bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation, distribution and γδ T cell-driven regulation of IL-22-producing T cells in tuberculosis (45.21)
Authors: Yao Shuyu , Huang Dan , Chen Crystal , Halliday Lisa , Zeng Gucheng , Wang Richard , Chen Zheng
Published: January 2023

Differentiation, distribution and immune regulation of human IL-22-producing T cells in infections remain unknown.We demonstrated that M. tuberculosis infection resulted in apparent increases in numbers of T cells capable of producing IL-22 de novo without in vitro Ag stimulation, and drove distribution of these cells more dramatically in lungs than in blood and lymphoid tissues. Consistently, IL-22-producing T cells were visualized in situ in lung tuberculosis (TB) granulomas by confocal microscopy and immunohistochemistry, indicating that mature IL-22-producing T cells were present in TB granuloma. Surprisingly, phosphoantigen HMBPP activation of Vγ2Vδ2 T cells down-regulated the capability of T cells to produce IL-22 de novo. Up-regulation of IFNγ-producing Vγ2Vδ2 T effector cells after HMBPP stimulation coincided with the down-regulated capacity of these T cells to produce IL-22 de novo. Importantly, anti-IFNγ neutralizing Ab treatment reversed the HMBPP-mediated down-regulation effect on IL-22-producing T cells, suggesting that Vγ2Vδ2 T-cell-driven IFNγ-networking function was the mechanism underlying the HMBPP-mediated down-regulation of the capability of T cells to produce IL-22. These novel findings raise the possibility to ultimately investigate function of IL-22 producing T cells and to target Vγ2Vδ2 T cells for balancing potentially-hyper-activating IL-22-producing T cells in severe TB.
Modulation of phosphatidylinositol 3-kinase (PI3K) as a tool to enhance anti-polysaccharide antibody response in the aged (45.23)
Authors: Fallah Mosoka , Chelvarajan R. , Bondada Subbarao
Published: January 2023

The elderly are at risk to increased infection with Streptococcus pneumoniae, gram positive bacteria. This has been shown to be due to a reduced production of pro-inflammatory cytokines (IL-6, IL-12, TNF-α) by splenic macrophages (SM), as well as a decrease in the B-cell class-switching to IgG3 and IgA, isotypes that are critical for the opsonization and clearance of the pathogen. Here we showed that PI3K expression and activity are increased in aged compared to young SM. Phosphorylation of AKT and glycogen synthase kinase-3 (GSK-3), downstream targets of PI3K, was also more in the aged than young SM and B-cells. The PI3K inhibitor, LY294002, enhanced TLR-2 and TLR- 4 dependent pro-inflammatory cytokine secretion by SM from the aged. Moreover, low concentrations of the PI3K inhibitor enhanced TLR-4 induced class-switching to IgG3 and IgA. Interestingly, inhibition of GSK-3 also enhanced TLR-4 induced IgG3 and IgA secretion as well as B-cell proliferation. Hence, a pharmacological modulation of the PI3K-AKT-GSK pathway could be a potential vaccine enhancement strategy against infection with S. pneumoniae in the elderly. (Supported in part by NIH Grants)
Stable expression of a foreign protein by a live rubella viral vector (45.25)
Authors: Berkower Ira , Virnik Konstantin , Ni Yisheng , Prutzman Kirk , Spadaccini Angelo
Published: January 2023

Live attenuated rubella virus has a number of desirable features for a live viral vector. It has a good safety profile, so it can immunize while growing exponentially in the host. It immunizes at a low dose, and one dose protects (against rubella) for life. It grows well in rhesus macaques, which allow a viral challenge. It has no DNA intermediate, cannot integrate, and usually does not persist in the host. A full length infectious cDNA clone of rubella was available for modification, but prior efforts to express foreign genes were frustrated by genetic instability and size limitations on the insert. A deletion of 507 bp in nonstructural gene P150 was permissive, so we asked whether this deletion could make room for insertion of zoanthus GFP (792 bp). By using the deletion/insertion strategy, zGFP was stably expressed for at least 12 passages. In the zGFP hybrid, rubella structural proteins were expressed at normal levels, while the vector grew to high titer (4 X 10^6/ml). Normal P150 function was detected by P150-GFP labeling of functional replication centers in living cells. Rubella may be useful for expressing vaccine antigens as large as most viral proteins, and it is stable enough for vaccine production followed by growth in vivo. This approach may extend the range and utility of rubella to include immunization against other viruses for which attenuation is not currently feasible.
SLAM activation induces ERK and CREB phosphorylation that leads to IFN-γ production during active tuberculosis (TB) (45.27)
Authors: Pasquinelli Virginia , Alvarez Ivana , Jurado Javier , Aspera Romina , Fernandez Do Porto Dario , Samten Buka , Barnes Peter , Garcia Veronica
Published: January 2023

IFN-γ is crucial for protection against M. tuberculosis (Mtb). Previously we reported that signaling through SLAM on Mtb-stimulated cells induced CREB activation increasing IFN-γ. Here, we deeply investigated the potential relationship between SLAM-CREB and IFN-γ. After Mtb-stimulation, most of the IFN-γ+ cells co-expressed SLAM and pCREB (48,68 ± 11,18), whereas a smaller percentage of T cells expressed one of the two molecules. These results demonstrate that the induction of IFN-γ mediated by SLAM in TB depends, at least in part, on CREB activation. It was reported that SLAM signaling would induce the activation of the protein kinase Erk. To analyze the signaling pathways induced by SLAM that contribute to CREB activation and IFN-γ secretion in TB, we studied Erk phosphorylation after SLAM activation. The addition of PD90059 (an Erk inhibitor) to Mtb stimulated cells inhibited the percentage of IFN-γ+pCREB+ T cells, indicating that Erk could be involved on CREB activation. Moreover, the addition of PD90059 to Mtb±α-SLAM stimulated cells significantly decreased IFN-γ production (% inhibition: 71,6 ± 7,4). Finally, α-SLAM induced Erk activation in Mtb stimulated cells. These results demonstrate that IFN-γ production induced by SLAM signaling on Mtb stimulated cells is, at least in part, mediated by Erk and CREB phosphorylation. These data contribute new information about the molecular basis operating during SLAM activation that leads to IFN-γ production in human TB.
Heat shock protein Gp96-Ig-peptide complexes based vaccine induce predominant immune responses at mucosal sites (46.13)
Authors: Strbo Natasa , Vaccari Monica , Pahwa Savita , Kolber Michael , Fisher Eva , Gonzalez Louis , Franchini Genoveffa , Podack Eckhard
Published: January 2023

