bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

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

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

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

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

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 >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

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<0.05) and Fluzone-only immunized monkeys (p<0.01). The cumulative level of viral replication as determined by an area under curve analysis were 7-8 fold lower (p<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

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

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

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.
Regulation of hematopoietic cell subsets in vivo following treatment with an anti-CD200 antibody in a murine autoimmune hemolytic disease (AIHD) model (83.12)
Authors: Yan Yan , Mickle Angela , Michalowski Christian , Hunter Jeffrey , Johnson Krista , Faas Susan
Published: January 2023

CD200 is an immunoregulatory protein, highly expressed on activated T and B cells and on various liquid and solid tumors. We have previously shown that treatment with an antagonist anti-CD200 antibody in vivo enhances anti-tumor immunity in a hematologic cancer model. The present studies evaluated whether in vivo anti-CD200 treatment modulates hematopoietic cell subsets and allo- and auto- antibody responses in an AIHD model. Mice were immunized weekly with rat red blood cells (RBC) to prompt robust anti-rat RBC allo- and cross-reactive auto- antibody responses. Immunized mice were randomized into groups receiving either vehicle alone, an anti-CD200 antibody (OX90mG2a), a non-reactive, isotype-matched control antibody (12B4mG2a), cyclosporine (CsA), or CsA plus OX90mG2a or 12B4G2a antibodies. Allo- and auto- antibodies were measured prior to and at the conclusion of treatment. Administration of OX90mG2a with or without CsA significantly impacted CD200+ cell subsets in treated mice. Decreased frequencies of CD200+ T cells, B cells, plasma cells and progenitor bone marrow cells were observed in OX90mG2a-treated mice relative to control mice. All CD200+ populations returned to normal levels after termination of anti-CD200 treatment. OX90mG2a administration also inhibited allo- and auto- antibody production in this model. These data suggest that anti-CD200 antibody therapy is a potent immunomodulator and may provide a therapeutic strategy for antibody-mediated diseases.
Sex hormone-independent capacities of female hematopoietic cells from lupus-prone mice (83.23)
Authors: Jorgensen Trine , David Alexandria , MacLeod Megan , Johnson Angela , Kappler John , Marrack Philippa
Published: January 2023

Many systemic autoimmune diseases are more commonly found in females than in males. This is evident in Systemic Lupus Erythromatosis (SLE), a prototypic systemic autoimmune disorder, where the ratio of females to males is 9:1. Our current understanding of the etiology of SLE implies important roles for genes and environmental factors such as sex hormones. Analyses of mouse models of SLE have proven good models for both; however, the relative importance of genes versus sex hormones is still largely unresolved. Using the (NZB x NZW)F1 mouse model system of SLE we have created a system in which we can segregate the effects of genes and hormones on the immune system in vivo. Briefly, male or female (NZB x NZW)F1 mice were lethally irradiated and reconstituted by age-matched male or female bone marrow (BM) or fetal liver (FL) cells. The resulting chimera mice were followed for the development of lupus-like disease. We found that female BM and FL cells express an intrinsic capacity to drive accelerated lupus-like disease development even in the presence of male pubertal sex hormones. Interestingly, chimera mice receiving female cells also presented with increased serum cytokine and immunoglobulin levels, suggesting that activation of both T and B cells happened more easily in female than male cells. These data represent a new system in which to investigate intrinsic differences between male and female immune mechanisms during the development of systemic autoimmunity.
Cathelin-Related Antimicrobial Peptide (CRAMP) regulates B cell IgG1 production (84.13)
Authors: Chen Yao , Kin Nicholas , Kearney John
Published: January 2023

CRAMP belongs to the cathelicidin gene family, which has direct antimicrobial activity against a broad spectrum of microorganisms. Previous studies have shown that CRAMP also modifies the local inflammatory response and regulates cellular and adaptive immunity. However, a possible direct effect of CRAMP on B cells has not been studied. Using CRAMP-deficient mice, we investigated whether B cells express and respond to CRAMP. We show that all mouse B cell subsets tested, including marginal zone, follicular, B1a, and B1b cells, produced CRAMP mRNA directly ex vivo and up-regulate CRAMP upon activation. Purified CRAMP-deficient B cells produced less IgG1 in response to CD40L or LPS plus IL-4 when compared to WT B cells, and the addition of exogenous CRAMP restored the level of IgG1. CRAMP-deficiency had no effect on proliferation, survival, or class switch recombination, but resulted in a decrease in the amount of mature IgG1 mRNA and single cell IgG1 production. Surprisingly, immunization with TNP-OVA with alum resulted in an enhanced TNP-specific IgG1 memory response in CRAMP-deficient mice compared to WT B6 mice. ELISpot and PCR analysis revealed increased numbers of TNP-specific IgG1-secreting B cells and increased CD4+ T cell IL-4 expression in CRAMP-deficient mice. Taken together these results show that B cells express and respond to CRAMP positively in vitro while in vivo CRAMP appears to also indirectly alter B cells through modulation of T cell cytokine production.
The CD4 memory T cell repertoire specific for a hemagglutinin influenza A virus peptide in DR1 individuals is clonotypically diverse (85.5)
Authors: Tutaj Monika , Gorski Jack
Published: January 2023

