cell infiltration into the CNS appears to be NK cell-dependent since this infiltration was abated in NK cell-depleted mice. Finally, enhanced apoptosis (30%) of infiltrating CD11c+ CD11b+ DCs was observed, underscoring the complexity of this system. * Authors contributed equally to the work; supported by NIH AI-78938.
Beta-Endorphin Neuron Transplant in the Hypothalamus Reduces Pro-inflammatory Cytokines and Inflammation in the Ankle’s Joint and Ameliorates the Course of Adjuvant-Induced Arthritis (96.15)
Authors: Govorko Dmitry , Boyadjieva Nadka , Ortiguela Maria , Sarkar Dipak
Published: January 2023

In our recent study, we demonstrated that implantation of stem cell-derived beta-endorphin producing (BEP) neurons into the rat hypothalamus is capable of suppressing the level of inflammatory cytokines in a cancer model. In this study, we are reporting the effect of the BEP cells transplantation into the hypothalamus on the systemic inflammation exemplified by adjuvant-induced arthritis (AIA). Female Lewis rats were transplanted with BEP cells or the neuronal cortical cells into the paraventricular nuclei. AIA was induced by intradermal injection of the oil suspension of Mycobacterium butyricum at the base of the tail. The severity of the AIA was assessed by evaluating clinical parameters such as ankle circumference, paw thickness, and the articular index. BEP neuron transplanted rats exhibited a statistically significant reduction in the articular index (P <0.05 at day 12), posterior paw thickness (P < 0.05 at day 16) and ankle circumferences (P < 0.01 at day 21) in comparison to cortical neuron transplanted rats. Concomitantly with the clinical parameters, the mRNA expression levels of pro-inflammatory cytokines such as TNF-alpha, Il-2, Il-6 and Il-17 significantly decreased in the ankle joints of BEP-treated rats in comparison to control. This study demonstrates for the first time that the central effect of BEP cell implantation into the rat hypothalamus reduces the inflammatory process development in the ankle joint and may ameliorate course of rheumatoid arthritis.
atRA restores the stability and functionality of nTregs in the inflammatory milieu (96.17)
Authors: Zheng Song Guo , Kong Ling , Zhou Xiaohui , Wang Julie , Shi Wei , Brand David , Fan Huimin , Liu Zhongmin , Zou Hejian
Published: January 2023

Natural Tregs play an important role in the maintenance of immune tolerance. Recent studies revealed that nTregs are plastic and unstable when stimulated with IL-6. They can be converted into either Th1, or Th17 or Th2 cells and lose phenotypic and functional characteristics in inflammatory milieu. all-trans Retinoic Acid (atRA) not only promotes Foxp3+ cell development, but restrains Th17 cell differentiation induced by IL-6+TGF-β. We here report that addition of atRA blocked Th17 conversion from nTregs. atRA-nTregs expressed lower levels of CD126 (IL-6Rα chain) and phosphorylated STAT3 compared to DMSO-nTregs when stimulated with IL-6. atRA- but not DMSO-nTregs maintained the suppressive activity in the presence of IL-6. Adoptive transfer of atRA- but not DMSO-pretreated nTregs to DBA/1 mice at day 28 after immunization with CII/CFA significantly reduced the severity of CIA. Using CFSE-labeled donor cells, we were able to observe that majority of nTreg cells lost Foxp3 expression, converted to IL-17-prodcuing cells, and >20% converted into Th2 cells in draining lymph nodes at one week after adoptive transfer to the established CIA. Conversely, atRA-nTregs maintained Foxp3 expression and did not convert to Th1, Th2 and Th17 cells in CIA. atRA alters Treg plasticity and restores the functionality of nTregs in the inflammatory milieu. This study may provide a novel therapeutic approach of nTregs for the treatment of Rheumatoid Arthritis and other autoimmune diseases.
CXCR7 antagonism prevents leukocyte trafficking into the central nervous system and treats ongoing disease during experimental autoimmune encephalomyelitis. (96.5)
Authors: Cruz-Orengo Lillian , Holman David , Wright Melissa , Dorsey Denise , Klein Robyn
Published: January 2023

