bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tchreg cells, a novel type of Treg cells possessing multifunctional activity and CD4+CD8+ phenotype, are developed as HOZOT cell lines by three-step coculture system from cord blood cells. (49.8)
Authors: Toraya Terumasa , Otani Takeshi , Okochi Ayumi , Sugimoto Akira , Suzuki Motoyuki , Harashima Akira , Inoue Toshiya , Tsuji-Takayama Kazue , Yamamoto Mayuko , Takeuchi Makoto , Yamasaki Fumiyuki , Nakamura Shuji , Kibata Masayoshi 
Published: January 2023

Abstract
Regulatory T cells (Tregs) play important roles in balancing immune responses in many aspects. However, most studies of Tregs have been focused on a type of CD4+FOXP3+ Tregs. Previously, we have reported another type of human Treg cell line, HOZOT, which was generated from cord blood cells. Intriguingly, HOZOT comprises of CD4+CD8+, CD4+CD8dim and CD4-CD8+ subpopulations and exhibits not only suppressive activity in allogeneic MLR but also cytotoxic activity against tumor cell lines and, moreover, helper activity under anti-CD3 stimulation conditions. Therefore, we have designated such Tregs as “Tchreg”, cytotoxic, helper and regulatory T cells. Multifunctional property and CD4+CD8+ phenotype of HOZOT are induced during coculture with mouse stromal cells. To address underlying mechanisms of Tchreg generation, we developed a reproducible method suitable for mechanistic analysis. This culture method consists of three steps. 1) Purified T cells were first cocultured on a stromal cell layer alone without any cytokines. A week later, activated T cells emerged concurrently killing stromal cells. 2) These activated T cells were cultivated on a stromal cell layer together with autologous monocytes. 3) Blast T cells were further expanded on a stromal layer in the presence of IL-2. This method, termed three-step coculture, usually results in over 3000-fold expansion. By exploiting this method, we have gained new insights into Tchreg cell biology.
The role of p53 in antigen-specific and bystander T cell proliferative responses (50.15)
Authors: Watanabe Masashi , Hathcock Karen , Vacchio Melanie , Moon Kyungduk , Hodes Richard 
Published: January 2023

Abstract
p53 is a tumor suppressor that triggers cell cycle arrest or apoptosis in response to DNA damage or oncogenic signals. The role of p53 in regulating survival and proliferation of normal somatic cells such as T cells, which undergo extensive proliferation, has not been extensively characterized. We observed that the number of peripheral memory phenotype (CD44hi) CD4 T cells is increased in p53 KO mice, suggesting that p53 might have a role in regulating this population. To investigate the role of p53 in regulating T cell activation and proliferation, we analyzed KLH-specific CD4 T cell clones from p53 KO and wild-type mice. WT and p53 KO clones showed similar antigen-specific proliferative response to stimulation with KLH/APC + IL-2. In contrast, p53 KO clones proliferated extensively in response to cytokine in the absence of KLH/APC, while WT clones did not respond to cytokines alone. These results suggested that p53 might prevent antigen-nonspecific (bystander) responses to cytokine in the absence of concomitant antigen-specific signaling. In vitro experiments demonstrated that wild-type KLH-specific CD4 T cell clones do not proliferate as bystanders in co-culture with responding OVA-specific T cells. In contrast, p53 KO clones exhibit extensive bystander proliferation, indicating a role of p53 in preventing antigen-non-specific bystander proliferation. We are currently investigating molecular mechanisms mediating p53 regulation of responses to cytokine signaling.
Maintaining CD28 expression prevents replicative senescence in human CD 8 T cells (50.23)
Authors: Parish Stan , Wu Jennifer , Effros Rita 
Published: January 2023

