bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor IL-10 forms an immediate non-redundant blockade to CpG-induced activation of tumor-infiltrating DC and macrophages (41.61)
Authors: Stewart Charles A , Scheetz Loretta , Trinchieri Giorgio
Published: January 2023

Toll like receptors (TLR) are a major class of pathogen recognition receptors that trigger potent dendritic cell DC activation and present an important immunotherapeutic target. Combination therapy of established tumors by local injection of CpG oligonucleotides (TLR9 agonists) and blockade of IL-10 signaling results in tumor rejection in mice. Using the MCA38 colon carcinoma and B16 melanoma models, CpG treatment alone drastically reduces tumor growth for the duration of treatment, but does not lead to tumor rejection. Local tumor injection of MCA38 tumors with CpG oligonucleotides and intra-peritoneal anti-IL10R antibody led to rapid production of CCL2, TNF- ƒÑ and IL-6 that were detectable in serum after 2 hours. 24 hours after CpG treatment, IL-10, IL-12, and IFN-ƒx were also detectable in serum. Cytokine production at both 2 hour and 24 hour time-points was partially dependent upon blockade of IL-10 signaling. To examine activation of tumor-infiltrating leukocytes, ex vivo cultures of tumor-isolated cells were set up at time-points following CpG injection. Significant production of TNF- ƒÑ, IL-6 and IL-10 was observed only at 2 hours with blockade of IL-10 signaling. The transient period of cytokine production was matched at the cellular level: Prior to treatment MCA38 tumors comprise of ~40% tumor-infiltrating leukocytes (TIL) which are mostly CD11b+CD11c+ cells characteristic of the macrophage or DC lineage. 24 hours following treatment, TILs were predominantly CD11b+Ly6G+ cells. Our results show that CpG treatment of tumors induces rapid activation of tumor-infiltrating cells that is highly potentiated at early time-points by the blockade of IL-10 signaling.
Novel phytol-based adjuvants in vaccine formulations: Evaluation of safety and efficacy in induction and persistence of specific immune responses (41.64)
Authors: Aachoui Youssef , Ghosh Swapan K
Published: January 2023

Adjuvants function as "immunologists' dirty tricks" for enhancement of immune response. However, no two adjuvants work in a similar fashion. Adjuvant efficacy depends on how they influence microenvironment, antigen presentation and retention of immunogens. We are developing water-in-oil emulsion-type adjuvants from phytol (J Immune Based Ther Vaccines 2006, 5:4; ibid 2007; 5: 3). Phytol is a diterpene present in chlorophylls, alpha-tocopherols etc. Phytol derivatives we developed are hydrogenated/nitrated and glycosylated products (US patent pending). Our previous study showed that one phytol derivative PHIS-01(hydrogenated) is an excellent non-toxic adjuvant. In this study, we investigated the safety and efficacy of two other phytol compounds, PHIS-02 and PHIS-03 relative to PHIS-01 and other adjuvants. Using phthalate- keyhole limpet hemocyanin (KLH) conjugate as the immunogen emulsified with these adjuvants, we assessed titer, specificity, and isotypic profiles of anti-phthalate antibodies induced. In addition, we studied their effects on cellular immunity such as T cell proliferation and cytokine production. Preliminary results show that (1) phytol adjuvants significantly augment antibody response of isotypes IgG1 and IgG2a, (2) promote effective T cell proliferation, and (3) exhibit low toxicity with no adverse autoimmune anti-DNA response. We also observed that these adjuvants exert apoptotic/necrotic effects on mouse lymphoma cells 2C3 and A20 in a concentration-dependent manner.
Rapid isolation of pure, functional and bead-free T cells directly from whole blood using Dynabeads® FlowCompTM technology (42.11)
Authors: Moeller Anne-Sophie W , Kierulf Bente , Reed Berit , Neurauter Axl A , Schjetne Karoline W
Published: January 2023

BACKGROUND: Fast and gentle isolation of pure cells directly from whole blood is beneficial for both basic research, drug discovery, patient monitoring (e.g. analysis of circulating tumor cells, prenatal diagnostic, T cell numeration in HIV, chimerism analysis in transplantation) and would render the time-consuming gradient centrifugation step superfluous.

AIM: Develop a fast and gentle protocol for isolation of functional and bead-free human CD3+, CD4+ or CD8+ T cells directly from whole blood.

METHODS: Peripheral blood was drawn from healthy donors. Antibody (specific for human CD3, CD4 or CD8) conjugated to a modified biotin was added to label the cells. In the next step, Dynabeads coated with modified streptavidin were added and target cells were captured when placed on the magnet. After resuspension with FlowCompTM Release Buffer the bead-free cells were analyzed using flow cytometry and tested in functional assays.

RESULTS: Highly pure and viable human T cells were isolated in less than 50 minutes directly from whole blood using Dynabeads® FlowCompTM. Isolated T cells readily proliferate in response to stimulation with Dynabeads® CD3/CD28 T Cell ExpanderTM as measured by incorporation of EdU (EdU Click-iT). We demonstrate that isolated T cells were activated both in a polyclonal and antigen-specific manner, and that isolated cytotoxic CD8+ T cells kill target cells.
ImmPort FLOCK: Automated cell population identification in high dimensional flow cytometry data (42.17)
Authors: Scheuermann Richard H. , Qian Yu , Wei Chungwen , Sanz Inaki
Published: January 2023