Multiple Sclerosis (MS) is a neurological disorder characterized by the pathological trafficking of autoreactive leukocytes into the central nervous system (CNS). We previously showed that removal of CXCL12 from abluminal surfaces of the CNS microvasculature facilitates the entry of CXCR4-expressing leukocytes within MS tissues and in mice with experimental autoimmune encephalomyelitis (EAE), a model for MS. Recently, the orphan receptor CXCR7 was determined to bind CXCL12/11. Evidence suggests that CXCR7 mainly regulates CXCR4 signaling by sequestering CXCL12 from extracellular milieu. Using MS and EAE specimens, we tested the hypothesis that inhibition of CXCR7 would prevent leukocyte migration into the CNS and induction of autoimmunity. CXCR7 is expressed by both human and murine CNS endothelium and CXCR7 antagonism completely blocks CXCL12 internalization in brain microvascular cells in vitro. In vivo antagonism of CXCR7 led to dose-dependent inhibition of EAE and ameliorated clinical symptoms in mice with ongoing disease. Immunohistochemical analysis revealed increased leukocytes within meningeal vessels, decreased perivascular infiltrates and parenchymal entry of leukocytes compared with controls. Abluminal CXCL12 expression persisted only in mice treated with high doses of CXCR7 antagonist. The benefits of persistent abluminal CXCL12 location during EAE suggest that pharmacologic targeting of CXCR7 may have therapeutic utility for the treatment of MS.
Helminths abrogate antigen-specific gut inflammation in a novel murine model of non-IgE mediated food hypersensitivity (97.13)
Authors: Leung John , Hang Long , Blum Arthur , Setiawan Tommy , Stoyanoff Karynn , Weinstock Joel
Published: January 2023

Non-IgE mediated (NIE) food hypersensitivities (FH) affect 2 million Americans. Helminths protect from IgE-mediated FH. Their effect on NIEFH has not been studied. We developed a murine model of NIEFH featuring severe antigen-specific enterocolitis with Th1/Th17 polarization. We used this unique model to test if helminths protect against NIEFH. METHODS: Rag1-/- mice, IL10-/- T cells and ovalbumin (OVA)-specific, TCR transgenic OT2 T cells were used to develop the NIEFH model. 6-wk-old Rag mice were reconstituted with IL10-/- and OT2 T cells. To determine if helminths protect, mice were infected with H. polygyrus before feeding low dose NSAID for 2 wks followed by OVA for 1 wk. RESULTS: On a 4-pt scale, histology score of duodenum, ileum and colon were 0.15±0.3, 0.85±0.8 & 1.9±1.1 in helminth-infected mice (n=23) versus 3.8±0.5, 2.9±1.2 & 3.0±0.8 in non-infected group (n=10) [p<0.02]. Laminar propria mononuclear cells (LPMC) from helminth-infected mice produced more IL10 in vitro than those from non-infected mice (1.4 vs 0.7 ng/ml, p<0.01). IL10 production was enhanced by OVA stimulation, but not by anti-CD3/28. LPMC from infected mice displayed less IL17 and IFNg when stimulated by anti-CD3/28 in vitro (p<0.01). CONCLUSION: Our study provides proof of concept that helminths protect against antigen-specific NIEFH. Abrogation of gut inflammation associates with suppressed IL17 & IFNg, and elevated IL10 production probably from non-T cell source. NIH, Broad Foundation
Hilar node CD5+ B cells from tolerant mice inhibit allergic airway disease in sensitized recipients (97.14)
Authors: Schramm Craig , McNamara Jeff , Singh Anurag , Secor Jr Eric , Guernsey Linda , Thrall Roger
Published: January 2023

Ovalbumin (OVA)-sensitized mice develop allergic airway disease (AAD) with 7-10 days of OVA aerosol exposure but then have resolution of disease and development of local inhalational tolerance (LIT) after 6 weeks of continued aerosols. We have shown that B cells isolated from hilar lymph nodes (HLN) of mice at LIT and adoptively transferred to OVA-sensitized mice inhibit AAD in the recipient animals due to induction of CD4+Foxp3+ regulatory T cells (J Immunol 2008; 180:7318-26). The present study continued to address the phenotype of these suppressive B cells. CD19+ cells were isolated from LIT HLNs and were separated into CD5+ and CD5- populations by flow cytometry. Cells were adoptively transferred to OVA-sensitized C57BL/6 mice (6-8 in each group), which were then exposed to 1% OVA aerosols for 7 days. Mice receiving LIT HLN CD5+ B cells had significantly less airway eosinophilia (61 ± 2%; 17 ± 3 x 105 cells) than mice receiving saline (74 ± 2%; 31 ± 4 x 105 cells) or LIT HLN CD5- B cells (76 ± 3%; 30 ± 4 x 105 cells; p < 0.05 for each by ANOVA). The number of airway CD4+Foxp3+ T cells was increased in animals receiving HLN CD5+ B cells (7.0 ± 0.7 x 104 cells) compared to those receiving saline (4.9 ± 0.7 x 104 cells) or HLN CD5- B cells (4.7 ± 0.7 x104 cells; p < 0.05). Thus, regional CD5+ B cells in HLNs of LIT mice increased CD4+Foxp3+ expression and inhibited AAD in adoptively transferred animals.
Opioid receptors are targets for alleviation of allergen-induced diarrhea and modification of immune parameters in mice (97.3)
Authors: Duncker Swantje , Philippe David , Mercenier Annick , Nutten Sophie
Published: January 2023

