bluuebunny/crossref_metadata_2025 huggingface dataset:
A critical role for c-kit on dendritic cells in regulating in T helper cell differentiation and allergic asthma (79.3)
Authors: Krishnamoorthy Nandini , Oriss Timothy , Fei Mingjian , Paglia Melissa , Yarlagadda Manohar , Vanhaesebroeck Bart , Ray Anuradha , Ray Prabir
Published: January 2023

Dendritic cells are integral in instructing the differentiation of T helper cells into Th1, Th2 and Th17 subsets and IL-6 is a key regulator of CD4 T cell differentiation .In this study, we investigated pathways in DCs that promote IL-6 production. We show exposure of DCs to house dust mite (HDM) or the mucosal adjuvant cholera toxin (CT) promotes cell surface expression of c-Kit and its ligand, stem cell factor (SCF), on DCs. This dual upregulation of c-Kit and SCF results in sustained signaling downstream of c-Kit, thereby promoting IL-6 secretion. Intranasal administration of antigen into c-Kit-mutant mice or neutralization of IL-6 in cultures from the lung-draining lymph nodes of immunized wild-type mice blunted the Th2 and Th7 responses. DCs lacking functional c-Kit or those unable to express membrane-bound SCF secreted lower amounts of IL-6 in response to HDM or CT. Expression of the Th2-Notch ligand Jagged-2 was also reduced in DCs from c-Kit-mutant mice in response to CT or HDM and the Th1-inducing adjuvant, CpG oligodeoxynucleotide, did not promote either c-Kit or Jagged-2 expression. DCs expressing nonfunctional c-Kit were unable to induce a robust Th2 or Th17 response and elicited diminished allergic airway inflammation when adoptively transferred into WT mice. DCs generated from mice expressing a catalytically inactive form of the p110delta subunit of phosphatidylinositol-3 (PI3) kinase secreted lower amounts of IL-6 upon stimulation with cholera toxin. Taken together, these results reveal the importance of the c-Kit-PI3 kinase-IL-6 signaling axis in DCs in the regulation of T cell responses.
ROG, repressor of GATA, regulates Th2-driven allergic airway inflammation and airway hyperresponsiveness (79.8)
Authors: Nakayama Toshinori , Hirahara Kiyoshi , Yamasahita Masakatsu
Published: January 2023

Studies of human asthma and of animal models of allergic inflammation/asthma highlight a crucial role for type 2 helper T (Th2) cells in the pathogenesis of allergic asthma. Repressor of GATA (ROG) is a POZ (BTB) domain-containing Kruppel type zinc finger family (or POK family) repressor. A repressive function to GATA3, a master transcription factor for Th2 cell differentiation is indicated. The aim of this study is to clarify the regulatory roles of ROG in the pathogenesis of Th2-driven allergic diseases, such as allergic asthma. We examined allergic airway inflammation and airway hyperresponsiveness in three different mouse models, which use either ROG-deficient (ROG-/-) mice, ROG transgenic mice or adoptive transfer of cells. In ROG-/- mice, Th2 cell differentiation, Th2 responses, eosinophilic airway inflammation and airway hyperresponsiveness were enhanced. In ROG transgenic mice, the levels of eosinophilic airway inflammation and airway hyperresponsiveness were dramatically reduced. Furthermore, adoptive transfer of Th2 cells with increased or decreased levels of ROG expression into the asthmatic mice resulted in reduced or enhanced airway inflammation, respectively. These results indicate that ROG regulates allergic airway inflammation and airway hyperresponsiveness in a negative manner, and thus ROG may represent another potential therapeutic target for the treatment of asthma patients.
Cigarette smoke suppresses in vitro allergic activation of mouse mast cells (80.1)
Authors: Mortaz Esmaeil , Folkerts Gert , Engels Ferdi , Nijkamp Frans P , Redegeld Frank A
Published: January 2023

Abstract

Background: Mast cells are important effector cells in innate or acquired immunity that contribute to host defense. Excessive activation of mast cells can result in the development of allergic diseases, including atopic asthma. Mast cell activation by IgE and specific antigen induces the cells to release spasmogenic, vasoactive, and proinflammatory mediators, which enhance airway smooth muscle contraction, vascular permeability, and inflammatory cell recruitment. Recently, we have demonstrated that exposure of mast cells to cigarette smoke medium (CSM) triggered mast cells to produce chemokines. On the other hand, smoking may decrease the risk of allergic sensitization, which could be explained by a reduced IgE production or a diminished response of mast cells to activation of the IgE receptor.

Objective: In this study, we investigated the effect of CSM on allergic activation of mast cells through IgE and antigen.

Methods: Primary cultured murine mast cells were exposed to CSM and activated with IgE and antigen or lipopolysaccharide (LPS). The releases of granules, production of leukotrienes, chemokines and cytokines was determined in supernatants by ELISA. The effect of CSM exposure on intracellular signaling, especially the NF-κB and Erk1/2 pathways, was analyzed by Western blotting.

Results: CSM suppressed IgE-mediated degranulation and cytokine release, but no effect was observed on leukotriene release. CSM induced phosphorylation of Erk1/2 in mast cells. In CSM-exposed mast cells ATF-1 was phosphorylated after stimulation with IgE/Ag. LPS activated mast cells were not influenced by CSM.