We have developed a vaccine design that uses the unique ability of the endoplasmic reticulum (ER) chaperone, heat shock protein gp96, to bind antigenic peptides and deliver them to antigen-presenting cells. Secreted form of gp96, gp96-Ig, was generated by replacing the ER retention sequence KDEL with the IgG1-Fc domain. To generate a cellular SIV vaccine, HEK-293 cells were transfected with gp96-Ig and the cDNAs encoding the SIV antigens gag, env and retanef. Irradiated, transfected 293 cells that secrete 1, 5 or 50 micrograms gp96-Ig-SIV complexes in 24h, were injected intraperitoneally (IP) in Mamu-A*01+ Rhesus Macaques at 0, 4 and 25 weeks. To determine whether gp96-Ig vaccination induces mucosal immunity, we used the 3T3-OVA-gp96-Ig/OT-1 model. Our data indicate that the best route of vaccination for induction of mucosal CD8-CTL immunity is the IP route. Gp96-Ig immunization increases frequency of CD11chighMHC classIIhighCD103+ cells in peritoneal cavity and efficiently induces CCR9 on responding T cells. In nonhuman primate model, the frequency of SIV-specific cells in the rectal mucosa reached 30-50% after the third immunization. We conclude that gp96-Ig vaccination startegy induces antigen-specific effector memory CD8T cells migrating to the intestinal mucosa and thus could be extremely useful for improving protection against a range of mucosal pathogens.
Effect of Bacillus-based direct-fed microbials on macrophage functions and Eimeria maxima infection in broiler chickens (46.18)
Authors: Lillehoj H. , Lee Kyungwoo , Guangxing Li , Lee Sung Hyen , Jang Seung Ik , Babu Uma , Lillehoj Erik , Siragusa Gregory
Published: January 2023

The present experiment was conducted to study the effect of dietary Bacillus-based direct-fed microbials (DFMs) on macrophage functions and resistance against experimental coccidiosis in broiler chickens. Birds were fed diets containing one of eight Bacillus subtilis strains (designated Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278) or one multiple-strain DFM product (AVICORR™) for 21 days, and then the chickens were uninfected or orally infected with 5000 Eimeria maxima (EM) oocysts. At day 21 (before EM challenge), strong phagocytosis ability using BSA-coated fluorescent latex beads and GFP-labeled salmonella was seen in animal given Bs18, 15AP4, Bs27, or Bs278 compared with the no DFM control group. At day 27 (6 days post infection), EM-induced reduction of body weight gain was significantly decreased by adding 15AP4 or Bs27 into broiler diets compared with no DFM/EM inoculated control. Serum Nitric oxide levels were increased in infected animals fed with Bs27 compared with the no DFM/EM inoculated control. The levels of Eimeria-specific antibody were increased in infected chickens fed with LSSAO1 compared with the no DFM/EM inoculated control. Finally, all experimental diets increased concanavalin A-induced splenocyte mitogenesis in infected broilers compared with the no DFM/EM inoculated control. In summary, dietary DFMs reduced the clinical signs of experimental avian coccidiosis and increased various parameters of immunity in broiler chickens.
Enterotoxigenic E. coli CFA/I Fimbriae Abate Collagen-Induced Arthritis (CIA) via Regulatory CD39+ T Cells and IL-35 (47.1)
Authors: Kochetkova Irina , Thornburg Theresa , Callis Gayle , Crist Kathryn , Pascual David
Published: January 2023

Colonization factor antigen I (CFA/I) expressed by Salmonella vaccine vectors can prevent experimental autoimmune encephalomyelitis and CIA attributed to suppression of Th1 and Th17 cells by regulatory and immune deviation mechanisms. We hypothesize that soluble CFA/I fimbriae may confer similar protection against CIA as Salmonella-CFA/I. Single dose, mucosal CFA/I application 14 days post-CIA induction abated clinical development of arthritis similar to Salmonella-CFA/I. IFN-γ, IL-6, and IL-17 responses by CD4+ T cells were suppressed in protected mice via the induction of IL-10 and regulatory FoxP3+ CD4+ T cells. Such regulatory T cells were both CD25- and CD25+, each conferring protection upon adoptive transfer to CIA mice. Further evaluation revealed the protective CD25- CD4+ T cells also expressed CD39, ectonucleoside triphosphate diphosphohydrolase, an enzyme responsible for extracellular ATP hyrolysis. Flow cytometry analysis revealed that CFA/I fimbriae nearly doubled the CD39+ CD4+ T cell subset, ~50% of which were CD25-negative. Moreover, mucosal CFA/I delivery enhanced by 2-fold IL-35+ FoxP3+ CD4+ regulatory T cells. CD39+ CD4+ T cells accounted for most (~70%) of TGF-β production by CD4+ T cells. Upon restimulation, these CFA/I -induced CD4+ T cells did not produce IFN-γ or IL-17, but IL-10 levels were enhanced. In conclusion, CFA/I fimbriae alone can augment regulatory T cells for protection against autoimmune diseases. Work supported by P01 AT-04986.
Th17/Treg immunoregulatory balance in human periapical lesions (47.5)
Authors: Rodrigues Denise , Marçal Juliana Romanelli , Samuel Renata , Fernandes Danielle , Araujo Marcelo , Napimoga Marcelo , Lima-Pereira Sanivia , Napimoga Juliana , Alves Polyanna , Mattar Rinaldo , Rodrigues Virmondes
Published: January 2023