So far, allergen avoidance is the most efficient treatment for individuals suffering from food-allergies. Since this almost always results in impairment of quality of life, new therapeutic concepts are needed. As opioid receptor (OR) expression has been demonstrated on various immune cells and as they have been shown to influence intestinal inflammation and motility, we were interested to study if OR are also involved in allergen-induced diarrhea. Ovalbumin-sensitized (OVA) Balb/c-mice were orally challenged with OVA, resulting in transient diarrhea and changes of plasma total IgE, OVA-specific IgE and mouse-mast-cell-protease-1(MMCP-1). Animals were pre-treated with kappa- or mu-OR-agonists (U50’488, DAMGO) and/or OR-antagonist and results were compared to controls. U50’488 and DAMGO significantly delayed the onset and decreased the severity of OVA-induced diarrhea. Animals receiving U50’488 showed lower total IgE and MMCP-1 concentration compared to controls, whereas DAMGO only showed a minimal influence on MMCP-1 release. The positive effect on diarrhea severity was partially reversed by pre-treatment with OR-antagonists. To our knowledge this is the first time that ORs are implicated as targets in modulation of food allergy. The changes in immune parameters with one of the OR-agonist suggest mechanisms extending beyond local intestinal motility and secretory regulation. Targeting of kappa- and/or mu-OR might prove valuable in alleviation of food allergies in the future.
Ischemia-reperfusion induces interferon-regulatory-factor-4 in renal dendritic cells which suppresses postischemic inflammation and prevents acute renal failure. (98.1)
Authors: Lech Maciej , Lassen Saraswati , Römmele Christoph , Mittruecker Hans-Willi , Mak Tak , Anders Hans-Joachim
Published: January 2023

Acute renal failure is still a serious medical problem that causes substantial morbidity and mortality. Ischemia-reperfusion (IR) activates TLRs causing inflammation and kidney injury. We hypothesized that certain factors suppress dendritic cell activation and thereby limit sterile renal inflammation, e.g. interferon-regulatory-factor (IRF)-4. Bi- or unilateral renal clamping was carried out for C57BL/6 (WT) and IRF4-knockout (IRF4-/-) mice. Known functions of the transcription factor IRF4 are the maturation of B cells, T cells and macrophages. Oxidative stress induced IRF4 expression in myeloid cells in-vitro as well as in the postischemic kidney. Lack of IRF4 aggravated acute renal failure after renal artery clamping together with increased intrarenal expression of TNF-α, IL-6, CXCL2, and CCL2 as well as excessive tubular necrosis and neutrophil influx. Consistent with these results we observed higher creatinine and urea levels in the serum of IRF4-/-mice after IR. This effect almost entirely depended on the role of IRF4 to suppress TNF-α release by intrarenal dendritic cells. Both clodronate depletion of these cells and TNF-α blockade with etanercept entirely abrogated the aggravation of cytokine expression and acute renal failure in Irf4-deficient mice. Thus, loss-of-function mutations in the IRF4 gene predispose to IR injury because the postischemic induction of IRF4 in resident dendritic cells suppresses them to secrete TNF-α and thereby limits immunopathology.
Docosahexaenoic acid inhibits cytokine production in dendritic cells and modulates T cell differentiation (98.6)
Authors: Kong Weimin , Yen Jui-Hung , Adhikary Sabina , Toscano Miguel , Ganea Doina
Published: January 2023

Protective anti-inflammatory effects of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were reported in clinical studies and animal models of colitis, sepsis, and stroke. In the present study we investigated the effects of DHA on the function of bone marrow-derived dendritic cells (DC) and CD4+ T cells. Our studies show that pretreatment with DHA prevents LPS-induced DC maturation, maintaining an immature phenotype characterized by low expression of MHCII and costimulatory molecules, and high levels of endocytosis. Exposure to DHA inhibits the production of proinflammatory cytokines and chemokines such as IL-6, TNFα, CCL-4, and IL-12p70. In addition, we report here for the first time that DHA has a similar inhibitory effect on IL-23 and IL-27, two other members of the IL-12 family. DHA exerted a similar inhibitory effect on IL-12 and IL-23 expression in vivo in LPS-inoculated mice maintained on a DHA-enriched diet. We also investigated the effects of DHA on T cells and found that DC treated with DHA (DC-DHA) have poor stimulatory capacity for T cells. T cells activated by DC-DHA also showed low levels expression of IFNα and IL-17, and high levels of Foxp3. Based on the inhibition of proinflammatory cytokine release and reduction in T cell stimulatory capacity, we conclude that DC are important targets for the anti-inflammatory activity of n-3 fatty acids.
The rheumatoid arthritis shared epitope is a potent helper T cell-polarizing ligand (98.8)
Authors: Almeida Denise , Ling Song , Pi Xujun , Hartmann-Scruggs Anne , Pumpens Paul , Holoshitz Joseph
Published: January 2023

