bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-15 (IL-15) enhances, as well as increases the sensitivity of human CD8+ memory T cell subsets to stimulation by the pro-inflammatory cytokines IL-12 and IL-18 (95.3)
Authors: Smeltz Ronald B
Published: January 2023

Human memory CD8+ T cell subsets, termed central memory and effector memory, can be identified by expression of CD45RA, CD62L, and CCR7. Accordingly, functional differences have been described for each subset, reflecting unique roles in immunological memory. The common gamma chain cytokines IL-15 and IL-7 can induce proliferation and differentiation of human CD8+ T cell subsets, as well as increased effector functions. Here, we observed that addition of IL-15 or IL-7 to cultures of human CD8+ T cells profoundly enhanced the IL-12+IL-18 pathway of IFN-γ production. Importantly, IL-15 and IL-7 lowered the threshold concentrations of IL-12 and IL-18 required for induction of IFN-γ by 100-fold. Comparison of IL-15 and IL-7 demonstrated that IL-15 was superior for its ability to enhance IL-12+IL-18-induced IFN-γ, without evidence of synergy between IL-15 and IL-7. We also observed that IL-15 and IL-7-mediated enhancement of IL-12+IL-18-induced IFN-γ production was a functional property of effector-memory CD8+ T cells. Despite a lack of association between cell division and IFN-γ, loss of CD62L expression correlated well with acquisition of IL-12+IL-18-induced IFN-γ. Indeed, purified central memory T cells stimulated with IL-15 and IL-7 developed into effector memory T cells with potent IL-12+IL-18-induced IFN-γ. Thus, in addition to its known role in development of T cell memory, IL-15 may amplify CD8+ T cell effector functions by increasing sensitivity to pro-inflammatory cytokine stimulation.

This research was supported by a grant from the Jeffress Memorial Trust Fund and the Department of Microbiology at VCU.
IL-15 acts as a potent inducer of CD4+CD25high cells expressing forkhead box protein P3 (95.6)
Authors: Imamichi Hiromi , Sereti Irini , Lane H. Clifford
Published: January 2023

IL-15 is a member of the cytokine family that utilizes a common γ chain. While initially characterized as a T cell growth factor, predominantly involved in CD8+ T cell proliferation and survival, IL-15 is a pleiotropic cytokine that can affect both innate and adaptive immune responses. In the present study, we demonstrate that IL-15 increases expression of the alpha chain of the IL-2 receptor (CD25) and foxP3, a master control gene for the development and function of regulatory T cells, in human peripheral CD4+CD25− T cells without a need for additional antigenic stimulation signals. Side-by-side comparisons involving two other γc-cytokines: IL-2 and IL-7 revealed that the induction of CD25high and foxP3 expression was most prominent with IL-15 (median-fold increase: 46 and 53, respectively for CD25high and foxP3, p=0.0005) and IL-2 (32 and 42, p=0.0004). More modest effects were seen with IL-7 (18 and 12, p=0.0105). Despite levels of foxP3 expression comparable to that of conventional Tregs, these cytokine-induced CD4+CD25+foxP3+ cells exerted only weak suppressor activity. The current study has uncovered an unexpected function of IL-15 in the induction of CD4+CD25+foxP3+ cells from human peripheral T cells, thus helped to delineate and provided further insights into the pleiotropic nature of IL-15 beyond the regulation of CD8+ T cells.

Funded in part by NCI Contract N01-CO-12400.
TNF-α and oligodendrocyte apoptosis after cervical spinal cord injury (SCI) (96.11)
Authors: Schaal Sandra Marie , Marcillo Alexander Eduardo , Stagg Monica , Pearse Damien D.
Published: January 2023