Historically, investigators have analyzed flow cytometry (FCM) data using manual gating strategies and two-dimensional dot plot visualization to identify and compare specific cell populations between samples. However, this approach is subjective and does not scale well as the number of parameters detectable with different fluorochrome reagents increases. We have developed a software system for automated population discovery in multidimensional FCM data - FLOw Clustering without K (FLOCK), which specifically takes into account the unique features of FCM data, including non-spherical parameter distributions and sparse cell populations. FLOCK includes the following steps: data cleansing, data shrinking, data normalization, informative dimension selection, dense hyper-region identification, histogram partitioning, centroid calculation, clustering, visualization, population statistics calculation, cell ontology mapping. As a result, FLOCK automatically and objectively determines the number of cell populations detectable in a sample and assigns population membership to each cell event, from the multi-parameter flow cytometry data, in the absence of manual gating. Our study of normal human peripheral blood has identified 17 unique B lymphocyte populations based on the expression of IgD, IgG, CD24, CD27, CD38 & B220. Supported by NIH N01AI40076.
PGC nanocarrier-formulated native GLP-1 prevents diabetes in NOD mice (42.3)
Authors: Reichstetter Sandra , Reed Lynn , Lai Man-Shun , Castillo Gerardo M , Bolotin Elijah M
Published: January 2023

Formulation of peptides in a nanocarrier made up of protected-graft-copolymer (PGC) can greatly enhance pharmacokinetics and biodistribution of the peptides in vivo. Glucagon-like peptide 1 (GLP-1) is β-cell protective and decreases the severity symptoms of diabetes in Type 2 and streptozotocin models. We hypothesized that PGC-formulated GLP-1 can prevent Type 1 diabetes in NOD mice. 4-5 week old female NOD mice [n=25] were treated for eight weeks with 3mg/kg PGC-formulated GLP-1 administered s.c. either once or three times per week. After the treatment period, the mice were monitored for signs of diabetes until they reached 36 weeks of age. A group of NOD mice treated with anti-CD3 antibodies (0.5mg/kg i.p. once at treatment week 0 and once at treatment week 1) was used as a positive control for diabetes prevention. At 36 weeks of age, 48% of the mice treated with saline had developed diabetes. Anti-CD3 treatment prevented 50% of the diabetes cases in the mice with only 24% of the mice in this group having developed diabetes at 36 weeks of age. Once a week GLP-1 treatment (3mg/kg s.c.) was as effective in preventing diabetes as anti-CD3 treatment with only 28% of mice being diabetic at 36 weeks of age. Surprisingly, there was no difference between the three times per week GLP-1 treatment group (3mg/kg s.c.) and the saline control: 54.1% of the mice treated three times per week became diabetic, which was not statistically significantly different form the 48% diabetes incidence in the saline group.
T regulatory and prostate cancer cell-specific drug targeting using novel XPclad© nanoparticles (42.7)
Authors: Singh Rajesh , Singh Shailesh , Sharma Praveen K , Lillard, Jr. James W
Published: January 2023

In our study, we determined the utility of cisplatin-loaded biodegradable XPclad nanoparticles coated with polycaprolactone-polyethylene glycol copolymers conjugated to folate. We hypothesized that these 28 ± 5 nm sized particles would enhance the therapeutic efficacy of cisplatin through targeted delivery to both prostate tumors and CD4+CD25HiFoxP3+ T cells (Treg) cells. PCa cell lines (PC3 and LNCaP), but not normal prostatic epithelial cells (PrEC and RWPE-1), expressed high levels of folate receptor α (FRα/FR1). Similarly, Treg, but not CD4+CD25LoFoxP3- T cells (T helper), cells expressed FRδ/FR4. Treatment using 20 μM of cisplatin solution resulted in > 90% cell death of PCa cells, PrEC, and RWPE-1 cells as well as purified Treg and T helper cells after 24 hr of culture. However, XPclad nanoparticles, containing cisplatin (equivalent to a 200 nM dose) induced the same level of apoptosis in PCa and Treg cells as cisplatin solution, but did not affect PrEC or RWPE-1 cells or T helper cell death. We also show that PC3 tumor-bearing mice that received either cisplatin solution (8 mg/kg/week) or cisplatin-encapsulated folate-coated XPclad nanoparticles (equivalent cisplatin dose = 0.08 mg/kg/week) had significant tumor regression. While mice receiving cisplatin solution had significant higher kidney and liver toxicities, XPclad nanoparticle-treated mice did not. In conclusion, our studies show that novel XPclad nanoparticles enhance tumor therapy efficacy with lower toxicities and suggest that FR-targeted nanotherapy cause the destruction of prostate tumor cells and Treg cells.
TNFR-TNFL superfamily member interactions induced by combined TLR/CD40 stimulation act as Signal 3 for enhanced CD8+ T cell immunity (44.10)
Authors: Sanchez Phillip J , Kedl Ross M
Published: January 2023

Previously, we have shown that antigen-specific CD8+ T cell expansion following TLR agonist/anti-CD40 immunization is dependent on the enhanced expression of CD70 on dendritic cells. The dependence on CD27-CD70 interactions was confirmed by using a blocking anti-CD70 antibody. We now show that in CD27-/- mice, the antigen-specific CD8+ T cell response to TLR agonist/anti-CD40 immunization is intact. However, administration of a blocking anti-OX40L antibody during immunization attenuates the CD8+ T cell expansion. Thus, similar to the wild-type C57BL/6 mice, the enhanced primary expansion of antigen-specific CD8+ T cells in response to TLR agonist/anti-CD40 vaccination is dependent on TNF receptor (TNFR) - TNF ligand (TNFL) superfamily interactions. Furthermore, TLR agonist/anti-CD40 vaccination is not affected by depletion of IL-12 using a neutralizing anti-IL-12p40 antibody in either wild-type or CD27-/- mice. Together, these data suggest that TNFR-TFNL interactions, facilitated by combined TLR agonist/anti-CD40 vaccination, can act as Signal 3 for CD8+ T cell immunity in the absence of IL-12.