Recent evidence strongly implicates IL17-producing helper T (Th17) cells in the pathogenesis of rheumatoid arthritis (RA) and other autoimmune conditions. Susceptibility to RA and disease severity have long been shown to associate with a HLA-DRB1-encoded 5 amino acid sequence motif, called the shared epitope (SE), however, the mechanistic basis of SE-RA association remains unknown. We have previously sown that the SE acts as an allele-specific ligand that triggers innate immune signaling in other cells. In order to gain better insights into the impact of the SE on immune regulation, we have studied its effect on helper T cell polarization in vitro. Here we show that when added to the CD8+ subset of dendritic cells (DCs), the SE ligand inhibits the enzymatic activity of indoleamine 2,3 dioxygenase (IDO), a key tolerogenic enzyme. When SE-activated DCs were co-cultured with naïve CD4+ T cells, they enhanced the differentiation to Th17 cells, and inhibited generation of Foxp3+ T regulatory cells (Tregs). The SE effect could be seen with peptidic SE ligands, but even more potently, when the SE was presented in its natural tri-dimensional conformation as part of HLA-DR tetrameric proteins. Our data demonstrate a previously unknown immune regulatory effect of the SE ligand that may provide important clues about its functional role in RA.
Transmembrane domain interaction regulates HLA-DO inhibition of HLA-DM (100.20)
Authors: Yoon Taejin , Macmillan Henriette , Roh Sujin , Mellins Elizabeth
Published: January 2023

Peptide-loading of MHC class II (e.g. HLA-DR) is regulated by the peptide exchange catalyst, HLA-DM (DM). HLA-DO (DO) inhibits DM, but the mechanism is not established. We found that a DMB point mutant with aberrant glycosylation at DMβ108 failed to co-precipitate with DR3 at pH 5, but was able to bind DO at this pH, and conversely, precipitated with DR3, but not with DO, at pH 7. Additional mutants implicated a unique region of DMβ in DO binding, distinct from but near the putative DR interface in human B cell line. However, FRET assay results with soluble recombinant proteins are inconsistent with these observations and argue that DO binds to a surface overlapping with the DR binding surface on DM. These contradictory results suggested that the transmembrane (TM) regions influence the interaction between DM and DO. We found that TM regions of DM and DO contain not only αβ dimerization motifs, but also intermolecular interaction motifs. A DM TM mutant with reduced DO interaction showed increased DM function in cells, suggesting that TM interaction between DM and DO regulates DO inhibition of DM. Taken together our results suggest DO inhibition is noncompetitive in cells and are consistent with previous evidence for a trimolecular complex (DR/DM/DO) in a B cell line.
Presentation of immunodominant epitopes from mixture of protein antigens is hierarchial (100.21)
Authors: Kim Ae , Hartman Isamu , Lanar David , Boronina Tatiana , Cole Robert , Sadegh-Nasseri Scheherazade
Published: January 2023

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. We have established a reductionist cell-free antigen-processing system for MHC class II that constitutes five purified components: HLA-DR1, HLA-DM, and three cathepsins. This system successfully identified the physiologically selected epitopes from two model antigens. When applied for de novo epitope identification to LSA-NRC of malaria, or HA1-H5N1 (Avian Flu), the system selected epitopes that were confirmed to be immunodominant by their capacity to activate CD4+ T cells in HLA-DR1 positive human volunteers or transgenic mice (Hartman/Kim, et al. Nat Med 16, pp1333-1340 (2010). To evaluate our system to a mixture of the above antigens, the system identified the epitopes from both antigens. We tested the immunogenicity of epitopes and the parent proteins in HLA-DR1 transgenic mice immunized with the mixture of both HA1 and LSA-NRC in a recall T cell proliferation assay. T cells responded only to the HA1 and its immunodominant epitope, while little, if any stimulation against LSA-NRC and its dominant epitope were measured. These observations did not change even when the ratio of LSA-NRC:HA1was 2:1. These findings show a hierarchy for the selection of immunodominant epitopes of different proteins during antigen processing, which can be important in design of combination vaccines.
HLA class II (HLA-DQ) protein stability profile and disease association (100.23)
Authors: Miyadera Hiroko , Tokunaga Katsushi
Published: January 2023