Conclusion: Our study suggests that exposure to cigarette smoke may lead to a

reduced allergic activation of mast cells without affecting their response to activation via e.g. bacterial derived LPS.
Interactions between eosinophils and sensory neurons in atopic dermatitis (80.11)
Authors: Fitch Erin L , Nie Zhengying , Fryer Allison , Jacoby David
Published: January 2023

Atopic dermatitis (AD) is a disease characterized by extreme itch, increased density of nerve terminals in the skin, and a large number of circulating eosinophils. Itch is transmitted by sensory neurons whose cell bodies are located in the dorsal root ganglia (DRG). Extracellular eosinophil products, indicating eosinophil activation, are present at high levels in atopic dermatitis lesions. We used immunostaining to visualize eosinophils and nerves in normal and atopic human skin. Using antibodies to eosinophil major basic protein (MBP) and to the nerve-specific protein, PGP9.5, demonstrated that 20-60% of nerves are physically associated with MBP in atopic skin, while normal human skin contains no MBP. We cultured DRG neurons from mice and showed that they produce eotaxin-1, a chemotactic factor for eosinophils, as well as the adhesion molecules ICAM-1 and VCAM-1. When co-cultured with mouse eosinophils for 24 hours, DRG neurons bind eosinophils. This interaction dramatically alters the morphology of DRG, causing increased branching and longer neurites. Thus, eosinophils and nerves interact in the skin of atopic dermatitis patients. This interaction may mediate increased nerve growth and contribute to the increased sensation of itch in AD.
Peanuts can contribute to anaphylactic shock by activating complement (80.2)
Authors: Khodoun Marat , Strait Richard , Orekov Tatyana , Hogan Simon , Karasuyama Hajime , Herbert De'Broski R. , Köhl Jörg , Finkelman Fred D.
Published: January 2023

Peanut allergy is the most common food-related cause of lethal anaphylaxis, typically persists into adulthood, and can reappear in individuals who have become peanut tolerant. These observations suggest that peanuts have special characteristics that increase their allergenicity. We evaluated whether i.v. injection of water-soluble peanut extract (PE) into unimmunized mice made more susceptible to shock by pretreatment with a β-adrenergic blocker and long-acting IL-4 can induce shock through an innate immune mechanism. We found: 1) LPS-free PE induces dose-dependent shock; 2) PE activates complement in vivo in mice and in vitro in mice and humans; 3) Tree nut extracts, which also cause severe anaphylaxis, also activate complement, while milk and egg white, which cause relatively mild food allergy, do not; 4) C3a, and, to a lesser extent, FcγRs contribute to PE-induced shock; 5) PE-induced shock depends more on macrophages and basophils than on mast cells and is T and B cell-independent; 6) platelet activating factor and, to a lesser extent, histamine contribute to PE-induced shock; and 7) PE and IgE-mediated mast cell degranulation synergistically induce shock in mice that have not been pretreated with IL-4 or a β-adrenergic agonist. These observations suggest that complement activation by peanuts may be a reason for the severity of peanut-induced anaphylaxis.
Bovine γδ T cell response in foot-and-mouth disease virus infection (FMDV) (81.12)
Authors: Toka Felix N. , Golde William T.
Published: January 2023

In cattle, ?d T cells are a major population of the peripheral blood T cells but there is no precise description of their role in viral infections. We examined the phenotype and function of bovine ?d T cells during infection with FMDV. The early phase of infection was examined to test for a possible role for ?d T cells in innate responses against FMDV. Gamma delta T cells were sorted from PBMC of naïve or FMDV infected Holstein calves. The cells from naive animals highly expressed CD62 and CD45RO and about 50% expressed CD25, but not CD335. Gamma delta T cells did not produce IFNγ and no lytic activity could be demonstrated against a tumor cell target in vitro, although a very low level of perforin was detected. Cells from infected animals had phenotypic alterations on γd T cells between 2 and 6 days post infection (dpi). Expression of CD62 and CD45RO was down regulated and CD25 expression moderately increased. Unexpectedly, CD13 was expressed on days 2 and 3 after infection and were cytolytic against K562 cells. In addition, γδ cells isolated on these days expressed CD335, perforin and produced IFNγ. However, the isolated γd T cells produced less IFNγ and expressed less perforin compared to γd T cells assayed from non-sorted PBMC, indicating that the function of γd T cells may depend on factors derived from other cells. Overall, bovine γd T cells are activated upon infection with FMDV and adopt NK cell phenotypical and functional properties. Therefore, bovine γd T cells may likely participate in the innate response to infection with FMDV and targeting activation of γδ T cells could lead to rapid protection against FMDV infection.
Immunological animal models for the immunotherapy against Alzheimer’s disease

(81.13)
Authors: Vasilevko Vitaly , Head Elizabeth , Lemere Cynthia A. , Cribbs David H.
Published: January 2023

The pioneering work by Dale Schenk established that antibodies specific for the beta-amyloid (Aβ) peptide could reduce the level of Aß pathology in the brains of transgenic mouse model of Alzheimer's disease (AD), overexpressing a human mutant form of the amyloid precursor protein (APP). Unfortunately, the first immunotherapy clinical trial in AD patients, the AN1792, was halted due to meningoencephalitis in a subset of the patients that received the anti-Aβ vaccine. Efforts to develop next generation vaccines are now being pursued by various groups to eliminate or reduce the potential for adverse outcomes in patients.

To further explore the diversity of immune responses by different mammal species to Aß1-42 immunization and to choose the adequate immunological animal model we immunized several strains of mice, as well as rats, guinea pigs, rabbits, and dogs with the AN-1792 vaccine.

We found that the dominant anti-Aß B-cell epitope in mice, rats and guinea pigs is located in an internal sequence of Aß1-42, and serum from these species recognizes APP on a Western blot. Conversely, anti-Aβ antibodies from monkeys, canines and rabbits all closely resembled the reported immune response in humans by recognizing only the free N-terminus of Aß1-42, and did not recognize APP by Western blot.

Thus, careful selection of animal models for testing new vaccine candidates may facilitate effective translation to human clinical trials.