The mechanism of SCI induced oligodendrocyte apoptosis is poorly understood. Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is involved in inflammatory mediated cell death and thought to play a pivotal role in secondary pathology after SCI, including oligodendrocyte cell death. Utilizing a contusive SCI model in rat, we characterized the spatial, temporal, and cellular profiles of tnf-α expression and production acutely post-injury. Temporally, tnf-α mRNA, as assessed by RT-PCR, increased within 1 h and remained elevated through 24 h, albeit to a much lesser extent. In situ hybridization performed at 1 h revealed tnf-α mRNA throughout a cross-sectional area of the spinal cord. Though the tnf-α mRNA was diffusely distributed in the white matter, it was predominantly expressed in the gray matter. On the other hand, TNF-α protein increased by 1 h post injury and peaked at 4 h as measured by ELISA. At 1 h, 4 h, or 24 h, TNF-α immunoreactivity was present both within and adjacent to the injury epicenter; in the ventral gray matter and throughout the majority of the white matter. Using a cell specific marker (CC1) we found that oligodendrocytes were the principle TNF-α immunoreactive cell type after SCI as analyzed by stereological methods. These oligodendrocytes were also TUNEL positive, suggesting that TNF-α might be cytotoxic to oligodendrocytes acutely following cervical SCI.
CNS-Derived IL-4 is Essential for the Regulation of Autoimmunity and Induces an Alternative Activated Macrophage Phenotype in the CNS (96.20)
Authors: Ponomarev Eugene , Maresz Katarzyna , Dittel Bonnie N
Published: January 2023

Regulation of inflammation in the CNS is essential to prevent irreversible cellular damage that can occur in neurodegenerative diseases such as multiple sclerosis (MS). We investigated the role of IL-4 in regulating CNS inflammation using the animal model of MS, experimental autoimmune encephalomyelitis (EAE). We found that CNS-derived IL-4 was a critical regulator because mice with a deficiency in IL-4 production in the CNS, but not the periphery, had exacerbated EAE associated with a significant increase in the absolute number of infiltrating inflammatory cells. We also found that CNS-resident microglial cells in both the resting and activated state produced the protein Ym1/2, which is a marker of alternatively activated macrophages (aaMΦ), in an IL-4-dependent manner. This aaMΦ phenotype extended to the lack of nitric oxide (NO) production by activated microglial cells, which is a marker of classically activated macrophages (caMΦ). We also show that IL-4 induced the expression of Ym1/2 in peripheral macrophages, which also produce NO. Thus macrophages that migrate into the CNS exhibit a dual phenotype. These data indicate that IL-4 production in the CNS is essential for controlling autoimmune inflammation by inducing a microglial cell aaMΦ; phenotype. Macrophages that have undergone alternative activation have been shown to be important in tissue repair, thus our results suggest a new role for microglial cells in the regulation of inflammation in the CNS.
Differences in the expression of chemokine receptors by intrathecal B cells in B cell-dependent vs. B cell-independent EAE (96.25)
Authors: Gerber Kimberly , Lyons Jeri-Anne
Published: January 2023

B cells are implicated in the pathogenesis of multiple sclerosis. Data from the Experimental Autoimmune Encephalomyelitis (EAE) model are contradictory. We previously showed that myelin oligodendrocyte glycoprotein (rMOG)-induced EAE was B cell-dependent, while MOG35-55-induced disease was B cell-independent. Differences in the intrathecal B cell population may be relevant to the role of B cells in these two models. Higher numbers of B cells were detected in the CNS of rMOG-primed mice compared to MOG35-55-primed mice. Analysis of chemokine receptor expression by B cells isolated from the CNS of rMOG- and MOG35-55-primed mice was performed to further characterize the recruitment of B cells to the CNS during disease. Differences in the expression of CXCR1 and CCR4 was initially suggested by RT-PCR and confirmed by Q-PCR. Preliminary analysis of the cognate chemokines for these receptors indicated differences in the absolute expression as well as the expression profile of these ligands. Thus, differences in expression of factors important in B cell migration in rMOG- and MOG35-55-induced EAE may be relevant to the recruitment of pathogenic B cells in B cell-dependent EAE.
Latent Membrane Protein 1 of Epstein Barr Viruses Activates the PI3K Pathway to Induce Production of IL-10 (96.5)
Authors: Lambert Stacie L , Martinez Olivia M
Published: January 2023