Human memory T cells are important for protection against recurrent pathogens. Complex memory T cell repertoires may be indicative of cross-protection against new influenza strains. Unfortunately, the complexity of memory repertoires and the extent to which complex repertoires are the norm in humans is largely unknown. This lack of understanding limits our ability to optimize vaccine strategies. Here, we investigated the CD4 T cell recall response to an HA peptide, conserved in most H3 flu strains, in three middle aged and three older healthy HLA-DR1 blood donors. The HA307-319 peptide was used to stimulate peripheral T cells in short term culture and the repertoires studied by using TCR CDR3β spectratyping and clonotyping. We sorted multimer-labeled and activated CD4 T cells and assessed the advantage of using both methods in repertoire analysis. We found that HA-specific clonotypes used as many as 18 TCR BV families and all BJ genes. The most prominent and shared responses were observed in TCR BV9, 25 and 28 families. Analysis of four thousand sequences revealed essential structural similarities among different individuals including public clonotypes, conserved CDR3 sequences embedded within different BV-J combinations, and several CDR3 amino acid motifs. This study describes the complexity of antigen-specific CD4 T cell response and indicates that only broad population studies can provide a comprehensive knowledge of memory repertoire selection in influenza infection.
Unique populations of lung mast cells are required for antigen-mediated bronchoconstriction (86.11)
Authors: Cyphert Jaime , Kovarova Martina , Koller Beverly
Published: January 2023

Several studies implicate mast cells as important effector cells of anaphylaxis. Much of our understanding of the functions of mast cells emanates from the study of mice lacking theses cells, particularly mice carrying mutations in c-Kit. Definitive evidence for the role of mast cells in these mice requires the normalization of altered immune responses by reconstitution with cultured mast cells (BMMC). While many niches can be restored with BMMC, this has not been demonstrated for mast cells present in the airways of the lung, cells poised to mediate bronchoconstriction during allergic responses. To determine if mast cell reconstitution is an appropriate model for the study of bronchoconstriction associated with allergic responses, KitWsh/Wsh mice were engrafted with either whole bone marrow (WBM) or BMMC and responses to IgE-mediated anaphylaxis determined. Engraftment of KitWsh/Wsh mice with WBM or BMMC results in reconstitution of the central airways with mast cells. While the treatment of both groups results similar systemic changes in a model of passive anaphylaxis, bronchoconstriction was observed only in animals which received WBM. While BMMC can populate the lung, they cannot restore IgE/Ag-mediated bronchoconstriction to mast cell-deficient animals. This suggests the mast cell population mediating this function may be unique, and to fill this niche in the lung cells must undergo a specific developmental program that is no longer available to cultured mast cells.
Expression of the human basophil (BASO) activation marker, CD63, may reflect the extent of degranulation by the AND (anaphylactic degranulation) mechanism. (86.24)
Authors: MacGlashan Donald
Published: January 2023

A variety of studies of suggested that expression of two commonly studied BASO activation markers, CD203c and CD63 (a protein associated with the granule membrane), do not always reflect the extent of histamine release (HR). While exploring the role of PKC in the expression of CD63 and CD203c using the PKC inhibitors Ro-31-8220 (RO) or bisindoylmaleimide II (BIS), it was noted that at concentrations (> 3µM) well above the IC50 for inhibition of PKC (100 nM), there was marked enhancement of CD63 expression with only modest enhancement of CD203c expression or HR. Previous studies have shown that BASO either shuttle granule contents out of the cell via small vesicles (piecemeal degranulation, PMD) or by fusion of granules to each other and the plasma membrane (anaphylactic degranulation, AND), these results suggested the hypothesis that high concentrations of RO or BIS induced a switch from PMD to AND. Morphological imaging of stimulated BASO showed a dramatic increase in the appearance of the degranulation sacs associated with AND in the presence of 3 µM RO. While the cytosolic calcium (Ca) response and degranulation sac formation were correlated, 3 µM RO did not alter the Ca response. Therefore, it was proposed that two processes determine the extent of PMD vs. AND, Ca and an unknown RO-sensitive kinase. It is also proposed that CD63 expression occurs predominantly with AND and that the relative amounts of PMD and AND determine the relationship between CD63 expression and HR.
Estrogen regulates the expression and activity of serine proteases in mouse splenocytes (87.11)
Authors: Dai Rujuan , Khan Deena , Ahmed S. Ansar
Published: January 2023