This research is supported by a grant from the NIH.
Immunotherapeutics effects of IL-7 during a chronic viral infection (44.13)
Authors: Nanjappa Som Gowda , Kim Eui Ho , Sullivan Jeremy , Morre Michel , Suresh M
Published: January 2023

Viral persistence in chronic infections is associated with ineffective T cell responses. Here, we evaluated the therapeutic use of IL-7 to enhance T cell responses during a chronic infection with lymphocytic choriomeningitis virus (LCMV). Mice infected with Clone 13 strain of LCMV were treated with IL-7 at various intervals spanning the first 30 days after infection. Our studies show that the ability of IL-7 to enhance T cell responses to a chronic viral infection is constrained by high viral load. Early in the infection (days 0-15 PI), when the viral load is very high, IL-7 administration induced minimal enhancement of T cell responses to LCMV. However, with reducing viral load (after day 15 PI), IL-7 augmented virus-specific T cell responses, albeit temporarily. An extended duration of IL-7 treatment after the initial phase of T cell expansion (days 8-30 PI), markedly enhanced the number of virus-specific CD8 T cells. Moreover, extended IL-7 therapy altered the cell surface phenotype, and augmented the cytokine-producing ability of LCMV-specific T cells. Surprisingly, despite the augmentation of virus-specific T cell responses, IL-7 therapy had a modest effect on circulating viral load in chronically infected mice. In summary, these findings suggested that IL-7 administration could be used as an adjunct to anti-viral immunotherapy to bolster T cell responses to chronic viral infections. This work was supported by PHS grant AI68841 to M. Suresh.
MCMV induces salivary gland dysfunction in autoimmune-prone NZM2328 mice (44.17)
Authors: Carroll Virginia , Lundgren Alyssa , Brown Michael G
Published: January 2023

Loss of secretions from the salivary gland (SG) is a disease feature of Sjogren's Syndrome (SS) as well as certain viral infections, including HCV and HIV. We have found that murine cytomegalovirus (MCMV) infection of autoimmune-prone NZM2328 mice serves as a model for virus-induced exocrinopathy, displaying many SS-like disease features. Interestingly, MCMV can induce severe SG dysfunction within 2-7 days after intraperitoneal infection. MCMV replication within the gland was not directly responsible for dysfunction since NZM2328 mice secreted normally 7-14 days after low dose infection, despite high virus within the gland. Since the low dose regimen limited viral burden elsewhere in the animal, such as the spleen, SG dysfunction seemed to correspond with systemic viral load. Surprisingly, lymphocytes were not required for MCMV-induced dysfunction at day 7, including NK, T and B cells. At day 2, morphological alterations were noted in acinar epithelium and flow cytometric analysis revealed rapid changes in glandular monocytic populations. Ongoing experiments are examining a potential causal link between systemic immune responses and/or infiltrating inflammatory monocytes with morphological and functional changes within the salivary gland. Supported by NIH R01AI50072 and a Sjogren's Syndrome Foundation Student Fellowship
TLR4-Deficient C3H/HeJ Mice Exhibit Enhanced Disease and Mortality Following Intranasal MHV-1 Infection (44.23)
Authors: Haag Brayton A , Hartwig Stacey M , Khanolkar Aaruni , Harty John T , Varga Steven M
Published: January 2023

Severe acute respiratory syndrome (SARS) is characterized by substantial acute pulmonary inflammation and a high mortality rate. A novel human coronavirus, SARS-associated coronavirus (SARS-CoV), was identified as the etiologic agent of SARS. With the disappearance of SARS in the human population following the initial outbreak in 2003, limited information is available regarding the immunopathogenesis of SARS. Thus, an animal model that recapitulates the human disease would prove to be of great benefit, providing valuable insight into the pathology of the infection. Recent studies have demonstrated that intranasal infection of susceptible mouse strains with a related mouse coronavirus, mouse hepatitis strain 1 (MHV-1), produces acute respiratory disease and mortality that resembles human SARS cases. We find that intranasal infection with MHV-1 results in morbidity and some mortality in susceptible C3H/HeN (H-2k) mice. Interestingly, MHV-1 infection of C3H/HeJ mice, that harbor a natural mutation in their toll-like receptor 4 (TLR4) gene which renders TLR4 unable to properly signal, results in enhanced disease and increased mortality. In the absence of a functional TLR4 signal, we observe enhanced CD4 T cell responses, where as CD8 T cell responses remain unaltered. Our results indicate that TLR4 plays an important role in respiratory coronavirus pathogenesis.
Mast cells induce alternative activation and inhibit caspase-3 mediated apoptosis of Francisella tularensis infected macrophages (44.24)
Authors: Rodriguez Annette Rose , Yu JiehJuen , Murthy Ashlesh , Guentzel M. Neal , Klose Karl E. , Chambers James P. , Forsthurber Thomas G. , Berton Michael T. , Arulanandam Bernard P.
Published: January 2023

Francisella tularensis is a highly infectious Gram-negative bacterial pathogen and the causative agent of pneumonic tularemia. We recently demonstrated inhibition of F. tularensis LVS replication in macrophages via mast cell production of interleukin-4 (IL-4). We have further analyzed the interactions of mast cells and macrophages using a primary bone marrow derived mast cell-macrophage co-culture system and determined that mast cells inhibit F. tularensis-induced macrophage apoptosis. Specifically, mast cells co-cultured with macrophages significantly reduced intramacrophage LVS growth and expression of apoptotic proteins caspase-3 (19.9%) and PARP (Poly-ADP ribose polymerase, 16.0%) compared to macrophages cultured alone (48.2% and 56.4%, respectively). The inhibition of LVS-induced apoptosis within infected macrophages also was seen by the direct addition of recombinant IL-4 and corresponded with up-regulation of mannose receptor expression and arginase activity which are associated with the alternative activation of macrophages. Moreover, IL4R-/- mice were more susceptible to intranasal challenge and exhibited greater caspase-3 activity in lung macrophages (12.9%) compared to similarly challenged wild-type (4.7%) animals. These results support a role for mast cells and IL-4 in control of bacterial replication and apoptosis following pulmonary LVS infection.