Abstract
The irreversible loss of expression of the CD28 costimulatory receptor is one of the signature changes in human T cells that are driven to the end stage of replicative senescence by chronic stimulation in cell culture. Besides activation, CD28 is also required for glucose metabolism, stabilization of various cytokine mRNAs, and, importantly, for optimal upregulation of telomerase, the enzyme that stabilizes telomeres. Increased proportions of senescent CD8 T cells are associated with decreased vaccine responsiveness and early mortality in the elderly, and, during HIV disease, with more rapid progression to AIDS. Previous work showed that inhibition of TNFα, which allowed for prolonged CD28 expression, delayed the onset of senescence. In the present study, CD8 T cells were stably transduced with CD28, and compared to empty-vector transduced control cultures. Maintaining continuous CD28 expression led to the significantly increased proliferative potential and sustained telomerase activity. Furthermore, IL-2 and IFNγ message levels were increased, and TNFα and IL-6, cytokines that are significantly increased at replicative senescence, were undetectable by ELISA at 25 population doublings, the time-point at which the control cultures reached senescence. These data underscore the central role of CD28 in memory CD8 T cell biology, and suggest a possible new therapeutic approach for enhancing immune function during aging and AIDS. (Supported by NIH AG023720 & AI060362).
A mutational analysis of CD28 utilizing targeted knock-in mice (50.24)
Authors: Boomer Jonathan , Deppong Christine , Dodson Lindzy , Shah Dulari , Bricker Traci , Green Jonathan 
Published: January 2023

Abstract
T cell activation requires signaling through the TcR and engagement of a costimulatory receptor. CD28 is the most important costimulatory receptor; however, the signaling pathways initiated by CD28 remain highly controversial. To examine this, we generated gene targeted knock-in mice that express mutations in either the 1) proximal YMNM (Y170F), 2) distal PYAP (AYAA), or 2) both the YMNM and PYAP (Y170F/AYAA) motifs of the cytoplasmic tail of CD28. Mutation of the PYAP motif resulted in impaired proliferation, cytokine secretion and B cell help. In contrast, the Y170F mutant had normal to slightly increased proliferation and IL-2 production in vitro, as well as a prolonged duration of inflammation in vivo. T cells from both mutant knock-in mice upregulated the activation markers CD25, CD69 and CD95. Interestingly, the CD28 knock-out and CD28-AYAA knock-in mice both fail to induce the expression of ICOS on T cells, a critical component for germinal center production. To determine if additional motifs mediate CD28-dependent functions, we tested the responses of the double mutant. The phenotype of the Y170F/AYAA double mutant was not substantially different than the single AYAA mutant. These data strongly suggest that the residual T cell responses present in the AYAA mouse are not due to signaling initiated by the YMNM motif, and that additional signaling pathways mediate the remaining CD28-dependent effects.
On the mechanisms of sensitivity and efficiency of cytolytic effectors (50.3)
Authors: Sykulev Yuri 
Published: January 2023

Abstract
Successful elimination of virus infected and cancer cells requires CTL to exercise highly sensitive and efficient cytolytic activity. There are two principal mechanisms responsible for the sensitivity and the efficiency of cytolysis. The sensitivity is determined by focused delivery of cytolytic granules to the secretory domain located within the cSMAC of IS. The MTOC polarization to the synaptic interface is a critical event of the granule delivery and is mediated by the ADAP-associated dynein motors attached to the pSMAC. A stable pSMAC also responsible for the confinement of released granules at the CTL/target cell interface that enables killing with a minimal amount of released granules. Thus, segregation on adhesion molecules and the pSMAC formation play an important role in the mechanism controlling the sensitivity of cytolysis. Another essential quality of the CTL-mediated cytolysis is the kinetics of target cell destruction that reflects efficiency of the cytolytic activity. The efficiency is regulated by the kinetics of the granule delivery to the secretory domain that occurs via “short” or “long” paths. The choice of the path is determined by the kinetics of the TCR early signaling, but not by the magnitude of the signaling. Thus, variations in the TCR signaling kinetics constitute the mechanism controlling the efficiency of the cytolytic activity. Whether similar mechanisms are utilized by other cytolytic effectors such as NK cells remains will be discussed.
Intrinsic IL-21 signaling is critical for CD8 T cell memory formation in response to Vaccinia viral infection (50.32)
Authors: Novy Patricia , Yang Yiping 
Published: January 2023