Cysts and granulomas are chronic periapical lesions mediated by a set of inflammatory mediators that develop in order to contain a periapical infection. This study analyzed the inflammatory infiltrate, mast cells and in situ expression of cytokines, chemokines and FoxP3 in human periapical granulomas and cysts compared to controls. Fifty-five lesions (25 cysts, 25 granulomas and 5 controls) were analyzed. The type of inflammatory infiltrate was evaluated by HE staining and the presence of mast cells was analyzed by toluidine blue staining. Indirect immunohistochemistry was used to evaluate the expression of cytokines, chemokines and FoxP3. The inflammatory infiltrate mainly consisted of mononuclear cells. In cysts, mononuclear infiltrates were significantly more frequent than mixed (polymorphonuclear/mononuclear) infiltrates (p=0.04). Mixed inflammatory infiltrates were significantly more frequent in patients presenting fistulae (p=0.0001). The number of mast cells was significantly higher in granulomas than in cystic lesions (p=0.02). A significant difference in the expression of IL-17 (p=0.001) and TGF-β (p=0.003) was observed between cysts and granulomas and the control group. Significantly higher IL-17 levels were also observed in cases of patients with fistulae (p=0.03). Mast cells, TGF-β and IL-17 probably participate in the modulation of the inflammatory process in established periapical lesions, especially in granulomas.
Biodegradable dissolving microneedle arrays effectively deliver antigens and adjuvants to skin DCs for the induction of antigen specific immune responses. (48.12)
Authors: Erdos Geza , Donahue Cara , Williams Mellissa , Ozdoganlar Burak , Falo Louis
Published: January 2023

The skin contains a high density of antigen presenting cells making it an attractive target for vaccine delivery. However, the remarkable barrier function of the skin continues to present a formidable obstacle to topical vaccine delivery. To overcome this barrier we designed carboxy-methyl cellulose microneedle arrays (MNAs) to topically deliver antigens and adjuvants into the skin. MNAs were designed and fabricated to enable advantages in penetration, delivery, and diversity of biocompatibility over existing skin delivery technologies. Initially we fabricated MNAs with integrated tracer antigens. Following MNA immunization labeled antigens were identified in dendritic cells in human and murine skin, and in the draining lymph nodes of immunized animals. To evaluate immunogenicity, we incorporated the model antigen OVA in combination with various adjuvants into MNAs. MNA-mediated delivery of low dose antigen/adjuvant induced potent and durable CTL responses compared to those generated by traditional intradermal immunization. Further, MNA immunization utilizing B16 tumor cell lysates as a source of antigen induced B16 specific DTH reactions and inhibited tumor growth in a B16 tumor model. Taken together, these results demonstrate the feasibility of the use of CMC microneedle arrays for cutaneous immunization and suggest potentially broad applicability for the co-delivery of structurally diverse antigens and adjuvants.
Human decidual tissue contains disarmed CD8+ Effector-Memory T cells (48.2)
Authors: Tilburgs Tamara , Schonkeren Dorrith , Eikmans Michael , Strominger Jack , Roelen Dave , Scherjon Sicco , Claas Frans
Published: January 2023

During pregnancy maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Recently, CD4+CD25bright regulatory T cells have shown to be concentrated in decidual tissue where they are able to suppress fetus-specific immune responses. Decidual CD8+ T cells form the largest fraction of T cells at the fetal-maternal interface but limited data is present on the characteristics of these cells. In this study we examined decidual and peripheral CD8+ T cells for CD45RA, CCR7, CD28 and CD27 expression using nine-colour flowcytometry. Our data demonstrate that decidual CD8+ T cells mainly consist of differentiated CD45RA-CCR7- effector-memory (EM) cells while unprimed CD45RA+CCR7+ naïve cells are absent. Unlike peripheral blood EM T cells, the decidual EM T cells do not express perforin and have a reduced expression of granzyme B, which was confirmed by immunohistochemistry of decidual tissue sections. Interestingly, quantitative PCR shows an increased perforin and granzyme B mRNA content in decidual EM T cells in comparison to peripheral blood. The presence of high levels of perforin and granzyme B mRNA in decidual EM T cells suggests that decidual CD8+ T cells pursue alternative means of EM cell differentiation that may include a blockade of perforin and granzyme B mRNA translation into functional proteins. Regulation of decidual CD8+ T cell differentiation may play a crucial role in maternal immune tolerance to the fetus.
Both systemic and mucosal LCMV immunization generate robust viral specific IgG in mucosal secretions, but elicit poor LCMV-specific IgA (48.3)
Authors: Zeng Junwei , Rajini Bheemreddy , Onami Thandi
Published: January 2023

Immunoglobulins in secretions play a critical role in protection at mucosal surfaces. We examined the generation of viral specific IgG and IgA in plasma and mucosal secretions of mice following systemic or mucosal immunization with LCMV, a widely used experimental model of viral infection. While there are early differences in humoral responses depending on the route of immunization, we show that both routes generate comparably robust viral specific IgG in plasma, vaginal, lung, and nasal secretions of immune mice. In contrast, LCMV elicited poor viral specific IgA responses. Mice that were infected i.n. showed elevated viral specific IgA in nasal and lung washes compared to i.p. infected mice, however, LCMV-specific IgG overwhelmingly contributed to the humoral response in all mucosal secretions examined. Thus similar to HIV-1, and several other mucosally encountered microbial infections, these data suggest that LCMV infection fails to induce vigorous viral specific IgA responses.
Long term overexpression of IL-10 increases recruitment of fibrocyte into the lung and induces fibrosis (48.5)
Authors: Sun Lei , Louie Marisa , Vannella Kevin , Moore Bethany , Shanley Thomas
Published: January 2023