National Institutes of Health grant PO1 AI057986; NIH/NIGMS MBRS-RISE GM60655
Protection of cynomolgus macaques from Ebola Sudan infection (45.10)
Authors: Dye John M , Muhammad Majidat , Bakken Russell , Barth James , Kuehne Ana , Olinger Gene G
Published: January 2023

Ebola virus, a filovirus, is a highly pathogenic human pathogen for which there is no vaccine or treatments licensed for human use. The Sudan subtype of the Ebola virus (SEBOV) was first identified in 1976 after a hemorrhagic fever outbreak in that region of Africa. There were reemergences of the SEBOV in sporadic outbreaks from 2000 to 2004 with a 40-65% case fatality rate in humans. In this study, we evaluated the use of the Venezuelan equine encephalitis replicon (VRP) vaccine vector expressing the glycoprotein (GP) of Ebola Sudan to protect cynomolgus macaque nonhuman primates (NHP) against a uniformly lethal challenge with SEBOV. We found that six of six NHP vaccinated with VRP expressing GP of Sudan were protected against infection with 1000 PFU of SEBOV. Furthermore, when we back challenged these Sudan survivors with another strain of Ebola, Zaire, there was only a partial protection (two of six survived). When we vaccinated NHP with VRP expressing GP of Sudan and GP of Zaire, we found that all animals were protected from either infection with Sudan or Zaire (three of three survived both infections). Taken in total, these data indicate that vaccination with only one subtype of Ebola virus will not protect against other subtypes. However, there is no interference when using multiple VRP vaccinations against different subtypes of Ebola viruses. These results are encouraging for the development of a multivalent vaccine platform against multiple strains of filoviruses.
The antigen-specific CD4 host response to Plasmodium yoelii 17XL infection after protective MSP-8 immunization alters multifunctional cytokine profiles (45.13)
Authors: Smigiel Kate , Darrah Patricia , Fogg Chris , Harvey Reid , Tullo Greg , Seder Robert , Burns James , Long Carole
Published: January 2023

Multifunctional CD4 T cells, which produce combinations of interferon gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and IL-2, have been shown to be immune correlates of protection after vaccination in a mouse model of L. major and for some models of M. tuberculosis. In this study, we have used multi-parameter flow cytometry to study a merozoite-surface protein-based (MSP-8) vaccine model against lethal murine Plasmodium yoelii (Py) infection, which we have shown to be dependent on IFN-γ and which also requires an active immune response during challenge infection for protection and survival. Our data supports previous studies, which have found that the patterns of multifunctional responses are established after the first immunization but the frequencies of CD4 responders and the amount of IFN-γ produced per multifunctional cell increase with subsequent immunizations. We further show that antigen-specific memory CD4 cells in the lymph nodes are functionally distinct from those in the spleen and liver before and after Py challenge. Also, the nonlymphoid-tissue (liver) responses occur at the greatest frequency and show the greatest plasticity in cytokine profile during infection. We conclude that this protective immunization induces multifunctional 3+ antigen-specific CD4 T cells (10-25% of MSP-8 specific CD4s) but that the host response to lethal malaria infection triggers a shift in multifunctionality towards more IFN-γ production, especially in non-lymphoid tissue.

Funding Sources: J. Burns (NIH #AI35661), C. Long (Intramural Program of NIH-NIAID)
Cytolytic Potentials of CD8+ T Cells Persisting in Genital Skin and Mucosa after HSV-2 Reactivation (45.16)
Authors: Zhu Jia , Phasouk Khamsone , Peng Tao , Woodward Amanda , Klock Alexis , Corey Lawrence
Published: January 2023

We have previously demonstrated that CD8+ T cells, including HSV-2 specific CD8+ T cells, persisted in genital skin for months after the herpes lesion completely healed, and preferentially accumulated at the dermal-epidermal junction where sensory nerve endings were enriched. These persisting CD8+ T cells have been postulated in the containment of the reactivating virus before clinical symptomatic lesion occurs. To understand functions of persisting CD8+ T cells in human, we have combined immunofluorescent (IF) staining, laser capture microdissection (LCM) and RT-PCR to analyze the gene expression of CD8+ T cells at the dermal-epidermal junction in post-healed lesion biopsy. CD8+ T cells were captured at a single cell level. The purity was validated by the lack of CD4 and a 100-fold more enriched CD8 expression in LCM captured cells than in whole tissue sections. In captured CD8+ T cells, we detected up-regulation of genes encoding cytolytic granules, such as perforin and granzyme B, as compared to keratinocytes in the epidermis. The expression of cytolytic granules was further confirmed by IF staining of persistent CD8+ T cells, even in post-healed biopsy of acyclovir treated individual. Our data strongly indicated the cytolytic potentials of CD8+ T cells persisting at the dermal-epidermal junction and implicated their role in immune surveillance against HSV-2 reactivation in skin and mucosa.