HLA (Human Leukocyte Antigen) is the most important genetic loci in relation to disease predisposition with autoimmune, infectious, and numerous other disorders, as revealed by genome wide association studies. Although allele specific antigen presentation has been believed as the major functional determinant implicated in diseases, the highly promiscuous antigen binding to disease associated and non-associated alleles in many autoimmune disorders suggest the contributions of multiple other factors in HLA-predisposition to diseases. The precise mechanisms by which particular HLA alleles exert disease condition thus remain uncovered for most of these disorders. In this study we have established a cell-based, highly quantitative assay to measure the cell surface protein expression levels of HLA class II, that might reflect their protein stability levels. The comprehensive picture on the HLA-DQ protein stability profile will be presented and discussed with regard to the allele specific polymorphisms and disease associations, as well as the haplotype evolution of HLA-DQ loci.
N-glycosylation of MHCII alters presentation of commensal carbohydrate antigens (100.24)
Authors: Ryan Sean , Cobb Brian
Published: January 2023

In conditions of dysregulated inflammatory responses, such as inflammatory bowel disease, recent studies have suggested that oral treatment with the capsular polysaccharide A (PSA) from the intestinal commensal Bacteroides fragilis promotes regulatory CD4 T cells and reduces disease pathology, yet the endogenous mechanisms of PSA immune activation remain unclear. Unlike traditional T cell-independent polysaccharide antigens, we have characterized a group of T cell-dependent antigens (T-glycoantigen) including PSA that are processed and presented within the MHCII pathway for CD4 T cell recognition. However, unlike prototypical peptide antigens, we have found that T-glycoantigen presentation is sensitive to the N-glycosylation state of MHCII. Furthermore, we have defined a key glycosyltransferase in antigen presenting cells that is necessary for T-glycoantigen presentation, but is dispensable for peptide antigen. We have translated this discovery into a novel mouse model to define the mechanisms behind the potential therapeutic role of PSA-specific T cell responses in health and disease. Our results provide evidence for the role of B. fragilis colonization in immune homeostasis and its potential use as a “probiotic” therapy to modulate dysregulated immune systems in inflammatory diseases. (Supported by grants OD004225 and GM082916 to BA Cobb)
Antigen presentation in the CNS during EAE (100.26)
Authors: Sosa Rebecca , Murphey Cathi , Cardona Astrid , Forsthuber Thomas
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) shares many similarities with the acute inflammatory episodes of human Multiple Sclerosis (MS) both clinically and histopathogically and has therefore been instrumental in defining the sequence of events that occur in MS. It is well established that CD4+ T cells specific for myelin antigen (Ag) can induce EAE in susceptible animals. Dendritic cells (DCs) and microglia are both capable of priming CD4+ T cells against myelin Ag via presentation by MHC class II molecules present on their cell surface. However, it has remained unclear precisely how infiltration of inflammatory cells into the CNS during EAE relates to the kinetics of myelin antigen uptake and presentation by specific APCs. In this study we have investigated which APCs are loaded with myelin antigen in the naïve brain as well as in the immunogenic context of EAE. Our results reveal that a low percentage of microglia are already pre-loaded with myelin Ag even in naïve mice and are capable of presenting Ag immediately for T cell activation following immunization. Several days after immunization a large influx of myelin-loaded DCs appears, coinciding with the onset of clinical disease. This data suggests that low-level presentation of myelin Ag by CNS-resident microglia may play a role in the induction phase of EAE, although the mechanism by which these cells have acquired Ag remains unresolved.
Peptide-MHC stability regulates the efficiency and duration of cross-priming (100.4)
Authors: Watson Alan , Mylin Lawrence , Thompson Megan , Schell Todd
Published: January 2023

Mechanisms that regulate the recruitment and the accumulation of T cells can impact the magnitude of determinant-specific responses. In this study, we demonstrate that peptide-MHC stability is a regulator of CD8+ T cell cross-priming. We find that increasing the kinetic stability of pMHC for an immunorecessive determinant from SV40 large T antigen, designated site V, overcomes the immunorecessive nature of the determinant. Using an in vivo assay to measure the proportion of naïve T cells that are triggered by cross-priming, we found that enhanced immunogenicity was due to two independent mechanisms regulated by pMHC-stability. 1) Enhanced cross-priming resulted in more efficient recruitment of site V-specific T cells, and 2) extended duration of cross-priming was associated with greater accumulation of site V-specific T cells. Thus, our results suggest that pMHC-stability regulates the magnitude of the CD8+ T cell response by influencing the number of naïve T cells recruited and the extent of T cell expansion. These findings have implications for the design of multivalent CD8+ T cell vaccines targeting subdominant determinants.
Crystal structure of human CD4 complexed with MHC class II, peptide and T cell receptor (100.47)
Authors: Yin Yiyuan , Wang Xin , Mariuzza Roy
Published: January 2023