In addition to digesting proteins, serine proteases also play critical roles in regulation of inflammatory responses, innate and adaptive immunity via proteolytic cleavage of protease-activated receptors (PARs) and/or signaling molecules such as NF-κB. Dysregulation of serine proteases activity may result in the development of variety of human diseases. We and others have shown that estrogen, an important immune modulator, promoted the production of proinflammatory molecules in different types of cell including murine splenocytes and peritoneal macrophages. Our recent report has demonstrated that serine proteases mediated proteolysis play a critical role in regulation of STAT1 and NF-κB activity in splenocytes from estrogen-treated mice. Inhibition of serine protease activity decreased the production of inflammatory molecules IFNγ and nitric oxide in splenocytes from estrogen-treated mice. In this study, we found that estrogen treatment increased the serine proteases activity in splenocytes. Further, we confirmed that neutrophils-derived elastase mRNA expression level is increased, and serine protease inhibitor SerpinB7 is decreased in estrogen-treated splenocytes when compared to placebo control. Together, our data suggest a novel mechanism underlying estrogen-mediated promotion of inflammatory responses. These studies may form the basis for developing new alternative therapies for estrogen-influenced autoimmune diseases by intervening the activity of serine proteases.
Innate-adaptive interactions provide stability in the homeostatic lung environment (87.16)
Authors: Cavanagh Mary , Hussell Tracy
Published: January 2023

The air is full of innocuous, but highly antigenic particles that are inhaled with every breath. In order to avoid the damage associated with an inflammatory response, lung-resident immune cells are kept under strict regulation and fail to respond to most inhaled antigens. Whilst the majority of cells in the airspaces are alveolar macrophages, many other cell types are present in the airspaces and interstitium. Interactions between lung-resident cells provide the basis for the suppression of immune responses; and the cytokines, chemokines and other factors that they secrete provide further levels of regulation. Using sphingosine-1-phosphate receptor agonists to block the entry of T cells into the lungs, we have been investigating the effects of T cell absence on lung immune homeostasis. FTY720 blocks lymphocyte egress from the thymus and lymph nodes by inducing internalisation of the S1P1 receptor which is required for exit into the blood and lymph. We find that exclusion of T cells from the lungs affects macrophage responses to TLR agonists, both in vitro and in vivo as a result of altered alveolar macrophage activation status. By dissecting the mechanisms involved in maintaining lung homeostasis and resolving inflammation, we hope to further our understanding of lung immunity both in terms of homeostatic disruption and immunopathology during or following infectious disease.
Timing of corticosterone treatment and its effects on the CD8+ T cell response to cell-associated antigen (87.18)
Authors: Yorty Jodi
Published: January 2023

Exposure of T cells to glucocorticoids alters their normal program of activation and expansion. In vitro work, often with the synthetic analog, dexamethasone, demonstrated a direct effect of glucocorticoids on T cell proliferation after antigen exposure. In vivo studies have also often utilized dexamethasone. In the current study we evaluated the effect of in vivo administration of the natural glucocorticoid, corticosterone (CORT), on the CD8+ T cell response to SV40 large T antigen (Tag)-expressing cells. C57BL/6 mice respond with a hierarchical CD8+ T cell response to Tag in which epitope IV (amino acids 404-411) is dominant and epitope I (206-215) is subdominant. Exposure to CORT over the course of the immune response to Tag, decreased the number of splenocytes by ~83%. The CD8+ T cell response to epitope I and epitope IV was almost completely abolished during the primary response. Tag-specific memory T cells were also severely affected by CORT treatment. Even when CORT was provided after cross-priming had occurred, the primary T cell response to Tag was still largely inhibited. The ratio of epitope I:epitope IV-specific CD8+ T cells was slightly increased in CORT-treated mice, suggesting that epitope IV-specific T cells may be more susceptible to CORT. Further studies will provide insight regarding when CORT exposure is most detrimental to CD8+ T cell responses that rely on both cross- and direct presentation to achieve maximal expansion.
In vitro characterization of novel anti-human CD27 mAbs (87.23)
Authors: Ramakrishna Venky , Vitale Laura , Sundarapandiyan Karuna , Zhao Biwei , Crocker Andrea , Widger Jenifer , O'Neill Tom , Storey James , Marsh Henry , Keler Tibor
Published: January 2023