Support by NIH grants RO-1 AG20241, AG-00538, P50 AG16573 to DHC, AG20242 to EH
Serological analysis of equine antibody responses to the facultative intracellular bacteria Corynebacterium pseudotuberculosis (81.14)
Authors: Abdel-Massih Dina , Valle Luca , Bucknam Alexis , Spier Sharon , Pollock Roberta
Published: January 2023

Corynebacterium pseudotuberculosis is a gram positive facultative intracellular bacteria that causes pigeon fever or dryland distemper in horses. Most infected horses develop large pus-filled external abscesses, which resolve with a low mortality rate. Some infected horses develop internal abscesses, and about 40% of these animals die. Our hypothesis is that the TH1 toTH2 balance determines the form of the disease: TH1 dominance leads to external abscesses and TH2 dominance leads to internal abscesses. We developed an ELISA that measures the antibody response to phospholipase D (PLD), the major antigen and exotoxin of the bacteria, and screened 50 infected and uninfected horses. Current diagnostic criteria state that horses with internal abscesses have high level of antibodies to PLD and horses with external abscesses have low antibody titers. Our results show that while most horses with internal abscesses show high antibody responses to PLD, many horses with external abscesses and some uninfected horses also have high titers to PLD. The class and subclass of anti-PLD antibodies are being measured to test whether TH1 or TH2 induced antibodies predominate in horses with different forms of the disease.
Is IgT the evolutionary equivalent of IgA? Insights into its structure and function (81.21)
Authors: Sunyer Oriol , Zhang Yong-An , Li Jun , Parra David , LaPatra Scott E.
Published: January 2023

From an evolutionary perspective, immunoglobulins emerged for the first time in fish. Teleost fish are known to contain at least three immunoglobulin (Ig) isotypes, IgM, IgD and the recently discovered IgZ/IgT. Thus far nothing is known about the protein structure, function and distribution of IgZ/IgT. We have raised monoclonal antibodies against IgT that have enabled us to purify it from trout plasma. Our analysis show that IgT is a 180 kDa monomeric Ig and represents the first monomeric immunoglobulin to be shown in teleost fish. Thus far, IgM is the only known teleost fish Ig involved in eliciting antigen-specific immune responses in teleost fish. However, here we present for the first time exciting data showing that rainbow trout produces IgT-specific responses upon antigenic stimulation. In addition we have identified a unique B cell subset expressing exclusively surface IgT. Importantly, the distribution of IgT+ B cells is predominant in the fish mucosal surfaces, suggesting a possible role of IgT in mucosal immunity.
IgD+ cell populations in channel catfish, Ictalurus punctatus (81.8)
Authors: Edholm Eva-Stina I , Stafford James , Sahoo Manoranjan , Bengtén Eva , Miller Norman , Wilson Melanie
Published: January 2023

Only two immunoglobulin (Ig) molecules, IgM and IgD have been identified in the channel catfish. While IgM has been extensively studied and in many aspects resembles mammalian IgM, catfish IgD is quite different. It is a chimeric molecule consisting of a rearranged VDJ region spliced to the Cμ1 domain, followed by seven Cδ domains and either a transmembrane or secreted tail. Through the use of anti-IgM and anti-IgD monoclonal antibodies three different B cell populations were identified that differentially express IgM and IgD; IgM+/IgD-, IgM+/IgD+ and IgM-/IgD+. PCR and RACE protocols amplified appropriate Ig rearranged message from these sorted populations indicating that they contained functional B cells. Furthermore, co-immunoprecipitation, Western blot analyses, and RT-PCR showed that IgD in the IgM-/IgD+ sorted cell population preferentially associates with the catfish Ig light (L) chain isotype sigma (IgL σ?. In contrast, the IgM+/IgD-, IgM+/IgD+ B cell populations express all four of the catfish IgL isotypes, IgLσ, IgLλ??and the IgL¿? which includes F and G IgL chains. In addition a third IgD+ cell population was identified consisting of IgM-/IgD+ granular-like cells of unknown origin, which do not express IgD message, and thus appear to be armed with exogenous IgD via a putative IgD binding receptor.

Supported by NIH grant R01 AI19530 and UMMC IRSP 59908.
TSLP and IL-7 use two different mechanisms in regulating human CD4+ T cell homeostasis (82.1)
Authors: Lu Ning , Wang Yui-Hsi , Liu Yong-Jun
Published: January 2023

Homeostatic proliferation of CD4+ T cells depends on both self-peptide-MHC ligands and cytokines such as interleukin 7 (IL-7) and Thymic Stromal Lymphopoietin (TSLP). Although it is well established that IL-7 directly acts on CD4+ T cells, data on whether TSLP directly induce human CD4+ T cell proliferation and TH2 differentiation are conflicting. We report here that resting and activated CD4+ T cells express high levels of IL-7 receptors, but very low levels of TSLP receptor when compared with that expressed by myeloid dendritic cells (mDC). While IL-7 induce strong STAT1, 3 and 5 activation and promote the proliferation of naïve CD4+ T cells in the presence of anti-CD3 and anti-CD28, TSLP only induce a weak STAT5 activation which is associated with the marginally improved cell survival, but fail to induce CD4+ T cell proliferation and TH2 differentiation. The effect of TSLP on enhancing human T cell proliferation could be observed only when sorted naïve CD4+ T cells were cultured with contaminated mDCs at as low as 0.5% in culture. TSLP could only induce purified naïve CD4+ T cells to differentiate into TH2 cells in the presence allogeneic mDCs. This study demonstrates that IL-7 and TSLP use different mechanisms in regulating human CD4+ T cell homeostasis and underscores the importance of cell purity in immunology research.