The recognition of agonist peptide and MHC class II by T cell receptor (TCR) triggers the activation and development of CD4+ T cells. CD4, as the co-receptor, plays an important role in T cell signaling. Wild-type CD4 contacts the non-polymorphic domains of MHC class II with remarkably low affinity. By using an affinity-matured CD4 molecule, we determined the X-ray crystal structure of the ternary complex between the four Ig-like domains of human CD4 (D1-D4) and a TCR bound to HLA-DR4 bearing an agonist peptide (pMHC). The structure precludes direct CD4-TCR contacts, but provides ample room for the placement of CD3 subunits. Importantly, the CD4-pMHC-TCR complex is incompatible with models for TCR triggering involving MHC or CD4 dimerization. On the other hand, it is compatible with a recent model for T cell activation involving TCR dimerization via the Cα domain. The structure also suggests that non-canonical TCR docking modes, such as those observed in certain autoimmune TCR-pMHC complexes, may affect the association with CD4 and thereby alter T cell triggering. Collectively, our results provide a picture of the CD4-pMHC-TCR signaling complex that will be useful for understanding the initiation events of T cell activation.
Cryptic tRNAi shapes the pMHC I repertoire (100.5)
Authors: Starck Shelley , Jiang Vivian , Pavon-Eternod Mariana , Prasad Sharanya , Pan Tao , Shastri Nilabh
Published: January 2023

MHC class I molecules present peptides on the cell surface for immune surveillance of viruses and cancer. Interestingly, the peptides are encoded not only in conventional AUG-initiated translational reading frames but also in non-AUG initiated cryptic reading frames. Whether the same or distinct translational machinery is used to produce cryptic peptides at non-AUG start codons, such as CUG, is not known. Here, we show that translational initiation of antigenic precursors at cryptic CUG codons is differentially regulated by ribosomal initiation complexes that contain novel initiator tRNAs. This tRNA is distinct from Met-initiator tRNA and enhances initiation at CUG start codons. Thus, a novel tRNA -based mechanism can supply peptides for presentation by MHC I.
Oxidative stress compromises endosomal antigen processing in aging APC (100.50)
Authors: Santambrogio Laura , Cannizzo E. Stefania , Clement Cristina
Published: January 2023

In aging, higher production and defective clearance of reactive oxygen species leads to the accumulation of oxidatively damaged biomolecules. Herein we demonstrate that splenic APC purified from old mice accumulate aggregates of oxidatively damaged proteins in endosomal compartments that are not found in young mice. MS/MS analysis determined that oxidized aggregates comprised both endogenous and exogenous proteins post-translationally modified by side chain carbonylation (mono and di-oxidation), nitrosylation and advanced glycation end products. Following in vivo immunization, aggregate accumulation interfered with efficient endosomal degradation and antigen processing due to interaction between the immunizing antigen and the misfolded carbonylated proteins. Treatment with anti-oxidants partially restored the adaptive immune response to the immunizing antigen. We propose that endosomal inefficiency, due to deposits of aggregated, misfolded and heavily oxidized proteins contributes to adaptive immunosenescence associated with aging, and that targeting drug, promoting the clearance of oxidized moieties, lead to partial reversion of this process.
IL-4- and IL-10-producing invariant NKT cells inhibit Th1 but not Th17 cells, while IFN-γ-producing invariant NKT cells minimally regulate Th1/Th17 response in experimental autoimmune encephalomyelitis (101.10)
Authors: Oh SaeJin , Chung Doo Hyun
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is regulated by Th1 and Th17 response. Invariant (i) NKT cells prevent EAE, depending on IL-4, IL-10, and IFN-γ. However, which cytokines produced by iNKT cells regulate Th1 and/or Th17 response in EAE remains unclear. To address this issue, B6 and Jα18-/- mice were immunized with MOG35-55 peptide. Jα18-/- mice showed higher clinical scores, IL-17 and IFN-γ transcript, and intracellular IL-17 and IFN-γ+CD4+ T cells in CNS than B6 mice, which was reduced by adoptive transfer of wild type or IFN-γ-deficient iNKT cells. Adoptive transfer of IL-4- or IL-10-deficient iNKT cells into Jα18-/- mice decreased IL-17 transcript, and intracellular IL-17+CD4+ T cells in CNS but not clinical scores, IFN-γ transcript, and intracellular IFN-γ+CD4+ T cells. Taken together, IL-4- and IL-10-producing iNKT cells inhibit Th1 but not Th17 response in CNS during EAE. Moreover, IFN-γ-producing iNKT cells minimally regulate Th1 and Th17 in EAE.
Loss of CIKS/Act1, the signaling adaptor for IL-17 family cytokines, significantly ameliorates development of fatal disease in the Fcgamma RIIB-deficient mouse model for lupus (101.18)
Authors: Pisitkun Prapaporn , Ha Hye-Lin , Claudio Estefania , Tivy Caitlyn , Wang Hongshan , Siebenlist Ulrich
Published: January 2023