Pulmonary fibrosis is characterized by the progressive deposition of extracellular matrix components such as collagen and fibronectin by fibroblasts and myofibroblasts. Three potential sources of fibroblasts and myofibroblasts include: expansion of resident lung fibroblasts, epithelial-mesenchymal transition, or recruitment and differentiation of circulating mesenchymal precursors (fibrocytes). IL-10 is a well-known immunosuppressive cytokine and in some studies has an inhibitory effect on fibrosis, however, this is less well studied. In our study, long-term overexpression of IL-10 (IL-10 OE) induced fibrosis. The total cell number from bronchoalveolar lavage (BAL) increased ten-fold in the IL-10 OE mice, with significant infiltration of T, B lymphocytes and collagen-producing cells. Fibrocytes from collagenase digested lung were identified by flow cytometry with dual staining of CD45 and collagen 1. IL-10 OE increased whole lung fibrocyte numbers by two-fold. Q-PCR analysis on an array of chemokine/chemokine receptor genes showed that only CCL2 and CCL2 receptor (CCR2) were highly upregulated in IL-10 OE mice, suggesting that IL-10 induced fibrocyte recruitment is CCL2/CCR2 specific. We also examined M2 macrophage activation and found significant numbers of M2 macrophages in both BAL and whole lung tissue from the IL-10 OE mice. Taken together, long term overexpression of IL-10 may induce fibrosis, in part, by fibrocyte recruitment and M2 macrophage activation.
Local autoantibodies in Chronic Rhinosinusitis (CRS) with Nasal Polyps. (48.8)
Authors: Tan Bruce , Li Quan , Suh Lydia , Kato Atsushi , Conley David , Chandra Rakesh , Peters Anju , Norton James , Harris Kathleen , Grammer Lelie , Kern Robert , Mohan Chandra , Schleimer Robert
Published: January 2023

Abstract Introduction: Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory condition of the nasal passage and paranasal sinuses. It is characterized by a Th2 biased inflammatory infiltrate not currently attributed to autoimmunity. Recent studies from our laboratory demonstrate elevated levels of BAFF, B-lymphocytes, and IgA. The role of these B-cells and the specificities of the immunoglobulins produced are still unknown. Methods: Protein extracts from nasal polyps of 11 patients were compared to nasal tissue obtained from 10 normal controls using autoantibody microarrays. A positive microarray result for anti-double stranded DNA (anti-dsDNA) was confirmed using 15 nasal polyp samples and 10 normal control samples on commercially available ELISAs for Total IgG, and anti-dsDNA IgG. Results: Total IgG levels were higher in nasal polyps relative to normal controls (35μg/mg Total Protein versus 17μg/mg Total Protein, p=0.02). Additionally, Anti-dsDNA IgG levels were higher in nasal polyps relative to normal controls on both the antibody microarray (188nfi/mg Total Protein versus 61nfi/mg Total Protein p<0.01) and ELISA (371U/mg Total Protein versus 66U/mg Total Protein p=0.03). Conclusions: Anti-dsDNA is present at higher levels in nasal polyp tissue than normal controls- suggesting that a local autoimmune reaction is present in nasal polyp tissue. Further studies are needed to test the role of this autoimmune response in the pathogenesis of CRS.
CCR4 improves phenotypic identification of T-regulatory cells; Validation and implementation of clinical test (49.26)
Authors: Gorski Kevin , Sitko John , Thomas John , Gavin Marc , Cottrell Susan , Cox Chris , Vincent Mike , Ferbas John
Published: January 2023

T-regulatory cells (Treg) are critically responsible for controlling unwanted immunological activity. Therefore, potential perturbation of Tregs can be important to consider when studying therapeutic intervention of inflammatory disorders with novel agents. Moreover, reliable assays are needed to allow detection of changes resulting from treatment. In the clinical setting intracellular staining for antigens such as Foxp3 can be unwieldy. Therefore, we chose to identify Treg using cell surface markers. Recent reports indicate that using a CD25hi and CD127dim phenotype on CD4+ T-cells allows identification of an enriched fraction of Treg using surface staining. On average, however, only 85 % of these cells are reported to be Foxp3+ Treg. We evaluated use of CCR4 together with CD25 and CD127 to refine detection of Treg. Foxp3 co-staining was performed as well to characterize the proportion of bonafide Treg. More than 90 % of cells in the CD25hi, CD127dim, CCR4+ CD4+ T-cell gate were found to co-express Foxp3. We then validated a 5-color method incorporating CCR4 as a marker for T-reg enumeration. 26 healthy volunteers and 13 asthmatic subjects were evaluated. Pre-analytical variables and assay performance characteristics will be discussed. We conclude that using CCR4 imparts additional specificity for phenotypic identification of Treg. Implementation is planned for clinical studies where therapeutic intervention has the potential to impact Treg.
Role of Smad and non-Smad signals in the development of Th17 and regulatory T cells (49.3)
Authors: Zheng Song Guo , Lu Ling , Wang Julie , Zhang Feng , Chai Yang , Brand David , Wang Xuehao , Horwitz David , Shi Wei
Published: January 2023