CD27, a member of the tumor necrosis factor receptor superfamily (TNFRSF7), is constitutively expressed on the majority of mature T cells, memory B cells, and a portion of NK cells. The interaction of CD27 with its ligand CD70 (CD27L) is important in T cell activation, proliferation, survival, maturation of effector and memory functions, in clonal B cell expansion and germinal center formation and in NK-dependent cytotoxicity. To explore the therapeutic potential of this target, a panel of fully human antibodies recognizing human CD27 was generated using human Ig transgenic mice immunized with recombinant human CD27. These human anti-CD27 mAbs showed specific and high affinity binding to CD27. The antibodies were sub-grouped into those that effectively compete with the binding of CD70 to CD27, and non-blocking antibodies. Several of the anti-CD27 mAbs tested induced pro-inflammatory cytokine and chemokine responses in human whole blood or PBMCs, which could be beneficial in the expansion of Th1 T cell compartment. In addition, some anti-CD27-mAbs mediated in vitro killing of CD27-expressing lymphoma cell lines by ADCC or CDC. The ultimate selection of CD27-activating and CD70-blocking mAbs could potentially complement and amplify existing therapeutic interventions in cancer.
Fucosylation regulates cell death in rheumatoid arthritis (87.24)
Authors: Li Jun , Hsu Hui-Chen , Yang Ping-Ar , Wu Qi , Mountz John
Published: January 2023

Abnormal inflammation and apoptosis are major pathogenic features of synovial fibroblasts (SF) in rheumatoid arthritis (RA). Fucosylation, an important type of glycosylation, has been identified in cancer and inflammation conditions. The role of fucosylation in RA was investigated. Real-time PCR indicated that between RASF and osteoarthritis (OA) SF (N=14 each), there was no difference in the expression of apoptosis related molecules, including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), its receptors DR4, DR5, DR6, DcR1, DcR2, and regulators of apoptosis FLIPL and cIAPs. However, there was a higher level of α1-2-Fucosyltransferase (Fut1, P=0.016), α1-3/1-4-Fucosyltransferase (Fut3, P=0.018) and α1-3-Fucosyltransferase (Fut6, P=0.038), but not α1-6-Fucosyltransferase (Fut8, P=0.910) in RA compared to OA synovial tissues. Interestingly, a strong positive correlation between TNFα and Fut1, Fut3 and Fut6 was identified. Fut1 also correlated with Il17a. Preincubation of RASFs with 2-Deoxy-D-Galactose (15 mM/ml), a FUT1 inhibitor, decreased the sensitivity to TRA-8, an anti-human DR5 antibody, mediated apoptosis and this effect can be reversed by fucose (15 mM). Similar results were also found on human monocyte/macrophage cell lines, HL60 and THP-1. Our studies suggest that fucosylation is strongly associated with abnormal inflammation and apoptosis in RA effector cells. Fucosylation might be a novel target for RA therapy.
Characterization of antitumor cytokine-induced killer cells generated from immature precursors (87.31)
Authors: Ryu Hwa Sun , Kim Yeon Jin , Lee Hong Kyung , Kim Jee Youn , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Cytokine-induced killer (CIK) cells have been explored for active immunotherapy of cancer. CIK cells gave strong anti-tumor effects and their cytotoxicity is MHC-unrestricted. In most clinical trials, CIK cells were generated from human PBMCs, mostly containing mature T cell precursors. However, whether immature T cells could be used as precursors of CIK cells was not clarified yet. The possible usage of immature T cells as sources of CIK cells was evaluated in this study. CIK cells were generated from immature T cells of bone marrow and thymus, and their functions were compared with CIK cells generated from mature T cells of spleen. Interferon gamma, anti-CD3 monoclonal antibody, and interleukin 2 (IL-2) were timely added to cultures to generate CIK cells. The absolute number of splenocytes, bone marrow cells, and thymocytes increased by 249, 54, and 14-fold, and CD3+NK1.1+ cells were 30%, 30%, and 33%, respectively. These three different cell populations showed similar profiles in cytokine gene expression, in that they highly expressed IFN-gamma and TNF-alpha. And they showed similar gene expression profiles of granzynme A/B, perforin, and FasL. In addition, they showed similar antitumor activity against B16F10 melanoma. In summary, although CIK cells generated from immature or mature T cells showed similar profiles in phenotypes, cytokine expression, and antitumor activity in vivo, clinical application of immature T cells might be impeded by low ex vivo expansion.
Phytol derivatives as novel immunoadjuvants (87.33)
Authors: Aachoui Youssef , Fitch Richard , Ghosh Swapan
Published: January 2023