This work was supported by NIH grants R37 AI042528 and PO1 AI030731.
IL-7 and IL-15 play a synergistic role in the development of CD8+ T cell response against an obligate intracellular parasite (45.17)
Authors: Bhadra Rajarshi , Guan Hongbing , Khan Imtiaz A.
Published: January 2023

IL-7 and IL-15 are believed to be important for homeostatic proliferation of CD8+ memory T cells. However, the simultaneous role played by these two cytokines in the generation of this response during acute phase of infection has not been described. Our results demonstrate that depletion of IL-7 in mice lacking IL-15 gene leads to poor development of both short-lived effectors as well as memory phenotype CD8+ T cells during Toxoplasma gondii infection. CD8+ T cells from these animals lack the ability to produce optimal levels of IFNγ upon antigenic-restimulation and their ability to exhibit cytotoxic response is severely hampered. Moreover, Bcl-2 and KI-67 expression on these cells is down regulated. To the best of our knowledge this synergism between IL-7 and IL-15 in generating a potent CD8+ T cell immunity in an infectious disease model has not been previously reported.
Differential immunomodulatory activity of soluble beta glucans from barley and yeast in antigen specific humoral immune responses (45.21)
Authors: Bagriacik Emin Umit , Uslu Kadriye , Imir Turgut
Published: January 2023

The main difference in chemical structure of (1 →3)-β-D-glucans from yeast cell wall and barley is the branching side chains. Yeast cell wall contains (1→3),(1→6)-β-D-glucans whereas barley contains (1→3),(1→4)-β-D-glucans. The purpose of this study was to compare adjuvant effects of two soluble beta-glucans from barley and yeast (Saccharomyces cerevisiae) in antigen specific humoral immune responses. In a conalbumin-immunized mouse model, mice were immunized by conalbumin at a relatively lower concentration (10 μg/mouse) in the presence of beta glucans. Anti-conalbumin antibodies in the serum specimens of immunized and control mice were quantified by ELISA.

At high doses, both glucans were effective in increasing circulating conalbumin-specific antibodies of IgM, IgG, and IgA types. However, 1 μg of â-glucan from barley resulted in lower yield in IgG1, IgG2a, IgG2b, and IgGA levels in comparison to the yeast-derived glucan at the same dose. We also found that antigen specific antibody levels enhanced by the β-glucan from yeast were always higher than those of the glucan from barley.

In conclusion, yeast-derived glucans might have better immunomodulatory or adjuvat activity in induction of antigen specific humoral immune responses over barley-derived glucans.β
The role of IL-12p40 during host cell infection with Yersinia pestis KIM D27 (45.22)
Authors: Parent Michelle A. , Joerger Torsten A. , Golwala Sohil N. , Smiley Stephen T. , Blumerman Seth L.
Published: January 2023

Yersinia pestis, a facultative intracellular pathogen, is the causative agent of plague. A previously developed vaccine model using Yersinia pestis KIM D27, B cell-deficient mice, and convalescent serum resulted in the generation of T cell-meditated protection against lethal pneumonic infection. In order to understand how this serum results in the development of protective T cells we investigated the interaction between the host immune response, convalescent serum and Y. pestis. First, we determined that IL-12p40 but not IL-12p35 was required for mice to survive this vaccination regimen. We then began to examine the early host response to infection by studying dendritic cells (DC) infected with Y. pestis in the presence of convalescent serum, since DC are cable of presenting antigen to naïve T cells. We infected cultures of bone marrow-derived DC and established that Y. pestis is able to infect DC. Interestingly, convalescent serum does not result in clearance of organisms but sustains infection. Currently, we are investigating cytokine production and cell surface maker expression profiles in Y. pestis infected DC. Preliminary data of infected DC in the presence of convalescent serum results in an increase in IL-12p40 gene expression. Our goal is to ascertain those events occurring early during the course of host infection that lead to generation of this cell-mediated protection. This research is supported by a grant from the University of Delaware Research Foundation.
Identification of novel HLA-A*0201-restricted CD8+ T-cell epitopes on Ebola Zaire glycoprotein (45.26)
Authors: Prugar Laura Irene , Rebekah James M , Gene Olinger G
Published: January 2023

Due to high morbidity and mortality rates, filoviruses are considered among the deadliest of human pathogens with case fatality rates that can reach 90% in some human outbreaks. Ebolavirus and Marburgvirus, the two genera of the family Filoviridae, pose a significant threat to military personnel, global security, and public health, and are classified as Category A bioterrorism agents by the CDC. While there is no licensed vaccine available, there are several promising vaccine candidates that have demonstrated immunogenicity and efficacy in animal models of disease. The vaccine candidates to date, have identified immunogens, usually the glycoprotein (GP), and most importantly,have demonstrated efficacy in the highly sensitive macaque models of filovirus disease. Human vaccine trials will need immunoassays that correlate with protection. Our lab has demonstrated the role of CTL in rodent models of Ebola Zaire. Here we report the first identified CTL epitopes of Ebola Zaire virus (EBOVZ) GP on HLA A*201. The binding of HLA-A*0201-restricted peptide in EBOVZ GP was determined by peptide binding alogorithiums, pentamer binding assays, and binding of overlapping or nonamers using T2 cells expressing HLA-A*0201. Potential HLA-A*0201-restricted peptides were selected from the SYFPEITHI database and screened by T2 cell-stabilization assay. In addition, overlapping peptides were used to identify potential epitopes that could be missed from the predicted algorithms. The various putative epitopes were winnowed and validated with T2-cell stabilization assays. Our results will provide a new insight into the development of therapeutic vaccines and the development of assays for future human vaccine trials for EBOVZ.
Dengue viral component triggering reactive nitrogen and oxygen species production leads to endothelial cell apoptosis (45.33)
Authors: Yen Yu-Ting , Wu-Hsieh Betty
Published: January 2023

Vascular leakage is one of the life-threatening complications occurring in dengue patients, however, the pathogenic mechanism is not well understood. In dissecting the complex interplay between the virus and the host, we found that dengue virus productively infects the endothelial cells and causes apoptotic cell death through triggering the production of reactive nitrogen (RNS) and oxygen radicals (ROS).