Abstract
The role of CD4 T cell help in primary and memory CD8 T cell responses to infectious pathogens remains incompletely defined. We showed previously that CD4 help provided during both the primary and secondary responses to vaccinia virus (VV) is required for the survival of activated CD8 T cells. Still unclear however, is the mechanism of CD4 help during the response to VV infection. Here we demonstrate that CD4 help is neither dependent on dendritic cell (DC) priming nor is contact dependent, indicating that CD4 help may be dependent on a soluble mediator. We determined that in vitro CD4 help for CD8 survival is dependent on IL-21, a cytokine produced predominantly by activated CD4 T cells. Furthermore, IL-21 signaling is required for the survival of activated CD8 T cells in vivo and CD8 T cells deficient in IL-21 signaling failed to develop a memory pool. In addition, we observed that IL-21 signaling in CD8 T cells can activate the STAT1 and STAT3 signaling pathways, both of which have been correlated to CD8 survival. Our study shows that direct IL-21 signaling is required for the formation of memory CD8 cells in response to VV and indicates that CD4-help may be mediated by IL-21 during VV infection. This suggests that differential IL-21 requirements during different pathogen infections may dictate the necessity of CD4 help in the primary CD8 response.
The TCR CD3 ζ subunit contains a charged inositol phospholipid-binding sequence that supports T cell interactions with antigen presenting cells (50.4)
Authors: van Oers Nicolai , DeFord-Watts Laura , Dougall David , Johnson Blake , Eitson Jennifer , Yan Li , Wuelfing Christoph 
Published: January 2023

Abstract
TCR-mediated intracellular signal transmission commences following the tyrosine phosphorylation of the CD3-encoded ITAMs. The CD3 ζ subunit, containing three ITAMs per chain, becomes one of the more heavily phosphorylated of the CD3 chains. The cytoplasmic tail of CD3 ζ contains multiple clusters of basic amino acids lysine and arginine. A number of transmembrane and cytosolic proteins, including the CD3 ϵ subunit, use these types of clusters to bind phospholipids. We report that the cytoplasmic tail of CD3 ζ contains a phospholipid-binding cluster with a relatively high affinity for PtdIns(3,5)P2, PtdIns(5)P, PtdIns(3)P, and PtdIns(4)P. The elimination of the phospholipid-binding function of CD3 ζ reduced the stability of TCR clustering at the immunological synapse. The function of this domain in TCR-mediated intracellular signaling responses will be described. These findings demonstrate a novel functional role for the CD3 ζ lipid-binding domain in T cell biology.
Activated NKT cells are depleted specifically in the liver and utilize different mechanisms for expansion that depend on TCR dependent vs independent mechanisms of activation (50.41)
Authors: Subleski Jeff , Hall Veronica , Wolfe Thomas , Scarzello Anthony , Weiss Jonathan , Chan Tim , Hodge Deborah , Back Timothy , Ortaldo John , Wiltrout Robert 
Published: January 2023

Abstract
Treatment of mice bearing implanted liver tumors with IL-18 plus IL-12 (IL-18+IL-12) elicited greater anti-tumor activity than IL-18 or IL-12 delivered as single agents. We found IL-18+IL-12-mediated anti-tumor activity was dependent on the reduction of hepatic NKT cells. Here we examine mechanisms regulating NKT cell modulation following TCR-dependent and independent activation using αGalCer or IL-18+IL-12 treatment of mice, respectively. We found acute treatment of mice with IL-18+IL-12 or αGalCer initially (24h) decreased NKT cells in the liver, but not in other lymphoid tissues. Specifically, treatment with αGalCer completely depleted the liver of NKT cells, while treatment with IL-18+IL-12 only depleted NK1.1(+) NKT cells. Following a loss of NKT cells at 24h with IL-18+IL-12 or αGalCer treatment, these cells reappear and expand by 72h. BrdU proliferation analysis revealed that IL-18+IL-12 and αGalCer expands the NK1.1(-) NKT cell subset in the liver. However, while NKT cell expansion induced with αGalCer requires an intact spleen and IFNγ, IL-18+IL-12-induced expansion does not. Finally, mice treated chronically with IL-18+IL-12 or αGalCer resulted in systemic NKT exhaustion which required an intact thymus for NKT cell repopulation of the liver. By characterizing the early response by NKT cells to various stimuli, these insights could better aid in understanding the role(s) NKT cells play in the control or treatment of infectious diseases, autoimmunity or cancer.
Novel Gene Expression Patterns in IFN-γ 3’Untranslated Region AU-Rich Element-Deleted Mice (51.7)
Authors: Young Howard , Hodge Deborah , Berthet Cyril , Subleski Jeff , Coppola Vincenzo , Schaughency Paul , Razzook Catherine , Buschman Matthew 
Published: January 2023