While TGF-β is essential for the development of peripherally induced Foxp3+ regulatory T (iTreg) and Th17 cells, the intracellular signaling mechanism by which TGF-β regulates development of both cell subsets is less well understood. Using Smad2 lymphocyte conditional knock out and Smad3 knock out mice, we report that neither Smad2 nor Smad3 deficiency abrogates TGF-β-dependent iTreg induction by a deacetylase inhibitor Trichostatin A (TsA) in vivo although loss of Smad2 or Smad3 partially reduces iTreg induction in vitro. Similarly, Smad2 and Smad3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis (EAE). In addition, ERK and/or JNK pathways were shown to be involved in regulating iTregs, while the p38 pathway predominately modulates Th17 and EAE induction. Therefore, selective targeting of these intracellular TGF-β signaling pathways during iTreg and Th17 cell development may lead to the development of therapies in treating autoimmune and other chronic inflammatory diseases.
Hepatic stellate cells regulate T cell responses in cooperation with Kupffer cells. (49.9)
Authors: Ichikawa Shintaro , Mucida Daniel , Kronenberg Mitchell , Cheroutre Hilde
Published: January 2023

Induced regulatory T cells (iTreg) are important regulators of mucosal effecter T cell responses. TGF-β is essential for iTreg differentiation, however, in the presence of interleukin-6, it promotes pro-inflammatory T helper 17 (Th17) cells. We have shown that vitamin A metabolite; retinoic acid (RA), acts as a key controller of TGF-β-dependent immune responses, inhibiting Th17 differentiation and promoting Foxp3+ Treg induction. Hepatic stellate cells (HSC) are liver resident cells that store the majority of vitamin A and RA in our body. Because the liver is a tolerogenic organ, in spite of its direct contact with intestinal antigens, we explored if HSCs are involved in the tolerogenic nature of the liver. Although HSC can prime naïve T cells we found they were less effective than the neighboring Kupffer cells (KC), which are major antigen presenting cells in the liver. Because KCs reside in the proximity of the HSCs within the liver sinusoide, we rendered it possible that HSCs could regulate the differentiation of KC-primed T cells. We show here that HSCs indeed promote Foxp3 induction by KC-primed OT-II T cells and interestingly, they inhibited Th17 differentiation, similar to the RA-mediated reciprocal regulation of TGF-β-dependent T cell polarization. These data suggest that liver resident KCs and HSCs synergize to cooperatively regulate liver T cell responses.
Reorganizing the signaling molecules in CD4+ T cells by TCR-MHC class II interactions in the thymic medulla prevents T cell hyperactivation (50.1)
Authors: Stephen Tom , Wilson Bridget , Laufer Terri
Published: January 2023

Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-dependent activation of single-positive thymocytes is attenuated in response to the self peptide-MHC molecules that will be encountered in the periphery. Mature “tuned” thymocytes lose responsiveness to low affinity ligands. We previously reported that tuning of CD4 T cells requires interactions with MHC II expressing thymic medullary epithelium and thymic DCs. Tuning is mediated by MHC II interaction with the TCR but not with the CD4-coreceptor, even though mature CD4 T cells depend on CD4 co-receptor associated Lck for TCR signaling. The increased threshold for TCR stimulation in tuned CD4SP thymocytes is associated with increased expression of the SHP-1 phosphatase and inhibition of ERK activation by weak ligands. Immature CD4 T cells contain a “free pool” of Lck that is not co-receptor associated. This pool of Lck localizes with TCR and may lower the activation threshold for weak ligands. Tuning limits this TCR associated pool of Lck. Thus, TCR signaling in mature T cells depends on CD4 associated Lck for activation. Taken together, our results suggest that reorganization of the TCR signalasome during thymic medullary residency defines the activation threshold of CD4 T cells and prevents hyperactivity to weak self antigens.
TcR-stimulation upregulates MS4a4B expression through induction of AP-1 transcription factor during T cell activation (50.14)
Authors: Yan Yaping , Zhang Guang-Xian , Rostami Abdolmohamad , Xu Hui
Published: January 2023

MS4a4B is a novel member of the MS4A family expressed in T-cells. We have shown previously that expression of MS4a4B in T cells is upregulated upon T cell activation. However, the mechanism that regulates MS4a4B expression is unknown. In this study, we explored the mechanism underlying TcR-stimulation-induced expression of MS4a4B by promoter analysis. We cloned 2495 bp of 5’-flanking region upstream of MS4a4B start coden and inserted the DNA fragment into pGL4.20 reporter plasmid. To analyze promoter activity, we transiently transfected EL4 thymoma cells and the T32 cell line with reporter plasmids. Expression of reporter gene was determined by dual-luciferase assay. Potential activator- and repressor-binding sites were analyzed by serial length of 5’-deletion. We have identified at least two potential activator binding regions and two repressor binding regions. The activator binding sites have been localized to two fragments, which are a 442-base pair region (region A) positioned from -1176 to -735, and a 119-base pair region (region B) positioned -188 to -70 respectively. MatInspector analysis showed that region A contains consensus binding motif of Jun/Fos (AP-1) family of transcription factors. EMSA showed that nuclear proteins extracted from CD3/CD28-activated T cells strongly bind to the AP-1 binding element, suggesting that TcR-stimulation may enhance MS4a4B expression through transactivation of AP-1 in T cells.
A critical role of GRAIL in T cell activation and tolerance (50.18)
Authors: Nurieva Roza , Zheng Shuling , Jin Wei , Sun Shao-Cong , Lozano Guillermina , Dong Chen
Published: January 2023

T lymphocyte activation is tightly regulated to ensure effective elimination of invading pathogens as well as maintaining tolerance against self-tissues. Gene related to anergy in lymphocytes (GRAIL) is a type I transmembrane protein localized to endosomal compartment with homology to RING finger proteins whose expression was previously associated with induction of T cell tolerance in vitro. In order to understand the physiological function of GRAIL in immune regulation, we have generated and analyzed a Grail-deficient mouse model. We found that naïve T cells lacking GRAIL exhibited greatly enhanced proliferation and cytokine production after TcR activation and did not depend on CD28 and ICOS for their effector cytokine expression. We also determined that lack of GRAIL abrogated suppressive function of regulatory T cells. Both naïve and regulatory T cells from Grail-deficient mice were less efficient in down-regulation of their TcR/CD3 expression. In vivo, Grail-deficient mice were resistant to immune tolerance induction and when compared to the wild-type mice, exhibited greater susceptibility to autoimmune diseases. Our results thus indicate GRAIL as an essential regulator of T cell tolerance by regulating naïve T cell tolerance and regulatory T cell function.
Sustained A1 and Mcl-1 expression from costimulatory signals promotes T cell survival (50.20)
Authors: Song Jim
Published: January 2023