Key words: interleukin 7, Thymic Stromal Lymphopoietin, T cell homeostasis, proliferation, survival
Cytomegalovirus (CMV) and human longevity: lessons from long-lived families (82.5)
Authors: Pawelec Graham , Maier Andrea B , Karin Haehnel , Westendorp Rudi JG , Derhovanessian Evelyna
Published: January 2023

Seropositivity for Cytomegalovirus (CMV) is one of the parameters comprising the "immune risk profile" (IRP) predicting mortality in the very elderly. However, as many centenarians are CMV-seropositive (CMV+), not infection per se, but the way in which the immune system controls the virus, might be important in ensuring longevity. To analyze this, we have investigated the CMV prevalence of individuals with 30% decreased mortality risk from long-lived families (offspring, n=200) and their partners as controls (n=200). 10-colour flow cytometry was used to determine the frequency of different adaptive and innate immune subsets in 100 offspring and 100 partners. CMV-seroprevalence was significantly less frequent in offspring, despite many sharing their daily living with an infected partner. The expected highly significant age-associated decrease in naïve T-cells and accumulation of late-differentiated memory T-cells in CMV+ individuals was confirmed only in controls whereas CMV+ and CMV-negative offspring showed similar immune profiles. Our data suggest that individuals genetically enriched for longevity are more resistant to becoming infected with CMV. Furthermore, even when they are infected, they seem to be less susceptible to CMV-driven changes to the immune system. We feel that these phenomena are likely to be related, perhaps causally.

This work was supported by EU 7FP LifeSpan (LSHG-CT-2007-036894), DFG-SFB685-B4 and DFG-PA 361-11/1
IL-2 signals are required for complete effector differentiation of CD8+ T cells upon acute infection in vivo (83.17)
Authors: Sacks Jilian A. , Bevan Michael J.
Published: January 2023

CD8+ T cells are critical for protection against viral and intracellular bacterial infections. They are characterized by their ability to acquire cytolytic activity to lyse infected target cells. This effector stage is associated with the expression of a variety of intracellular and cell surface markers. Here, we examined the role of direct signaling by the cytokine IL-2 on the acquisition of CD8+ T cell effector functions. IL-2 signals via its heterotrimeric high-affinity receptor composed of the IL-2Rα-chain (CD25), β-chain (CD122) and common γ-chain (CD132). Previous work from our lab and others has shown that direct IL-2 signaling on CD8+ T cells is imperative for their differentiation into memory cells capable of secondary expansion. Using a mixed bone marrow chimeric approach in which wildtype (WT) and CD25 knockout (KO) cells are responding in the same environment we now demonstrate that KO CD8+ T cells are defective in effector differentiation following primary acute infection. CD25 KO effectors show decreased lytic activity and expression of effector-associated proteins and transcription factors, such as Granzyme B and T-bet. In addition, these cells show increased expression of naïve/memory-associated factors, such as Bcl2, Eomesodermin and IL-2 production, despite their inability to expand upon rechallenge. Overall, we conclude that direct IL-2 signaling is required for complete CD8+ T cell effector differentiation following infection in vivo.

This work was supported by the Howard Hughes Medical Institute and the National Institutes of Health Grant AI-19335 (to M.J.B.)
Lack of LFA1 mediated signals promotes generation of memory precursor effector CD8 T cells during LCMV infection (83.19)
Authors: Peixoto Antonio , Iannacone Matteo , Alvarez David , Buchanan Ian , Flynn Michael , von Andrian Uli
Published: January 2023

Multiphoton intravital microscopy (MP-IVM) studies have revealed that T cell priming encompasses a prolonged phase of stable interactions between T cell and Dendritic cells (DCs). Although believed to promote T cell activation, the role of prolonged T-DC interactions remains poorly understood. Here we dissect the contribution of LFA1 mediated adhesion/signaling in two settings: in mediating P14 T cell interactions with GP33-39 peptide pulsed DCs and modulating the differentiation of P14 T cells after an LCMV infection. First, we show that the lack of LFA1 on P14 T cells significantly decreases the T-DC interaction time measured by MP-IVM. Second adoptive transfer of physiological numbers of P14 LFA1 -/- T cells into WT recipients generated reduced effector cell numbers at day 8 after LCMV i.p. infection, when compared to P14 LFA1+/+ T cells. Moreover, P14 LFA1-/- T cells were poor cytokine producers and showed a significant increase in the frequency of IL7Rhigh KLRG1low memory precursor effector cells. In summary, our results suggest that prolonged T-DC interactions mediated by LFA1 promote generation of short lived effector cells. In contrast, short lived T-DC interactions promote the generation of memory precursor effector cells.
Molecular signature of human virus specific effector CD8+ T cells (83.20)
Authors: Akondy Rama S , Miller Joseph D , Doho Gregory , Wu Hong , Zilliox Michael , de Rio Carlos , Mulligan Mark J , Edupuganti Srilatha , Haining W. Nicholas , Ahmed Rafi
Published: January 2023

Evaluation of the cellular immune response soon after an infection is difficult in humans. In consequence, most of our current understanding of the differentiation of an antiviral CD8+ T cell response derives from either total T cells or from cross sectional studies or from various persistent or recurring viral infections. Effective live viral vaccines like the 17D strain of yellow fever virus (YF-17D) model a primary acute viral infection and present an opportunity to study the attributes of an anti-viral CD8+ T cell response in humans. The activation markers HLA-DR and CD38 have been previously shown to identify effector CD8+ T cells after a primary vaccination (Miller et al, 2008). We use these markers to isolate YF-17D specific effectors from human vaccinees. This effector population is transient, appears only during an anti-viral response and contains mostly YF-17D specific CD8+ T cells. Gene chip analysis of these cells shows that effectors undergo dramatic reprogramming as evidenced by changes in the number of genes (~4000) and their expression (effector / naïve fold change from +100 to -26) when compared to naïve CD8+ T cells. This signature is distinct not only from naïve CD8+ T cells but also from the TCM, TEM, and TEMRA memory subsets. We also show that the effector gene expression profile is fairly conserved when compared to the mouse LCMV effectors. One of the many uses of this signature is to understand and identify factors involved in naïve to effector cell differentiation.