To determine the dengue virus component(s) that is responsible for the cause of cell death, we transfected human microvascular endothelial cell HMEC with pCR3.1 plasmids encoding Flag-tagged viral components Core, PrM, NS1, NS2A, NS2B, NS3, NS2B-NS3, NS4A, NS4B, and NS5, separately. Apoptotsis in cells transfected with NS2B-NS3 was 10-fold higher and that in cells transfected with NS1, NS3 and NS5 or core was only between 4- to 6-fold higher than cells transfected with vector alone. Confocal microscopy revealed that while NS2B expression remained in the cytosol, NS3 and NS2B-NS3 in both the cytosol and the nucleus at 24 h after transfection. Interestingly, the effect of NS2B-NS3 on endothelial cells was reversed in the presence of NO and ROS inhibitors. These results demonstrate that NS2B-NS3, being the major component that induces endothelial cell apoptosis, interacts with subcellular components in both the cytosol and the nucleus, and induces the production of RNS and ROS to cause apoptotic outcome.
CD4 T cell derived IFN-gamma protects from lethal encephalomyelitis by inhibiting neutrophil infiltration independent of IL-17 (45.6)
Authors: Savarin Carine , Bergmann Cornelia C , Stohlman Stephen A
Published: January 2023

Immunodeficient SCID recipients of virus-primed memory CD4 T cells survive CNS infection by neurotropic coronavirus (JHMV), whereas IFN-γ deficient (GKO) donor CD4 T cells substantially increase morbidity. Control of virus replication and demyelination are similar in both recipients compared to uncontrolled replication without demyelination in non-treated mice. However, distinct from wt CD4 T cell recipients, GKO recipients exhibit massive CNS neutrophil infiltration, prominent CD4 T cell localization to grey matter, and detection of IL-17 mRNA. To determine the relative role of IFN-γ, IL-17 and neutrophils in disease severity, equal populations of wt and GKO CD4 T cells were co-transferred to SCID mice. Despite the vast abundance of GKO versus wt CD4 T cells accumulating in the CNS at 8 days p.i. (80 vs 20 %), disease severity and survival was similar to protected recipients of wt CD4 T cells. Furthermore, the minor population of wt CD4 T cells in mixed recipients resulted in decreased CNS neutrophil infiltration, consistent with modestly elevated IFN-γ and reduced neutrophil-attracting chemokines. However, wt CD4 T cells did not reduce CNS IL-17 mRNA levels in recipients of mixed relative to GKO donor cells. The data suggest that IFN-γ dominates over IL-17 in controlling excessive neutrophil recruitment and thus protecting JHMV infected SCID recipients from premature death.
Determining the role of Ikaros in Th17 differentiation (46.18)
Authors: Wong Larry , Brown Melissa
Published: January 2023

CD4+ Th17 T cells contribute to the pathogenesis of a number of autoimmune/inflammatory diseases. Yet the molecular events that govern their development are still poorly understood. A reciprocal relationship between Th17 and T regulatory (Tregs) cells exists such that the relative expression of cell-specific transcription factors, RORγt and Foxp3, dictates whether CD4+ T cells differentiate into Th17 or Tregs cells, respectively. We have defined a role for Ikaros in these cell fate decisions. Ikaros is a site-specific DNA-binding transcription factor involved in the epigenetic regulation of several T cell cytokine genes. We demonstrate that Ikarosnull CD4+ T cells are developmentally impaired in vitro under Th17- skewing conditions. In addition to producing less IL-17, these cells exhibit reduced RORγt and higher Foxp3 expression than their wild type counterparts. Furthermore, all Foxp3+ T cells are also CD25+, suggesting that the lack of Ikaros predisposes Th17 T cells to a regulatory T cell phenotype. The effect of Ikaros on IL-17 production is not limited to CD4+ T cells. Ikarosnull CD8+ T cells also have dramatically lower IL-17 production. However, unlike Ikaros-deficient CD4 T cells, Ikarosnull CD8+ T cells do not express markers of Treg cells but show increased IFNγ production. Thus, our data suggest Ikaros is intimately involved in the differentiation of IL-17-producing T cells.

This research is supported by NIH RO1 NS047578
The mechanism of protection against influenza by transfer of Tc17, an IL-17 secreting CD8 T cell subset. (46.5)
Authors: Hamada Hiromasa , Garcia-Hernandez Maria de la Luz , Reome Joyce B. , Misra Sara K. , Dutton Richard W.
Published: January 2023

Interleukin (IL) -17 secreting CD4 T (Th)17 and CD8 T (Tc)17 effector cells are found in the lung following primary challenge with influenza A and blocking antibody to IL-17 increases weight loss and reduces survival. Tc17 effectors can be generated in vitro using naïve CD8 T cells from OT-1 T cell receptor (TcR) transgenic mice. T cell numbers expand 20 fold and a majority secretes IL-17, but little IFN-γ. Tc17 effectors are negative for granzyme B, for perforin message and lack cytolytic activity in vitro, in contrast to Tc1 effectors. Many of the Tc17 effectors become IFN-γ/IL-17 double producers after adoptive transfer and kill targets by a Fas dependent, perforin independent, pathway in an in vivo CTL assay. Adoptively transferred Tc17 cells expand greatly after transfer to naïve recipients following challenge and can protect them against lethal influenza infection. Transfer of Tc17 effector cell enhances the infiltration of neutrophils and monocytes to lung after influenza infection and protects against the development of respiratory distress much more effectively than transfer of equivalent numbers of Tc1 effectors. Additional mechansims of protection are being explored.