Abstract
Much research has focused on transcriptional control of interferon-gamma (IFN-γ); however, little is known about post-transcriptional control of this cytokine. Our studies focus on a highly conserved AU-rich region in the 3’untranslated region (UTR) of the IFN-γ mRNA. To study the impact of this sequence on IFN-γ gene expression and immune function, we created an IFN-γ 3’UTR ARE-deleted mouse. ARE elimination results in IFN-γ mRNA stabilization and chronic low levels of circulating IFN-γ protein that are not present in control mice. Upon stimulation, the ARE-deleted mice produce 3-5-fold more IFN-γ than wild-type mice, supporting the model that the ARE-region functions as a negative modulator of IFN-γ expression. Next, we conducted PCR array and Nanostring direct multiplexed assays on RNA from bone marrow, kidney, liver, lymph node, spleen, and thymus from ARE-deleted and WT mice. As expected, mRNA for IFN-γ and IFN-γ-responsive genes, CXCL9 and CXCL10, were elevated in all tissues from ARE-deleted mice. IFN-γ-induced tissue-specific gene expression patterns were associated with a variety of immune functions including chemotaxis, inflammation, Th1/Th2 development and B cell proliferation/antibody production. These results indicate that small changes in IFN-γ expression have far reaching effects on immune development and function. Thus, this mouse serves as a unique model for understanding the systems biology consequences of chronic IFN-γ expression.
B cell and peptide based immunotherapy vaccine for Respiratory Syncytial Virus (RSV) infection (52.16)
Authors: Lee Sujin , Currier Michael , Celis Esteban , Moore Martin 
Published: January 2023

Abstract
RSV is the most important pathogen for lower respiratory tract illness (LRI) in infants. There is no vaccine in use. As elicitation of antigen-specific CD8 T cell plays an important role in immune protection against many viruses, we sought to induce robust RSV-specific CD8 T cell immune responses. Ex vivo-activated B cells are an abundant source of antigen-presenting cells for immunotherapy. In the present study, we applied a novel approach using a combination of ex vivo-activated B cell prime and peptide boost to develop RSV vaccine, named as B-TriVax. TriVax uses a synthetic peptide representing a dominant RSV CD8 T cell epitope (M2 82-90), a toll-like receptor agonist (Poly-IC), and a costimulatory anti-CD40 antibody to generate large numbers of RSV-specific T cells. We primed BALB/c mice with M282-90-pulsed CD19+ B cells and boosted with TriVax at different time points after priming. We generated &gt;20% M282-90-specific systemic CD8 T immune responses. Currently, we are investigating M282-90-specific CD8 T cell mediated vaccine efficacy in a RSV challenge model.
A cationic lipid/DNA adjuvant (JVRS-100) dramatically improves the immunogenicity and protection after immunization of elderly rhesus monkeys with a licensed inactivated seasonal influenza vaccine (Fluzone®). (52.21)
Authors: Carroll Tmothy , Matzinger Shannon , Lay Marla , Chang Stella , Fairman Jeffery , Miller Christopher 
Published: January 2023

Abstract
To determine if an adjuvant can improve the efficacy of an inactivated seasonal influenza vaccine in immunosenescent primates, elderly rhesus monkeys were vaccinated with Fluzone. In a subset of the animals, Fluzone was mixed with a cationic lipid/DNA adjuvant (JVRS-100). After influenza A/Memphis/7/2001 (H1N1) challenge, the 9 monkeys vaccinated with Fluzone alone had no detectable reduction in viral shedding in tracheobronchial secretions relative to the 9 non-immunized control animals. However, the 9 animals immunized with JVRS-100 and Fluzone had a 4-fold lower peak level of virus in tracheobronchial secretions compared to unimmunized control animals (p&lt;0.05) and Fluzone-only immunized monkeys (p&lt;0.01). The cumulative level of viral replication as determined by an area under curve analysis were 7-8 fold lower (p&lt;0.05) in animals immunized with JVRS-100 and Fluzone compared to both Fluzone-only and non-immunized monkeys. Moreover, monkeys immunized with JVRS-100 and Fluzone had no evidence of fever or weight loss whereas both signs of illness were evident in the non-immunized and Fluzone-only animals in the first week after challenge. Thus, addition of JVRS-100 to Fluzone generates protective immunity that blunts virus replication and eliminates the clinical signs seen in the elderly monkeys that are not protected by Fluzone alone.
T-cell responses in mouse-adapted SARS-CoV infected mice (52.22)
Authors: Zhao Jincun , Perlman Stanley 
Published: January 2023