This research was supported by NIH/NIAID (U19 AI057266).
The tyrosine kinase PYK2 integrates TCR and LFA-1 signaling in CD8 T cells to promote effector versus memory fate (83.21)
Authors: Beinke Soren , Matloubian Mehrdad , Schlessinger Joseph , Weiss Arthur
Published: January 2023

Focal adhesion kinase regulates integrin-mediated interactions of non-hematopoietic cells. In T cells, the homologous proline-rich kinase 2 (PYK2) is activated by the T cell antigen receptor and the integrin LFA-1 but its function is not well understood. Studying T cells from PYK2 knockout mice, we have demonstrated that PYK2 is essential for LFA-1 mediated T cell polarity and activation of CD8 T cells by synergistic TCR / LFA-1 stimulation or antigen presenting cells in vitro. During the acute response to lymphochoriomeningitis virus (LCMV) infection in vivo PYK2-deficient CD8 T cells are impaired in their expansion and differentiation into short-lived effector cells but generate normal numbers of memory precursor cells. Interestingly, the LCMV specific CD8 T cell memory population that is maintained long-term after LCMV infection is increased in PYK2 knockout mice. These data indicate that PYK2 deficiency results in an alteration in CD8 T cell fate during priming leading to a loss of effector but gain of memory functions. Therefore, PYK2-mediated T cell polarity may be an important determinant of CD8 T cell fate.
Visualizing interactions of antigen, B cells and FDC in vitro (84.23)
Authors: Kim Young-A , Carroll Michael C
Published: January 2023

Follicular dendritic cells (FDC), which reside in the primary B cell follicles and germinal centers of secondary lymphoid organs, can trap and retain antigens (Ag) in the form of immune complexes (IC) through Fc and complement receptors. Ag presentation by FDC plays an important role in the positive selection of high-affinity B cells and possibly maintenance of memory B cells. Recent studies reveal that B cells transport IC from the splenic marginal zone and subcapsular sinus in draning lymph node to FDC via complement receptor (CR, CD21/35). How Ag is transferred to FDC and subsequently presented to cognate B cells is not clear. To investigate interactions between Ag, B cells, and FDC in vitro, spinning disk confocal microscopy was used. We found that IC taken up via CR on B cells form clusters on the cell surface and this may enhance transfer to CR on FDC. Moreover clustering of IC and CR is independent of CD19 based on analysis of B cells prepared from mice bearing a mutant CR.
Naive B cells participate in secondary antibody response via immune complex mediated activation (84.6)
Authors: Goins Chelsey L , Chappell Craig P , Murthy Shashidhara , Selvaraj Periasamy , Jacob Joshy
Published: January 2023

The adaptive immune system confers survival advantage to the host by its inherent ability to respond to a plethora of pathogens and its ability to never forget an antigenic insult. In the case of humoral immune responses, repeat antigenic exposure leads to a secondary antibody response that is 2-3 orders of magnitude greater than the primary. This increased efficacy is due to the production of amplified levels of antigen-specific antibody, as well as the accelerated kinetics of their production. Current dogma holds that this response results from activation of memory B cells, which are formed following primary exposure and can rapidly produce large quantities of specific antibody upon secondary exposure. Our research, however, suggest that memory B cells may not be the chief source of antibody during the secondary response. Using a hapten model system we have shown that the phenomena associated with secondary responses - increased antibody levels and accelerated kinetics of antibody production - can be recapitulated by primary immunization with immune complexes. This suggests that immune complexes are able to activate antigen-inexperienced naive B cells in such a manner that their response mirrors a secondary response. Additionally we have found that blocking the Fc receptor binding activity of immune complexes inhibits the secondary antibody response. We propose that immune complexes are formed upon secondary antigen exposure, and that these complexes enhance the secondary antibody response through naive B cell activation.
Proliferative and phenotypic characterization of human mesenchymal stem cells by flow cytometry (84.7)
Authors: Bradford Jolene A , Clarke Scott T , Boucher Shayne , Vemuri Mohan
Published: January 2023

Adult human mesenchymal stem cells (hMSCs) are rare fibroblast-like cells that can differentiate into numerous tissue types, such as bone, cartilage and fat. hMSCs can impact tumorigenesis and tumor progression, including lymphoid neoplasias. hMSCs have been defined as positive for CD73, CD90, CD105 and negative for CD14, CD19, CD34, CD45 and HLA-DR. Phenotypic and proliferation profiling of hMSC through multiple passages can reveal changes that are indicative of gradual lineage restriction. We characterized hMSC using flow cytometry and an eight-marker panel in combination with DNA content and cell proliferation analyses. Using two hMSC sources, adipocyte-derived (ADSC) and bone marrow-derived (BMSC) stem cells, lineage restriction was inferred from changes in marker expression and proliferation rate over multiple passages. Using XenoFree media, the proliferation rate for ADSC decreased at low passage number while BMSC maintained expected phenotypic and proliferation profiles over multiple passages. When a lipid supplement was added, ADSC maintained phenotypic and proliferation profiles similar to BMSC. Flow cytometry allows robust and rapid analysis of phenotypic and proliferative responses by hMSCs cultured under serum-free and/or xenofree conditions. This approach can have strong relevance for cell-based therapy and for studying interactions between normal stem cells and cancer.
Unique properties of CD4 T cells selected by MHC class II expressing thymocytes (85.1)
Authors: Chang Cheong-Hee , Sofi Hanief , King Phil , Schwartzberg Pam , Qiao Yu
Published: January 2023

We have demonstrated that MHC class II expressing thymocytes can efficiently mediate positive selection of CD4 T cells in mice. Human thymocytes express MHC class II and thymocyte-mediated CD4 T cells selection provides a mechanism for several documented observations made in humans that could not be explained otherwise. We named thymocyte-selected CD4 cells T-CD4 (Thymocyte-selected) and the other E-CD4 (Epithelial cell-selected) to reflect their selection pathway.