Costimulation is essential for mediating efficient T cell responses. In recent years, advances have been made in studies of costimulation as potential therapies in the treatment of diseases. However, the intracellular signals by which costimulation affects the function of T cells still needs to be elucidated. A1 and Mcl-1, two bcl-2 family proteins are up-regulated in effector T cells by ligation with an agonist antibody to the costimulatory molecule OX40 (CD134), which is essential for long-term T cell survival. To understand whether they are molecular targets of OX40, we examined A1 and Mcl-1 expression in primary T cells from wild-type and OX40-deficient mice, and found A1 and Mcl-1 were up-regulated after early T cell activation, but not be maintained in T cells from OX40-deficient mice. In addition, we used retrovirus-mediated transduction to introduce A1 or Mcl-1 into antigen (Ag)-responding T cells from OX40-deficent mice. We found that exogenous expression of A1 or Mcl-1 reversed the survival defect of OX40-deficient T cells both in vitro and in vivo. These results indicate that A1 and Mcl-1 are molecular targets of OX40, which regulate T cell survival. The data provide new insights into the mechanisms by which costimualtion regulates T cell survival.
Partial rescue of Itk-/- iNKT cell development by Txk/Rlk reveals a unique role for Itk in the survival of iNKT cells. (50.40)
Authors: Qi Qian , Bai Yuting , Schwartzberg Pamela , August Avery
Published: January 2023

Invariant natural killer T (iNKT) cells are a distinct population of innate T lymphocytes that play important role in the immune response. Itk and Txk/Rlk are Tec family kinases that are expressed in iNKT cells, with the expression level of Itk about 7-fold higher than that of Txk. It has been reported that the Itk null mice have reduced iNKT cell frequency and numbers, as well as defects in iNKT cell development and cytokine secretion, all of which are exacerbated in the Itk/Txk DKO mice. By contrast, the phenotype of iNKT cells in Txk null mice is similar to that from WT mice. In order to determine whether Itk and Txk plays distinct roles in iNKT cell development and function, we examined the iNKT cells in Tg(Lck-Txk)/Itk-/- mice, which over express Txk in T cells to the similar level as Itk. Over expression of Txk rescues the maturation and cytokine secretion of iNKT cells in Itk null mice. It also rescues the altered expression of transcription factor T-bet, eomesodermin and PLZF. By contrast, over expression of Txk does not rescue the reduced iNKT cell numbers in Itk null mice, which is due to the failure to rescue the increased apoptosis observed in Itk-/- iNKT cells. These data suggest that Txk plays an overlapping role with Itk in iNKT cell development and function, but that Itk also has a unique function in the survival of iNKT cells.
Evaluation Of The Immune Response Against The Newcastle Disease Virus When Co-Administered With Beta Subunit Of The Cholera Toxin (52.17)
Authors: Franco García Julio , Moreno Salcedo Yazmín , González Hernández Carlos , León Kempis Maria
Published: January 2023

METHODS:4 groups of 10 SPF chicken each were included: Positive control, vaccinated subcutaneously with a commercial ND vaccine, negative control -non-vaccinated- and two more groups were vaccinated with ND+CTB, one subcutaneously and the fourth group was vaccinated via intranasal. Vaccination at 3 wks of age and challenge 21 days post-vaccination. Sera were collected before vaccination and in a weekly manner during 6 wks post-vaccination in order to evaluate serological response and UHI titers. Mortality and clinical signs were evaluated during 21 days post-challenge. RESULTS. Chickens vaccinated subcutaneously with ND-CTB reached the highest antibody titers when compared to the other experimental groups followed by chickens vaccinated with the commercial vaccine, then the intranasally vaccinated group and then the negative control. The group immunized subcutaneously with CTB also showed an earlier onset of serological response against ND and the duration of immunity was similar to the positive control. Mortality post challenge could be observed in the negative control as well as in intranasaly immunized birds, where all birds died as opposed as in groups of the positive control and the subcutaneously vaccinated, where all the birds survived. CONCLUSIONS: CTB seemed to contribute towards an earlier onset of humoral response in the ND model
Infectious challenge of Plasmodium berghei γ-spz immunized mice rescues effector CD8+ T cells, thus assuring protracted protection (52.9)
Authors: Krzych Urszula , Dalai Sarat , Ehrler Lindsey , Jobe Ousman , Egner Lisa
Published: January 2023

Protracted protection against experimental Plasmodia sporozoite (spz) challenge has been achieved in human and murine malaria by multiple immunizations with radiation-attenuated Plasmodia sporozoites (γ-spz). In B6 mice, long-lasting protection induced by Plasmodium berghei (Pb) γ-spz is linked to liver CD8+ T effector memory (EM) and central memory (CM) cell subsets. Protracted protection also depends on the interval between the last boost immunization and the infectious spz challenge. An interval of 6 months or longer results in loss of protection. In this study we asked whether a delayed challenge causes a decline of either CD8+ TEM or TCM or both cell subsets. We measured IL-2 and INF-γ producing polyclonal CD8+ TEM (CD44hiCD62Llo KLRGintCD107+CD122lo) and CD8+ TCM cells (CD44hiCD62Lhi KLRG-CD122hi) and observed that the frequencies of cytokine producing KLRGintCD107+ CD8+ TEM cells fell precipitously at 210 days after the last boost with γ-spz and that these mice were not protected when challenged 7 months after the last boost immunization. In contrast, mice challenged 2 weeks after the last boost maintained high frequencies of both CD8+ TEM and CD8+ TCM subsets when tested at 210 days following the last boost immunization with γ-spz. These mice were also protected upon a re-challenge (210 days) and remained protected beyond day 548. We are currently investigating the rescuing mechanism of CD8+ TEM cells by infectious spz challenge.
Abnormal morphology of melanosomes in the autoimmune vitiligo-prone Smyth line chicken does not appear to be due to alteration in lipid composition (83.16)
Authors: Stepicheva Nadezda , Liyanage Rohana , Lay Jackson , Dienglewicz Robert , Erf Gisela
Published: January 2023