Adjuvants are widely used to influence the course of immune response. Improved adjuvants tailored to immunogens are much sought after. In order to achieve this, we are developing new adjuvants based on chemically modified phytol (J Immune Based Ther. Vaccines 2006, 5:4). We developed hydrogenated/nitrated or glycosylated derivatives of phytol (US patent pending). These compounds exhibit excellent adjuvanticity, and in some respects seem superior to Alum or Freunds’ adjuvants. In this study, we show using cytokine microarray how they influence host microenvironment in the peritoneal lavages of BALB/c mice after injection of phytol adjuvants alone or in combination with different antigens. Our results indicate that 1) within 2 hr after injection there was increase in levels of chemotactic factors MCP-1, KC, MIP-1, LIX and IL-6. 2) The levels of these cytokines were more pronounced with PHIS-01 compared to PHIS-02 and PHIS-03. At 24 hours after injection, the inflammatory cascade was amplified stimulating the production of other cytokines that could involve cells of acquired immunity such as B-lymphocyte chemo-attractant (BLC), T-cell activation-3 (TCA), IL-12 , TIMP-1; in addition, they induced innate immunity-related chemotactic factors. Unlike alum, there was no induction of IL-1β and IL-4 suggesting a different mode of action. We also noted that the cytokine profiles significantly changed when protein antigens were incorporated with the phytol-derived adjuvants.
Identification of variant isoforms of CD44 and their functions in murine immune cells (87.6)
Authors: Guan Hongbing , Singh Narendra , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

CD44 is known to be implicated in a variety of immunological processes such as T cell migration, activation and differentiation. CD44 comprises a standard form and a family of isoforms that vary by their degree of post-translational modifications and due to alternative splicing of their primary amino acid sequence encoded by up to 10 variant exons. The isoforms actually determine the differential functions of CD44 in various types of immune cells and immune responses. Upon activation, lymphocytes transiently express certain CD44 splice variants. However, the precise nature of CD44 variant isoforms expressed by various cells types of the immune system has not been studied thus far. In this report, we screened variant exons from exon 1 to 10 using Southern blot hybridization and identified CD44 isoforms on CD4, CD8, FoxP3+ Treg and dendritic cells, either naïve or activated with SRBC, OVA, LPS, anti-CD3 plus anti-CD28, or IL-2 respectively. The results showed distinct isoforms for each cell type that also varied based on naïve or activated status. In experimental autoimmune encephalomyelitis (EAE), targeted deletion of full CD44 sequence prevented EAE whereas sole deletion of variant 7 induced a progressive and non-remitting type of EAE. Our study, has for the first time, identified the unique CD44 isoforms expressed on murine CD4, CD8, Treg and dendritic cells and shown the differential role of CD44 variant isoforms in the regulation of autoimmune disease.
Cartilage remodeling genes on murine chromosome 15 involved in sex-affected pathways controlling the effector phase of arthritis (88.19)
Authors: Adarichev Vyacheslav , Kudryavtseva Elena
Published: January 2023

Gender differences in susceptibility to many autoimmune diseases including rheumatoid arthritis (RA) are well documented. To dissect sex-affected inflammatory mechanisms in murine models for RA, we generated BALB/c.DBA/2-Pgia8 congenic strain. The strain carries chromosome 15 sex-specific locus Pgia8 that controls arthritides induced with aggrecan, collagen type II, B. burgdorferi, and pristane. We report here that the locus also controls collagen antibody-induced arthritis (CAIA), which stresses the locus role in the effector inflammatory phase of the disease. CAIA severity in congenic males was 30% lower (p < 0.005) than in wild-type BALB/c males (locus effect). Severity in congenic males was only 50% of that in congenic females (sex-related effect). Differential expression analysis of total RNA isolated from paws showed that only dozen of genes within the locus were differentially expressed in wild-type versus congenic mice, and their expression was significantly affected by sex. In inflammatory conditions, expression pattern for the gene encoding for Cthrc1 (collagen triple helix repeat containing) was highly correlated with CAIA severity, r=0.84. In basal conditions, Cthrc1 expression correlated with the concentration of IL-6 message. Taken together, these data could indicate that Cthrc1 controls cartilage remodeling differently in males and females and might provide a converging point for regulation of different types of arthritides including RA.