T-CD4 T cells are distinct and different from E-CD4 T cells by exhibiting the effector function similar to innate T cells. T-CD4 cells can produce both Th1 and Th2 cytokines simultaneously shortly after activation. Remarkably, Stat6 that is critical for Th2 cell differentiation is not required for T-CD4 T cells to produce Th2 cytokines. Interestingly, these characteristics are found in NKT cells that are also selected on thymocytes. Consistent with these observations, development of T-CD4 T cells require SLAM/SAP signal that is essential for NKT but not E-CD4 T cells. To understand the molecular mechanisms and signaling pathways that are involved in T-CD4 T cell generation, we studied the role of ITK and RasGAP. ITK deficient cells were selected more efficiently by thymocytes than thymic epithelial cells. In contrast, RasGAP plays a more important role in the generation of T-CD4 T cells than E-CD4 T cells. Therefore, E- and T-CD4 T cells require different signaling mechanisms for their optimum development.

The study was partly supported by NIH.
A Y-chromosome factor impairs iNKT cell development (85.15)
Authors: Toussaint Leon , Terrizzi Stephanie , Teuscher Cory , Brossay Laurent
Published: January 2023

Invariant Natural Killer T (iNKT) cells are a fairly recently discovered unique subset of T lymphocytes. These granular cells are derived from the thymus and share properties of both NK and T cells. iNKT cell development is unique from mainstream T cell selection and the polygenic factors that influence NK T cell ontogeny are still unclear. Previous studies done in our laboratory has shown the absence of iNKT cells in male B6.IFN-αβR1-/- mice. Analysis of the iNKT cell compartment in a variety of mice demonstrated that the deficiency was linked to the Y-chromosome but independent of IFN-αβ. Here, we generated wild-type male B6 mice carrying the YNKT-chromosome as well as male B6.IFN-αβR1-/- mice that do not carry the YNKT-chromosome. We found that wild-type B6 (YNKT) male mice have a strong decrease in iNKT cell frequency. In contrast, the B6.IFN-αβ R1-/- male mice have a relatively normal repertoire, confirming that the deficiency is linked to the Y-chromosome and is independent of IFN-α/β In addition, preliminary data using two different strains of B6 consomic mice for the Y-chromosome show that they have a normal iNKT cell repertoire. This suggests that a consomic Y chromosome is not sufficient to influence iNKT cell development. We are currently investigating whether the phenotype observed is due to a Y chromosome mutation or an epistatic interaction with other genes. This work was supported by the National Institutes of Health Research Grant AI46709.
Glycerol-3-phosphate acyltransferase-1 Alters Thymic Development in Mice (85.16)
Authors: Jolly Christopher A , Gulvady Apeksha , Collison Lauren W
Published: January 2023

Our lab has recently shown that T-lymphocyte function ex vivo from glycerol-3-phosphate acyltransferase-1 (GPAT-1), first enzyme in de novo glycerophospholipid biosynthesis, knockout mice is similar to aged T-lymphocytes. In order to determine the mechanism(s) for this observation, we examined thymic development in GPAT-1 KO mice. Thymi were weighed then thymocytes isolated for determination of proliferation and thymic subset proportions. We found that the thymi from GPAT-1 KO mice were 30% smaller than the wild type controls and thymocyte proliferation was reduced. Furthermore, the thymic single positive CD4 and double negative subset proportions were increased while the double positive population was decerased. The changes in thymic weight and thymocyte subsets are most likely not linked to altered fetal thymic development since thymic weights from 3 week old mice were identical to that seen in wild type controls. Since T-lymphocyte GPAT-1 activity is reduced in aged T-lymphocytes these data give insight into a novel mechanism by which aging may alter thymic development and subsequent peripheral T-lymphocyte function. This work was supported by NIH AG20651 (CAJ).
Interplay between pre-TCR, CXCR4 and laminin10/11 during thymic β selection (85.8)
Authors: Trampont Paul C , Tosello-Trampont Annie-Carole , Shen Yueli , Duley Amanda K , Bender Timothy P , Sutherland Ann B , Littman Daniel R , Ravichandran Kodi S
Published: January 2023

The βselection checkpoint during thymocyte development is a key step when immature thymocytes at double negative 3 (DN3) stage are selected to progress to the DN4 stage and beyond. While the role for the pre-T cell receptor (pre-TCR) during βselection has been established, how other receptors contribute to βselection is not well understood. Here, we identify a previously unrecognized role for CXCR4 during βselection using mice with a conditional loss of CXCR4 expression in DN thymocytes. CXCR4 physically and functionally associates with the pre-TCR and influences βselection via at least three mechanisms: regulating steady-state localization of immature thymocytes within thymic subregions, providing survival signals, and influencing efficient thymocyte proliferation post-β selection. We also show co-localization of SDF-1α, laminin 10/11 and DN3 thymocytes at the subcortical thymic regions, where the βselection events are thought to take place. We characterize the adapter protein ShcA and ERK kinase as functionally relevant signaling molecules downstream of CXCR4/pre-TCR/integrins. These data identify CXCR4 as a novel costimulator during βselection that functions together with pre-TCR and integrin ligands to orchestrate thymic development. Taken together, these data identify a novel interplay between the pre-TCR, CXCR4 and integrin receptors in regulating the βselection checkpoint.
Altered CD4/CD8 cell fate decision in the absence of MAZR due to redirected differentiation of MHC class I-restricted thymocytes (85.9)
Authors: Sakaguchi Shinya , Hombauer Matthias , Naoe Yoshinori , Bilic Ivan , Schebesta Alexandra , Taniuchi Ichiro , Ellmeier Wilfried
Published: January 2023