Epstein Barr virus is a B lymphotrophic gammaherpes virus that is associated with multiple human malignancies. The EBV-encoded protein, latent membrane protein 1 (LMP1), is required for transformation of human B cells by EBV in vitro. LMP1 usurps multiple cellular signal transduction pathways to promote cell survival and to induce expression of cellular IL-10, an autocrine growth factor for EBV+ B cell lymphomas. However, the mechanisms by which LMP1 induces IL-10 are not understood. We previously showed that rapamycin, an mTOR inhibitor, could suppress IL-10 production by EBV-infected B cell lines. To test whether LMP1 activates rapamycin-sensitive signaling pathways to induce IL-10, we stably transfected EBV− BL41 cells to express a chimeric NGFR.LMP1 protein that mimics LMP1 signaling upon crosslinking. Treatment with either the p38 inhibitor SB203580 or with inhibitors that affect PI3K/AKT/mTOR signaling diminished (25–75% decrease) IL-10 production in EBV+ B cell lines. While induction of LMP1 signaling through NGFR.LMP1 resulted in phosphorylation of MAPK and PI3K pathway intermediates and production of IL-10, inhibition of either the p38 or PI3K/AKT/mTOR signaling axis markedly suppressed LMP1-mediated IL-10 production. These data indicate that p38 and PI3K/AKT/mTOR signaling contributes to LMP1-induced IL-10 production. Moreover, targeting these pathways may be an effective strategy to prevent IL-10 production and inhibit the growth of EBV+ B cell lymphomas.
Genetic variations in ITGA4 and susceptibility to MS (97.13)
Authors: O’Doherty Catherine , Ray Soma , Sun Chao , Carulli John , Vandenbroeck Koen , Antiguedad Alfredo
Published: January 2023

In recent years adhesion molecules have been studied in terms of their role in the pathophysiology and treatment of multiple sclerosis. Migration of leukocytes into tissues is facilitated by interactions between surface antigens such as Very Late Antigen (VLA4) (ITGA4/ITGA2 complex) and LFA1 and their endothelial ligands, VCAMs and ICAMs, respectively. Yednock et al (1) clearly pointed to a specific role for VLA4 in the pathogenesis of the animal model of MS, experimental autoimmune encephalomyelitis (EAE). VLA4 has been shown to be upregulated in patients and its expression has been related to both disease subtype and lesion development on MRI. Furthermore, Natalizumab, the monoclonal antibody to VLA4, has recently been licensed for use in MS. It reduced relapses and development of new lesions in clinical trials. The effective treatment of MS patients with Natalizimab points to the critical involvement of VLA4 in disease pathology and, lends further credence to the possibility that the benefits of IFNB may also be mediated to a large extent by suppression of VLA4 expression. We have examined 13 single nucleotide polymorphisms in ITGA4 (the gene encoding the á subunit of VLA4) in a cohort of 352 Basque MS patients and 235 healthy controls. Single marker and haplotype analysis were performed. Two SNPs, one in the promoter region and the other in intron 2, showed modest associations with susceptibility to MS.

This work was funded by Biogen Idec.
Microfluidic platform simulation of leucocyte adhesion to the endothelium (97.8)
Authors: Williams Vivienne , Kashanin Dmitry , O’Dowd Frank , Robinson Alexander John
Published: January 2023

Leukocyte adhesion to endothelial cell bound proteins, such as ICAM-1 and VCAM-1, is an initial step of the inflammatory response. We have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. Using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. 0–5 dynes/cm2).

The adhesion profiles of resting and PMA-stimulated peripheral blood lymphocytes (PBLs) were recorded, with respect to VCAM-1, ICAM-1, and BSA. Images at each shear stress level were captured using a digital camera, and analysed using our in-house Ducocell software package. Distinct morphological changes in PMA-stimulated PBLs, compared to non-stimulated cells, can be observed. These include a less rounded appearance of the PMA-stimulated PBLs, and evidence of “uropod” formation, which anchor the T cell to the endothelium as part of the migration process.

Levels of adhesion to VCAM-1 are high (80–90%, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and PMA-stimulated PBLs. However, there is a distinct difference between the adhesion levels of non-stimulated and PMA-stimulated PBLs to ICAM-1, with PMA-stimulated cells showing a higher affinity for ICAM-1 than non-stimulated cells (approx. 70% and 40%, respectively). PBL adhesion to BSA is negligible.
A microfluidic platform providing improved throughput for neutrophil rolling and cell adhesion assays (97.9)
Authors: Ianescu-Zanetti Cristian , Blatz Andrew
Published: January 2023