Abstract
It is generally believed that a dysregulated innate immune response and exuberant cytokine/chemokine expression contribute to the pathogenesis of Severe Acute Respiratory Syndrome. However, we recently showed inefficient immune activation and a poor virus-specific T cell response underly severe disease SARS-CoV infected mice. Here, we extend these results to show that virus-specific T cells, in the absence of activation of the innate immune response, were sufficient to significantly enhance survival and diminish clinical disease. We demonstrated that T cells are responsible for virus clearance as intravenous adoptive transfer of immune splenocytes to SCID-BALB/c mice prior to infection reduced virus titers in the lung. Further, transfer of splenocytes or lung-derived cells from SARS-CoV-infected mice or of in vitro generated CD8 T cells protected mice from lethal infection. Enhancement of the number of virus-specific T cells using SARS-CoV-specific peptide-pulsed dendritic cells prior infection also resulted in a robust T cell response, earlier virus clearance and 100% survival. These studies are the first to show that T cells play a crucial role in SARS-CoV clearance and that a suboptimal T cell response contributes to the pathological changes observed in SARS. They also provide a new approach to SARS vaccine design.
UNDERSTANDING IMMUNE PROTECTION MECHANISMS OF AN INFLUENZA A VIRUS M2 PEPTIDE VACCINE (52.3)
Authors: Fu Tong-Ming 
Published: January 2023

Abstract
M2 peptide is under clinical investigation as a target for universal flu vaccines. Studies have shown that Abs induced by M2 vaccines can provide cross-strain protection against flu A virus challenge. Because of the high level expression of M2 protein on the cell surface of flu virus infected cells and minimal neutralizing activities of M2 Abs in vitro, Ab-mediated cytotoxicity by NK cells has been implicated as a protection mechanism by M2 peptide vaccines. However, the studies were mostly based on the observations using M2 immune sera. Thus, to better understand immune protection mechanisms by M2 vaccines, we generated 4 mAbs. Although all mAbs showed little antiviral activity in vitro, adoptive transfer of 2 of 4 mAbs could confer protection against lethal flu A virus challenge in naïve mice. More importantly, the protection was not affected by depletion of NK cells in mice. Further characterizations revealed that the 2 protective mAbs recognize core epitopes located at the N-terminal 10 amino acids of M2 peptide, different from those reported previously, such as clone 14C2. Interestingly, both protective mAbs preferentially reacted with M2 peptides in multimeric form and in parallel orientation, while non protective mAb showed no such substrate preference. Our findings dispute the notion that M2 vaccine-induced protection is medicated by NK cells, and clearly show that the epitopes recognized by protective M2 mAbs are complex and conformational.
Designing a plague vaccine utilizing α-gal modified antigens (52.6)
Authors: Phanse Yashdeep , Carrillo-Conde Brenda , Huntimer Lucas , Ramer-Tait Amanda , Bellaire Bryan , Narasimhan Balaji , Flick Ramon , Mandell Robert , Wannemuehler Michael 
Published: January 2023

Abstract
Background: F1-V from Yersinia pestis has been shown to be a protective antigen against disease. We modified the F1-V antigen with α-galactose residues and immunized α galactosyl transferase (α-GT) KO mice in order to mimic the same defect in humans that results in circulating antibodies against α-gal epitopes enhancing immune responsiveness. Aim: To demonstrate that α-gal modified F1-V induces a more robust immune response than unmodified F1-V. Methods: α-GT KO mice were injected subcutaneously with different doses of α-gal-F1-V or F1-V. All mice were boosted with F1-V at 36 dpi and necropsied on day 40 in order to evaluate in vitro recall response of lymphocytes and to measure anti-F1-V antibody titers. Results: Mean IgG titer values were 50,000 and 10,000 for mice immunized with the α-gal-F1-V or F1-V, respectively. The relative avidity of the antibody response was also higher for the α-gal-F1-V immunized mice. Moreover, lymphocytes from α-gal-F1-V immunized mice demonstrated enhanced proliferation in response to F1-V compared to cells from F1-V immunized mice. Conclusions: α-gal modified F1-V increases both the avidity and amount of anti-F1-V produced following vaccination. As a functional assay, α-gal-F1-V antisera efficiently opsonized Y. pestis, facilitated phagocytosis by human monocytes and prevented Y. pestis-induced monocyte cytotoxicity.