Cell fate specifications during thymocyte differentiation are linked with the tightly controlled expression of CD4 and CD8. We have previously identified that the transcriptional regulator MAZR is an important regulator of the activation of the Cd8ab gene complex during the DN to DP transition. To investigate the role of MAZR in more detail, MAZR-/- mice were generated. MAZR-/- mice were smaller in size and were born at reduced mendelian frequency. MAZR-/- DN thymocytes did not show premature expression of CD8, indicating that the deletion of MAZR is not sufficient to activate CD8 expression in DN cells. However, variegated CD8 expression in DP thymocytes of Cd8 enhancer E8I,E8II doubly deficient mice was dramatically reduced in the absence of MAZR. In addition, we observed elevated CD4/CD8 ratios in thymocytes and in peripheral T cells of MAZR-/- mice. Using MHC class I-restricted TCR transgenic mice, we demonstrated that the altered CD4/8 ratio in MAZR-/- mice was, in part, due to redirected differentiation of a fraction of MHC-class I restricted thymocytes into the CD4 lineage. Thus, our data provide genetic evidence that MAZR controls CD8 expression during thymocyte development. In addition, our results may indicate that MAZR is part of the transcription factor network that controls CD4/CD8 cell fate decisions of DP thymocytes.

Supported by the Austrian Science Fund (P19930, SFB-F2305 and START Program Y-163).
MicroRNA profiling of thymocytes undergoing stress-induced atrophy (86.10)
Authors: Medeiros Jennifer J , de la Morena Maria Teresa , van Oers Nicolai S.C.
Published: January 2023

Acute thymic atrophy occurs in a number of clinical situations including pregnancy, irradiation, immunosuppressive therapy, and bacterial infections. Lipopolysaccharide (LPS), a component of Gram-negative bacteria, can induce thymic atrophy within 48 hrs. While cytokines, corticosteroids, and TNF can mediate various aspects of thymic atrophy, little is known about the possible role of microRNAs. MicroRNAs (miRNAs) are a class of non-coding, small RNAs that regulate gene expression in many biological systems including the immune system. To assess whether miRNAs might regulate LPS-induced thymic atrophy, we performed miRNA profiling. C57BL/6 mice were injected with LPS and RNA was extracted from the thymus at various time points post-injection. LPS induced an overall decrease in thymic cellularity within 72 hours, with a significant decrease in the CD4/CD8 double positive population (p=<0.05). Preliminary microarray analysis yielded 8 and 6 unique miRNAs that were up- and down-regulated, respectively. We are currently confirming the microarray data with Northern blots probing for selected miRNAs. MiRNA expression patterns unique to different diseases or disorders are crucial to not only elucidating the role of miRNAs in the innate and adaptive immune response, but also in developing novel therapeutic treatments.
Leptin protects against endotoxin-induced thymic remodeling (86.9)
Authors: Gruver Amanda L , Sempowski Gregory D
Published: January 2023

Thymic atrophy is highly inducible by stress. While prolonged thymus atrophy can contribute to T cell deficiency, no treatment is available to enhance thymopoiesis during stress events. Leptin deficiency in mice results in chronic thymic atrophy and suppressed immunity, suggesting a role for leptin in regulating immune homeostasis. Specific roles however for intrathymic leptin signaling are currently undefined. We report here the novel finding that leptin receptor expression in the thymus is not present on developing thymocytes, but restricted to medullary thymic epithelial cells. We found leptin protected against endotoxin-induced acute thymic atrophy by preventing the loss of medullary thymic epithelial cells, protecting DP thymocytes from endotoxin-induced apoptosis, and increasing proliferation in DN thymocytes. We demonstrated that these thymoprotective effects are mediated through a leptin receptor isoform b-specific mechanism, and that leptin treatment increased thymic steady-state mRNA levels for IL-7. Taken together, our data suggested a novel intrathymic role for leptin in indirectly modulating thymocytes during thymic injury via a direct effect on medullary thymic epithelial cells. Overall, these findings support the use of leptin as a potential therapeutic to enhance T cell reconstitution in settings of stress-induced thymic atrophy. Supported by AG025150.
PKCbeta-II expression promotes PKD2 activation in Malignant B Lymphocytes (88.19)
Authors: Sather Blythe D , Mikhalap Svitlana V , Moreno-Garcia Miguel E , Sommer Karen , Shlapatska Larysa M , Sidorenko Svetlana P , Rawlings David J
Published: January 2023