CIKS (Act1 or TRAF3IP2) is an adaptor protein required for signaling by receptors of the IL-17 family of cytokines. CIKS has been shown to be vital for disease in autoimmune mouse models of CIA and EAE. IL-17A may promote spontaneous germinal centers formation in the lupus prone BXD2 mice. However, the significance of IL-17 cytokines in the clinical outcome of lupus disease has never been addressed. Here we show that Th17 cell numbers are increased in Fcgr2b-deficient mice and IL-17 cytokine signaling is critically involved in disease development in this lupus model. Loss of CIKS significantly reduced mortality rates in Fcgr2b-deficient mice. Unexpectedly, the reduced mortality was not correlated with lower levels of antibodies to ANA or anti-dsDNA. Nevertheless, loss of CIKS on an FcgR2b-deficient background did reduce the formation of spontaneous germinal centers, which correlated with a diminished rate of isotype switching and plasma cell differentiation; furthermore, the generation of T-cell effectors was slowed. Most importantly, loss of CIKS profoundly reduced severe glomerulonephritis and inflammatory cell infiltrations in kidneys, key events in the fatal outcome of the lupus-like disease in Fcgr2b-deficient mice. These novel findings suggest that IL-17 family cytokines signaling via CIKS play critical roles in the pathogenesis of lupus disease in Fcgr2b-deficient mice; these findings also position CIKS as a potential new target for therapeutic intervention in SLE.
Location of immunization and interferon-γ are central to induction of salivary gland dysfunction in Ro60 peptide immunized model of Sjögren's syndrome (101.25)
Authors: Yin Hongen , Vosters Jelle , Roescher Nienke , D’Souza Anil , Kurien Biji , Tak Paul , Chiorini John
Published: January 2023

Introduction: Anti-Ro antibodies can be found in the serum of the majority of patients with sjögren's syndrome (SS), which is a chronic autoimmune epithelitis. Immunization with a 60-kDa Ro peptide (Ro60) has been shown to induce SS in mice. The aim of this study was to investigate the mechanism associated with loss of gland activity and optimal conditions and factors involved in salivary gland (SG) dysfunction in this animal model. Methods: Ro60 peptide immunization (either convention or multiple antigenic peptide (MAP)-Ro60) was tested in two strains of mice with or without secondary adjuvant and in different injection sites. Each group of mice was tested for loss of SG function, SG lymphocytic infiltration, anti-Ro and anti-La antibody formation, and cytokine production in cultured cells or homogenized SG extracts. Results: Ro60 peptide immunization in the abdominal area of female Balb/c mice led to impaired SG function, which corresponded with increased Th1 cytokines (IFN-γ and IL-12). Changing the immunization conditions to MAP-Ro60 in the abdominal area also led to impaired SG function in SJL/J mice. As was seen in the Balb/c mice, increased IFN-γ corresponded with SG dysfunction. Conclusions: Effective induction of SG dysfunction after Ro60 peptide immunization depends on the site of injection. The mechanism of action of Ro60 peptide immunization appears to involve an increase in Th1 cytokines, resulting in the induction of SG dysfunction.
CD45ROhighCD57+CD8high T cells in primary biliary cirrhosis (101.4)
Authors: Tsuda Masanobu , Ambrosini Yoko , Moritoki Yuki , Yoshida Katsunori , Zhang Weici , Yang Guo-Xiang , Ando Yugo , Rong Guanghua , Nakatani Toshio , Bowlus Christopher , Ansari Aftab , Lian Zhe-Xiong , Gershwin M. Eric
Published: January 2023