This work was supported by NIH AI046530 and the Trudeau Institute.
The effects of age on CD4 upregulation and Treg distribution in a TAChR peptide-specific immune response (47.1)
Authors: Stacy Sue , Standifer N. E. , Pasquali Amanda , Krolick K. A. , Infante A. J. , Kraig Ellen
Published: January 2023

Studies of age-related defects in T cell function have been complicated by the difficulty of identifying antigen-specific T cells early in the activation process. Thus, peptide-responsive T cells were identified and characterized by the upregulation of CD4, an early event following T cell receptor ligation. We focused on the Torpedo californica acetylcholine receptor (TAChR) peptide, p146-162, since responses to this peptide have been well defined in young mice. In old mice, the fine specificity of the TAChR response was preserved with age; p146-162 remained immunodominant. Yet, in vitro proliferative responses to p146-162 were diminished in a dose-dependent manner in lymph node cells (LNC) derived from old peptide-primed mice. When p146-162-specific Vβ6+CD4high T cells derived from young (2-5 months) and old (20-27 months) mice were examined, the upregulation of CD4 was maintained in old age but the frequency of Vβ6+CD4high T cells was often lower; reflecting fewer CD4+ LNC, changes in TCR repertoire selection and/or altered T cell regulation. Interestingly, Foxp3+CD25+CD4+ T regulatory cell (Treg) frequencies were elevated in the old peptide-responding populations. The Vβ6-CD4normal Treg phenotype predominated in both age groups, yet a unique "activated" CD4high Treg subpopulation was also identified. Future studies may help elucidate the potential role of these CD4high Tregs in promoting age-related immune dysfunction. Support: Myasthenia Gravis Foundation, Nathan Shock Aging Center, NIH R03 AG 14557 and San Antonio Area Foundation.
Curdlan induces Th17 polarization via Jagged1 activation in human dendritic cells (47.12)
Authors: Matsushita Sho , Hashimoto Kumiko , Takagi Rie , Higashi Takehiro
Published: January 2023

Th17-inducing activity is carried by certain polysaccharides such as ƒÀ-glucan derived from Candia albicans. Our previous studies have shown that Th1- and Th2-inducing activities can be qualitatively evaluated by the expression patterns of Notch ligand isoforms, using human monocyte-derived dendritic cells (DCs) and some leukemic cell lines. Thus, the Th1- and Th2-inducing activities correlate with the upregulation of Delta4 and Delta1 mRNA, respectively. However, the association of Th17-inducing activities with Notch ligand expression patterns has been unclear. The present study shows that curdlan, one of ƒÀ-glucans, has the ability to induce DC-mediated Th17 differentiation, by using a mixed lymphocyte reaction (MLR) between human monocyte-derived DCs and allogeneic naïve CD4+ T cells. It is also interesting to note that Jagged1 mRNA in DCs is up-regulated by curdlan. Furthermore, polyclonal anti-Jagged1 antibody inhibited such DC-mediated Th17 differentiation. These findings suggest that curdlan induces human DC-mediated Th17 polarization via Jagged1 activation in DCs.
A method for enrichment and detection of viable human and mouse IL-17-secreting T cells (47.16)
Authors: Assenmacher Mario , Niemoeller Michaela , Foerster Anna , Zyntek Claudia , Winkels Monika , Schmitz Jürgen , Richter Anne
Published: January 2023

IL-17 plays a key role to defend infections, but also in chronic inflammatory disorders. We developed tools for the enrichment and detection of IL-17-producing cells.

After T cell activation IL-17 was detected on the surface of IL-17-producing cells after first labeling the cells with a bispecific CD45-IL-17 antibody affinity matrix. Cells were again cultured for 45 min to allow IL-17 secretion. Secreted IL-17 was specifically caught onto the affinity matrix of IL-17-producing cells. Subsequently IL-17-secreting cells were fluorescently stained with a second IL-17 antibody or additionally labeled with magnetic beads.

Stimulation of mouse spleen cells with PMA/ionomycin showed a maximal IL-17 secretion after 4 h in <0.5% of CD4 and CD8 T cells, but in around 20% g/d T cells and 7% NKT cells. In contrast in human PBMC the IL-17 secretion was mainly restricted to CD4 T cells, with a maximum of 0.1-1% of them producing IL-17 after 4 h activation with the artificial superantigen CytoStim. Magnetic enrichment yields in IL-17+ cell populations with a purity of >90%. Nearly all of the enriched IL-17+ CD4 T cells co-expressed CD154, confirming the specificity of the detection and enrichment method.

The IL-17 secretion assays allow detection of IL-17-secreting cells and provides highly enriched populations of viable IL-17-producing cells to investigate its function and the role of IL-17 in host defense and disease development.
Different by Destruction: Unequal Inheritance of the Transcription Factor T-bet as a Mechanism to Diversify Daughter T Cell Fates (47.29)
Authors: Chang John T , Kim Jiyeon , Kinjyo Ichiko , Yin Catherine , Berg Leslie , Jordan Martha , Koretzky Gary , Reiner Steven L.
Published: January 2023

We have previously suggested that asymmetric cell division might represent a mechanism to ensure that appropriate diversity of cell fate arises from the descendants of a single lymphocyte during an immune response. We now show that the fate-determining transcription factor T-bet is asymmetrically inherited by dividing CD4+ and CD8+ T cells recruited into an immune response. T-bet is induced in interphase T cells within hours of activation. During mitosis, T-bet undergoes proteasome-dependent degradation. Mitotic destruction is mediated by T cell receptor-induced tyrosine phosphorylation of T-bet. Unequal inheritance of T-bet is associated with asymmetric segregation of the proteasomal degradative machinery during mitosis and cytokinesis. Mutations of T-bet at the critical tyrosine and those disabling the T cell receptor-associated kinase, ITK, both result in symmetric inheritance of T-bet without affecting asymmetry of the proteasome. These results suggest that two experimentally distinct mechanisms promote the unequal inheritance of T-bet by initial daughter T cells: one signal that targets T-bet for mitotic destruction and another signal that renders inequality in the inheritance of the cellular machinery that destroys T-bet. These findings offer a new framework for understanding how signaling to a single T lymphocyte can result in unequal fate determination of its daughter cells.
Notch ligand-Dll4 regulated T cell activation and IL17 production (47.30)
Authors: Mukherjee Sumanta , Schaller Matthew A. , Lukacs Nicholas W.
Published: January 2023