Development of therapies for diffuse large B-cell lymphomas (DLBCL) requires a precise knowledge of signaling aberrations essential for tumor development. In normal B cells, the B cell receptor (BCR) initiates intracellular signals crucial for the survival and proliferation and may contribute to pathogenesis. Previous studies identified PKCbII, PKD1 and PKD3 as key DAG regulated downstream targets of BCR induced signaling and many refractory DLBCL tumors exhibit elevated expression of PKCbII. This correlates with activation of the PKCbII-NF-kB signaling cascade; and inhibition of PKCbII blocks the survival of these tumors in vitro. In this study, we show PKD2 is the predominant PKD family protein that is expressed and activated in response to BCR engagement in normal and malignant human B cells. Following BCR engagement in naïve B lymphocytes, activated PKD2 initially co-localizes with the BCR and then shuttles from the cytoplasm to the nucleus. We show using immunohistochemistry that localization and activation of PKD2 in DLBCLs correlates with the level of PKCbII expression. Using several models, we show that while PKCbII is not required for BCR-mediated PKD2 phosphorylation, increased expression of PKCbII promotes both PKD2 activation and class II HDAC phosphorylation. Finally, we show that PKD2 and a key substrate for PKCbII in B cells CARMA1, synergize to drive antigen receptor-triggered NF-kB transcriptional activity. Together, our data imply that PKD2 and PKCbII may work in concert to control transcriptional events regulating B cell survival.
IMMUNE-DEFICIENT MOUSE STRAINS DISPLAY MARKED VARIABILITY IN GROWTH OF HUMAN MELANOMA LUNG METASTASES (88.6)
Authors: Carreno Beatriz M. , Garbow Joel R , Kolar Grant R , Jackson Erin N. , Engelbach John A , Becker-Hapak Michelle , Carayannopoulos Leonidas N , Piwnica-Worms David , Linette Gerald P
Published: January 2023

Immune-deficient mice are widely used in cancer research to study human cancer biology and evaluate new therapeutics. No comprehensive study has been published documenting differences in human tumor engraftment among immune-deficient strains. Using RhoC-expressing human (A375) melanoma cells, we evaluate scid, NOD-scid (NS), NOD-scid β2mnull (NSB), and NOD-scid IL2Rγnull (NSG) as xenograft tumor recipients. Bioluminescence, magnetic resonance imaging and histopathology were employed to monitor serial tumor growth. Melanoma metastases growth is delayed and variable in scid, and NS mice. In contrast, NSB and NSG mice show rapid tumor engraftment, although tumor growth is variable in NSB mice. NK cells were detected in all strains except NSG, and in vitro activated scid, NS and NSB NK cells kill human melanoma lines and primary melanoma cells. Expression of human NKG2D ligands, MICA and MICB, renders melanoma susceptible to murine NK cell-mediated cytotoxicity and killing is inhibited by antibody blockade of murine NKG2D. Murine NKG2D recognition of MICA/B is an important receptor-ligand interaction employed by NK cells in immune-deficient strains to limit engraftment of human tumors. The absolute NK deficiency in NOD-scid IL2Rγnull animals makes this strain an excellent recipient of melanoma and potentially other human malignancies.
Calcineurin Ab deficiency causes defective Treg-cell generation and enhanced effector T-cell differentiation (89.13)
Authors: Bommireddy Ramireddy , Chen Hwudaurw , Gard Connie , Molkentin Jeffery D. , Doetschman Tom
Published: January 2023

Calcineurin (CN), a protein phosphatase activated by Ca2+-calmodulin, is involved in activation of NFAT by its dephosphorylation. CN inhibitors such as cyclosporine A (CsA) and FK506 are potent inhibitors of T-cell responses and are commonly used to prevent graft rejection. Consistently, CNAβ-deficient mice exhibit defective thymocyte maturation in young mice. However, T cells undergo spontaneous activation in older Cnab-/- mice, which develop splenomegaly, thymomegaly, hepatomegaly and succumb to metastatic B-cell lymphomas. We have also observed that FOXP3+ Treg cells are decreased while mature activated CD4+ and CD8+ T cells (CD62L-CD44+) are increased in Cnab-/- mice compared to age-matched control mice. These data show that CNAβ is important for the development and homeostasis of mature T cells and Treg cells. Cytokine profile analysis revealed that CNAβ signaling is required to inhibit proinflammatory cytokines (IL-4, IL-10, IL-17 and IFNγ) by TGFβ1 and TGFβ1 induced FOXP3 expression in T cells, suggesting that CNAβ signaling is required for Treg-cell generation. The data suggest that in the absence of CNAβ, a defective T-cell regulatory environment leads to accumulation of activated T cells which produce proinflammatory cytokines and cause inflammation and cancer.

Grant support: This study was supported by NIH AI067903 and CA84291 to TD, and IRG7400128 to RB.
Deriving murine steady state dendritic cells in the presence of a potent AhR ligand produces modulatory dendritic cells (89.18)
Authors: Simones Tom , Shepherd David M.
Published: January 2023

TCDD is the prototypical aryl hydrocarbon receptor (AhR) agonist and a potent immunotoxicant. However, the specific mechanisms underlying the immunomodulatory effects of TCDD are currently undefined. TCDD adversely affects a number of immune cells including DCs. We hypothesize that TCDD alters the differentiation of steady state DCs and causes the formation of modulatory DCs. To test this hypothesis, steady state bone marrow-derived dendritic cells (BMDCs) were grown in the presence of Flt3 ligand, and vehicle or TCDD. Flt3 ligand growth factor is commonly used to derive BMDCs representative of non-inflammatory immature steady state DCs. Growth in TCDD caused decreased expression of both CD11c and MHC II and increased the basal CD86 expression as determined by flow cytometry. An increased frequency of TCDD-derived BMDCs expressed MHC II, CD86 and CD54 following LPS or CpG stimulation as compared to vehicle-treated control BMDCs. In response to LPS or CpG, TCDD-BMDCs produced less IL-6 and TNFα. TCDD-BMDCs also up regulated indoleamine 2, 3-dioxygenase 2 (IDO2) mRNA levels, while IDO1 mRNA levels remained unchanged. IDO1 and IDO2 are thought to play a role in Treg generation and immune suppression. Altogether these data suggest that TCDD-BMDCs display immune regulatory properties attributable to a novel modulatory DC population. This research was supported by grants ES013784 and RR17670.