The Smyth line (SL) chicken is an important animal model for autoimmune vitiligo. SL vitiligo is multifactorial in nature including genetic susceptibility, immune system components and environmental triggers. Genetic susceptibility to SL vitiligo appears to be manifested in part by an inherent melanocyte defect. SL melanosomes (pigment-carrying organelles) have irregular-shaped membranes with pigmented extensions that may be due to abnormal lipid composition. Lipids can affect protein topology and trafficking (and hence protein degradation and presentation), membrane stability and oxidative stress resistance. To compare melanosomal lipids from SL and control lines of chickens, melanosomes were isolated from growing feathers of 4-week-old SL, Brown line (parental control) and Light Brown Leghorn (vitiligo-resistant) chickens and lipids were extracted. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with thin-layer chromatography (TLC) was used to determine expression differences in different classes of lipids by using TLC-MALDI imaging technique. Total fatty acid composition was examined by gas chromatography-mass spectrometry (GC-MS) of fatty acid methyl esters. Statistical analysis of preliminary data shows no significant differences in total fatty acid and phospholipid composition in melanosomes from SL and control chickens. Hence, lipid composition may not explain the melanosomal abnormalities observed in SL melanocytes.
Global transcriptome analysis of feathers from Smyth line chickens revealed different gene-expression profiles at various stages of autoimmune vitiligo (83.17)
Authors: Shi Fengying , Lee Jeong Yoon , Song Joon Jin , Kong Byung-Whi , Dienglewicz Robert , Erf Gisela
Published: January 2023

The Smyth line (SL) chicken is an animal model for autoimmune vitiligo (SLV). SL chickens develop spontaneous, post-hatch, autoimmune melanocyte loss in growing feathers (GF). With repeatable access to GF and the high incidence (85-95%) of SLV, SLV development can be examined in the same bird. A microarray study was conducted to investigate the global transcriptomic profile in GF from SL and Brown line (BL, parental control) chickens. RNA pools were made from GF of BL chickens, SL chickens that never developed SLV (S0), and SL chickens before (S1), during (S2) and at complete (S3) SLV. Complementary RNA probes were generated from BL and SL (S0~S3) RNA, labeled with Cy3 and Cy5, respectively and hybridized on a chicken 4x44 K Agilent microarray. The slide was scanned and normalized signal intensities were analyzed to identify differentially expressed (DE, >2x) genes between BL and SL samples. There were 504, 518, 1124, and 1151 DE genes containing 262, 226, 658, and 680 unique DE genes in S0, S1, S2 and S3, respectively. S0 and S1 had similar proportions of up-and down-regulated DE genes while up-regulated DE genes predominated in S2 and S3. The highest expressed DE genes were related to immune activity and cell death. Ingenuity Pathway Analysis software was used to determine biological functions and pathways of DE genes. The study provides comprehensive information from global gene expression to functionalities and biological networks of DE genes in the process of SLV.
Pigmented growing feathers from autoimmune vitiligo-prone Smyth line chickens have heightened sensitivity to 4-tertiary butyl phenol (83.19)
Authors: Dong Lei , Dienglewicz Robert , Erf Gisela
Published: January 2023

The Smyth line (SL) chicken model for vitiligo exhibits spontaneous, autoimmune loss of melanocytes in growing feathers. Onset of SL vitiligo (SLV) can be observed between 6 and 20 wks of age and affects 85-95% of SL chickens. Multiple factors contribute to the melanocytes’ cell death, including genetic susceptibility to vitiligo as well as environmental factors that trigger autoimmune loss of SL melanocytes (e.g., live turkey herpesvirus (HVT) administration at hatch). In humans, phenolic derivatives, such as 4-tertiary butyl phenol (4-TBP), have been shown to trigger vitiligo in susceptible individuals. 4-TBP is structurally similar to tyrosine, the initial substrate of tyrosinase in melanin synthesis. In addition to interfering with melanin production, 4-TBP exposure results in oxidative stress. To examine the effect of 4-TBP in SL and control chickens, pigmented growing feathers were collected from 1, 4 and 7 week-old SL, Brown Line (BL, parental control, <1% vitiligo) and Light Brown Leghorn (LBL, vitiligo resistant) chickens that did or did not receive HVT. The newest three mm growth (the melanocyte-containing, active pigmentation site) of the feathers was cultured with 4-TPB (200 μM) and the production of oxidative radicals was monitored in vitro. Independent of HVT treatment, 4-TBP induced more oxidative stress (P < 0.05) in feathers from SL than BL and LBL chickens, suggesting heightened sensitivity of melanocytes to 4-TBP in vitiligo-prone SL chickens.
Regulatory T cells control CNS-infiltration of autoreactive T cells during viral infection without affecting the antiviral immune response (83.24)
Authors: Cervantes-Barragan Luisa , Firner Sonja , Züst Roland , Prodinger Carolin , Bechmann Ingo , Sparwasser Tim , Thiel Volker , Ludewig Burkhard
Published: January 2023