The activation and differentiation of T cells are dependent upon numerous initiating events that are influenced by the immune environment, nature of the antigen, as well as the activation state of APCs. The study evaluated how activation by a specific notch ligand, delta-like 4 (DLL4), expressed by dendritic cells relates to the development of specific aspects of T cell differentiation. In particular, our data have indicated that DLL4 is inducible by pathogen-associated signals through TLR activation but not by early response inflammatory cytokines, IL-1 and IL-18 that also activate cells via MyD88 adapter molecules. To investigate the ability of Dll4 to activate T cells, our observations from in vitro specific cultures with ovalbumin peptide specific TCR transgenic cells (DO11.10) confirmed earlier reports that Dll4 inhibits Th2 cytokine production but further indicated that it also influences the generation of IL-17 producing T cells. However additional skewing conditions were necessary to drive IL17 production. In the absence of notch signals IL17 production was significantly inhibited. We also identified that both RORγt and IL17 are direct transcriptional notch targets that further enhance the differentiation of Th17 cell populations. Thus, the regulation of T cell differentiation may depend upon the efficient activation of T cells by specific notch ligand stimulation by novel activation pathways.

Acknowledgement: This work was supported by the NIH RO1-AI073876 grant to Nicholas W. Lukacs.
Mammalian target of rapamycin complex 2 regulates helper T cell differentiation and function (47.31)
Authors: Lee Keunwook , Gudapati Prathyusha , Magnuson Mark , Killeen Nigel , Boothby Mark
Published: January 2023

Mammalian target of rapamycin (mTOR) is involved in cellular processes such as survival and proliferation. Regulation of the original rapamycin-sensitive complex, mTORC1, in signaling downstream from Akt is well defined. However, physiological roles in lymphoid cells of an alternative mTOR complex, mTORC2, are unknown. Using conditional deletion of rictor, an essential subunit of mTORC2, we recently uncovered a rate-limiting role of mTORC2 in T cell development and Akt activation. We now show that mTORC2 regulates helper T cell development and function by a pathway that is either Akt-dependent or largely independent of Akt, depending on the direction of differentiation. Specifically deleting rictor late in T cell ontogeny impaired T cell help to Ab class switching for both Th1- and Th2-regulated isotypes. mTORC2 relayed signaling of helper T cell help differentiation into both Th1 and Th2 effector cells, and proliferation distinct from pro-survival effects. Strikingly, a constitutive active Akt was sufficient to restore normal Th1, but not Th2 differentiation. Overall, our data indicate that mTORC2 plays fundamental roles in cellular differentiation while orchestrating differential signaling relays.
AhR-dependent changes in gene transcription induced by TCDD during naive CD4+ T-cell differentiation (47.5)
Authors: Rohlman Diana C , Pham Duy , Steppan Linda B , Kerkvliet Nancy I
Published: January 2023

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ligand for the AhR and a potent immunosuppressant in vivo, inhibiting Th1, Th2 and Th17 T-cell dependent responses. The AhR is an orphan receptor that functions as a ligand-activated transcription factor upon dimerization with AhR nuclear translocator (ARNT). In this study, AhR-dependent changes in expression of genes associated with T-cell differentiation or containing AhR-ARNT binding sequences were examined. Purified CD4+ T-cells were stimulated under Th0 or Th1 conditions for 24, 36 or 48 hours in the presence of TCDD or vehicle. RNA was isolated for real time PCR. Treatment with TCDD significantly increased the expression of AhR-regulated genes Cyp1a1 and Cyp1b1 under both Th0 and Th1 conditions, in AhR+/+ but not AhR-/- T-cells. Expression of the AhR gene itself was not altered. Under Th0 conditions, expression of Tbet, IL-10, CTLA-4 and IFNy was decreased, while expression of CD69, IL-12rb2, Gata3 and Nfe2l2 was increased. Under Th1 conditions TCDD-treated cells responded with increased expression of IFNy, IL-12rb2 and Tbet, but decreased Stat4. These results support the hypothesis that AhR activation by TCDD influences early CD4+ T-cell differentiation. (Supported by NIEHS Training Grant #T32ES007060)
The tyrosine kinase MerTK negatively regulates the function of murine natural killer T cells (47.7)
Authors: Flores Rafael R. , Wang Bo , Tisch Roland
Published: January 2023

NKT cells have multiple effector functions including the capacity to prevent the growth of tumors, and the ability to immunoregulate autoimmunity. The key events that regulate NKT cell activation and function remain ill-defined. Recently, NKT cells were shown to express the receptor tyrosine kinase (RTK) Mer (MerTK). MerTK belongs to the Tyro3 RTK family. These RTK mediate the clearance of apoptotic cells (AC) and regulate the activation of dendritic cells (DC) and macrophages. Recently, we demonstrated that DC lacking MerTK are resistant to the inhibitory effects of AC. In this study, we sought to determine the role for MerTK in regulating NKT cell activation and function. To address this issue C57BL/6 mice (B6) lacking MerTK expression (B6.mertkkd/kd) were employed. Our data demonstrates that unprimed B6.mertkkd/kd NKT cells exhibit a heightened level of activation in comparison to wild-type NKT cells, while long-term exposure to alpha-GalCer (αGC) activated both sets of NKT cells similarly. Previously primed B6.mertkkd/kd NKT cells secreted cytokines upon subsequent stimulation with αGC, whereas wild-type NKT cells failed to be activated and remained anergized. In addition, stimulation with αGC pulsed DC, IFNγ and IL-4 production was significantly increased in NKT cells prepared from B6.mertkkd/kd versus B6 mice. These results indicate that MerTK serves to down-regulate cytokine production by NKT cells.