Primary biliary cirrhosis (PBC) is a female predominant organ specific autoimmune disease with specific destruction of intrahepatic small biliary epithelial cells (BECs). Patients develop a multilineage response to the E2 component of pyruvate dehydrogenase (PDC-E2) involving autoantibody as well as autoreactive CD4 and CD8 T cell responses. Recent data from murine PBC models have suggested that liver-infiltrating CD8 T cells play a critical role in biliary destruction in PBC. Many studies suggest the augmented cytotoxicity of CD57+CD8 T lymphocytes. Therefore, we have studied the frequencies and phenotypic characterization of CD8highCD57+ T lymphocyte components in both patients with PBC and age matched controls. Our data indicate that CD45ROhighCD57+CD8high T lymphocytes from PBC patients, but not controls, have significant phenotypic alterations, including increased expression α4β7high and reduced expression of annexin V after TCR stimulation. Our data also indicate that patient CD45ROhighCD57+CD8high T cells express higher granzyme A, B, perforin, CCR5 and α4β7, reduced expression of CCR7 than other CD8high T cells. Furthermore, IFN-γ and IL-5 productions by CD57+CD8high T lymphocytes upon in vitro TCR stimulation are increased in PBC patients. In conclusion, our data demonstrate that CD45ROhighCD57+CD8high T cells are a subset of cytotoxic memory cells which play a critical role in the chronic and progressive destruction of BECs in PBC.
Functional expression of HLA-E by human oligodendrocytes (101.40)
Authors: Zaguia Fatma , Antel Jack , Saikali Philippe , Arbour Nathalie
Published: January 2023

Immune cells infiltrating the central nervous system (CNS) of Multiple Sclerosis (MS) patients contribute to the destruction of oligodendrocytes. We aim at characterizing mechanisms implicated in such CNS damage. HLA-E, a non-classical MHC class I molecule, is recognized by either the NKG2C or NKG2A receptors expressed on subsets of T cells and NK cells. While NKG2C is an activating receptor, NKG2A is described as an inhibitory receptor. Activated human immune effectors including NK and T cells can kill human oligodendrocytes in vitro, yet whether HLA-E could modulate such cytotoxicity is unresolved. We observed that human adult oligodendrocytes express basal HLA-E levels, which are upregulated upon pro-inflammatory cytokine treatments, mimicking the inflamed CNS observed in MS lesions. We performed in vitro functional assays using activated immune cells bearing either the NKG2A (NK cells) or NKG2C (CD4 T cells) receptor co-cultured with human oligodendrocytes. We observed that addition of blocking antibodies specific for NKG2A on NK cells increased the killing of oligodendrocytes compared to an isotype control. In contrast, we detected a decreased cytotoxicity against oligodendrocytes when we block the activating receptor NKG2C on a subset of CD56-positive CD4 T cells, which we previously identified as carrying cytotoxic properties. Our results support that human oligodendrocytes modulate via the expression of HLA-E, cytotoxic attacks mediated by several immune effectors.
Sequential effects of IL-21 and IL-17 to regulate spleen CXCR5+ follicular T helper cells in autoimmune BXD2 mice (101.44)
Authors: Ding Yanna , Hsu Hui-Chen , Wu Qi , Yang Pingar , Mountz John
Published: January 2023

CXCR5+ follicular T helper (Tfh) cells locate in the light zone (LZ) of germinal centers (GCs) to help differentiation of antibody producing B cells. Both IL-17 and IL-21 have been associated with the development of GCs but their effects on Tfh have not been clear. Autoimmune BXD2 mice developed spontaneous GCs and displayed increased number of Tfh in the spleen. In BXD2-Il21-/- or BXD2-Il17ra-/- mice, there were significantly reduced PNA+Fas+ GC B cells in the spleen and autoantibody titers in sera. This is not exclusively related to Tfh development since the percentage of CXCR5+ICOS+ Tfhs is 2-fold lower in BXD2-Il21-/- but is 1.5-fold higher in BXD2-Il17ra-/- mice compared to wild type. Confocal images, however, showed dramatically diminished CD4 T cells in GC LZ of BXD2-Il17ra-/- spleen. In vitro treatment of CD4 T cells with IL-21 increased the percent of CXCR5+ICOS+ Tfhs and the expression of IL17R on them. qRT-PCR confirmed that IL-21 treatment increased the expression of Cxcr5, Icos and Il21 on CD4 T cells. In contrast, IL-17 treatment increased Rgs16 but decreased Cxcr5 and diminished CXCL13-mediated chemotaxis on CD4 T cells. Our study suggests that IL-21 and IL-17 act sequentially on Tfhs in BXD2 mice. IL-21 first promotes differentiation of Tfhs and prepared these cells for IL17 stimulation by upregulating IL-17R. Subsequently, IL-17 is required to upregulated the expression of Rgs16 and stabilizes Tfh in GC LZ to promote spontaneous development of GCs.