Regulatory T cells are essential for suppressing immune responses to autoantigens and therefore help to prevent autoimmunity. During viral infections in the CNS, an indiscriminate regulation of T cells can prevent autoimmune diseases but could also impair the control of viral replication. We analyzed here the impact of regulatory T cells in the control of T cell infiltration to the CNS, virus-induced CNS pathology, and viral clearance using the mouse hepatitis virus (MHV) A59 intranasal infection model; a virus infection that leads to encephalitis and demyelination. MHV infection of “depletion of regulatory T cell” (DEREG) mice, revealed that the lack of regulatory T cells during MHV infection leads to an increased T cell infiltration and pathology in the CNS. However, antiviral T cells response were not affected by the depletion of FoxP3+CD4+ T cells indicating that regulatory T cells control infiltration of T cells to the CNS without impairing or delaying the antiviral immune response. Moreover, in MHV infected mice, adoptively transferred-myelin oligodendrocyte glycoprotein (MOG35-50)-specific CD4+ T cells proliferated in cervical lymph nodes and migrated to the CNS, suggesting that MHV infection in the CNS induces the activation of self-reactive T cells which are controlled by regulatory T cells to reduce the risk of developing inflammatory CNS disease.
ST2 deficiency enhances the severity of Th1/Th-17 mediated inflammation (83.27)
Authors: Lukic Miodrag , Volarevic Vladimir , Mensah-Brown Eric , Shahin Allen
Published: January 2023

ST2 selectively and stably expressed on the surface of T-helper 2 (Th2) but not Th-1 cells is a ligand for recently identified Interleukin-33 (IL-33). We have studied the susceptibility to and severity of the two T cell mediated inflammatory process: multiple low dose streptozotocin induced diabetes and Con-A induced hepatitis, in ST2 deficient (ST2-/-) and “wild-type: BALB/C mice. After diabetes induction (40mg STZ/kg b.w. for 5 days) ST2-/- mice developed glycemia and glycosuria. β cell loss and intra-islet mononuclear infiltrates while “wild type” mice did not develop any biochemical signs and histology revealed only peri-insulitis. When injected with Con-A (12 mg/kg b.w) ST2-/- mice developed significantly enhanced hepatitis as evaluated by liver function test 8 and 24 hours after induction and quantitative analysis of the hepatocytes necrosis. Analysis of cellular make up of the pancreatic lymph nodes and pancreata (in diabetes) and liver infiltrating cells (in ConA induced hepatitis) and their cytokines content by FACS analysis and cytokine expression by RT-PCR revealed the enhanced infiltration of Th-1 and Th-17 cells and higher content and expression of proinflammatory cytokines: IFN-γ, IL-17 and TNF. We therefore concluded that attenuation of IL-33-ST2 axis facilitates the induction of Th-1/Th-17 mediated inflammatory autoimmunity.
Regulation of dendritic cell function and autoimmunity by microRNA-155 (83.29)
Authors: Lind Evan , Dissanayake Dilan , Ohashi Pamela
Published: January 2023

While many of the mechanisms that control self tolerance in the immune system have been discovered, there are many unanswered questions in understanding the molecular mechanisms that maintain immunological quiescence. The studies outlined here aims to examine how a newly discovered class of small RNA molecules called micro-RNA control one of the checkpoints limiting autoimmunity, namely the maturation state of dendritic cells. Micro-RNA are a class of non-protein coding RNA that act by binding the 3’ UTR end of messenger RNA and inhibiting translation. We have evidence implicating that one specific micro-RNA, miR-155, plays a role in maintaining self-tolerance in the immune system. We find that miR-155 levels increase in dendritic cells after maturation induced by TLR stimulation. In a new murine model of autoimmune diabetes developed in our lab, dendritic cells lacking miR-155 are unable to break immune tolerance to pancreatic islet cells antigens. Using this model we will investigate whether the lack of miR-155 in dendritic cells affects the induction and expansion of cytotoxic T lymphocytes as well as the pro-inflammatory response. Furthermore, we will also explore the targets that are regulated by miR-155 that mediate the induction of autoimmunity.
Mixed Chimerism with MHC-Matched Bone Marrow Transplantation Results in Dominant Protection From Autoimmune Diabetes (83.8)
Authors: Gill Ronald , Coulombe Marilyne , Toews Carrie , Yu Liping , Eisenbarth George
Published: January 2023

Allogeneic mixed chimerim can prevent disease onset in Type 1 diabetes. While bone marrow (BM) allografts from MHC-mismatched donors can prevent disease in NOD mice, levels of mixed chimerism can be variable and unstable. Also, MHC-mismatched BM usually involve donor T cell depletion, increasing risk for graft-versus-host reactivity. Therefore, we tested the ability of MHC-matched donors to protect NOD mice from disease. Young female NOD mice were irradiated (2 x 450R) and grafted with the following BM donors: (1) Disease-prone NOD (H-2g7), (2) MHC-matched, disease-resistant C57Bl/6.H-2g7 (B6.H-2g7), (3) MHC-mismatched, disease-resistant C57Bl/6 (B6), or (4) Mixtures of disease-prone + disease-resistant BM. Importantly, donor BM was depleted of mature T and B cells. Recipients of only NOD BM recapitulated diabetes in 31/33 (94%) recipients by 30 weeks. Conversely, recipients of B6.H-2g7 BM alone did not develop diabetes (0/26). Mixed NOD + B6.H-2g7 BM grafts resulted in marked disease protection in 27/30 recipients (p <.001). However, recipients of mixed NOD + B6 BM grafts rejected the MHC-mismatched B6 donor and developed disease in 9/10 hosts (90%). Importantly, mixed NOD + B6.H-2g7 BM transplants resulted in remarkably stable, multi-lineage chimerism (>40-50% NOD) for ≥ 30 weeks. Thus, MHC-matched BM allografts permits dominant disease protection and stable macro-chimerism in NOD mice. Also, disease-prone MHC is not an intrinsic barrier to autoimmune regulation.