A large number of processes underlying immune system biology are dependent on leukocyte adhesion and neutrophil rolling phenomena. In vitro, the control and quantification of the shear stress applied during such experiments is performed using laminar flow chambers. Despite their wide use, flow chambers suffer from low throughput and significant sterilization and setup times. Here we report on the development of a microfluidic platform that offers significant advantages over existing systems. Microfluidic designs enable the simultaneous assaying of a number of different shear regimes, while reducing reagent consumption. We present experimental data for cell adhesion and rolling neutrophil assays comparing traditional flow chamber experiments to microfluidic experiments at multiple shear force points, including time-lapse images. Biomimetic flow patterns and a wide range of shear protocols are also presented.
Lung eosinophils homing in homeostasis (98.2)
Authors: Sung Sun-sang Joseph , Lannigan Joanne A. , Rose C. Edward
Published: January 2023

Severe lung eosinophilia is a significant feature of the inflammatory response in asthma. Besides producing various secretory products that mediate tissue injury and leukocyte activation, eosinophils from inflamed tissues have been shown to present antigens to T cells. However, the phenotype and function of eosinophils in normal tissues is poorly characterized and their antigen presentation capacity controversial. With magnetic microbead selection against the major eosinophil markers, eosinophils from normal lung tissues have been enriched to 50% of the selected cells. Surface marker staining showed that eosinophils in homeostasis expressed low to undetectable levels of MHC class II and CD11c, two markers expressed by antigen presenting cells. However, they expressed the costimulation molecules CD80 and CD86. Unactivated eosinophils expressed integrins α4, α5, and α6 chains of the β1 family, high levels of the αL (CD11a) and αM (CD11b) chains of the β2 family, and αvβ3 and α4β7. They also expressed high levels of other adhesion molecules including SiglacF, ICAM-1, ICAM-2, PSGL-1, CD24, and CD44. Besides CCR3, eosinophils expressed CCR2 and CCR2-deficient mice showed marked reduction in lung eosinophils. These data indicate that in homeostasis, eosinophil antigen presentation requires the upregulation of MHC class II, whereas costimulation molecules are already on the surface. During antigen-mediated allergic airway inflammation, MHC class II expression on eosinophils is induced, rendering the eosinophils presentation-competent. Besides CCR3, eosinophil homing to lungs is mediated by CCR2 and a wide array of adhesion molecules.
Differential modulation of NK and NKT cells in the liver and spleen following IL-18 + IL-12 treatment of mice (98.9)
Authors: Subleski Jeff John , Hall Veronica L , Weiss Jonathan M , Ortaldo John R , Wiltrout Robert H
Published: January 2023

IL-18 + IL-12 treatment elicited significant antitumor activity in the liver via an increase in NK cells and a concurrent decrease in the detectable number of NKT cells. To determine both the fate of NKT cells and the origins of NK influx into the liver following IL-18 + IL-12 treatment, we optimized liver leukocyte isolation techniques and determined that IL-18 + IL-12 was not causing NKT cells to down-modulate their TCR, as was previously reported with anti-CD3 or α-GalCer activation. Also, no discernable apoptosis was detected at 6 or 24 hours. However, IL-18 + IL-12 treatment caused an increase in NKT cells in the spleen that coincided with a decrease in these cells in the liver. We also observed a decrease in NK cells in the spleen that coincided with an increase of these cells in the liver. These findings suggest that IL-18 + IL-12 induces a well coordinated but opposing relocation of selected subsets. To identify potential genes involved in cell migration, we used a chemokine array and identified the differential gene expression in the spleen and liver. In the spleen, we found that IL-18 + IL-12 induced CCL19 in the spleen which is the ligand for CCR7 on NKT cells. In contrast, in the liver this treatment induced CCL9 and CCL20 which are respective ligands for CCR1 and CCR6 on NK cells. These data delineate potentially novel, redistribution mechanisms whereby proinflammatory cytokines may modulate the liver leukocyte repertoire.
Effects of TH17 cell transfer on the airways (99.1)
Authors: McKinley Laura , Magill Amy , Logar Alison , Kolls Jay
Published: January 2023

A distinct subset of CD4+ T helper cells characterized by the production of IL-17A, IL-17F, and IL-6 has recently been discovered. The differentiation of TH17 cells is dependent on transforming growth factor-β and IL-6 while IL-23 is involved in function of TH17 cells in host defense. TH17 cells have been shown to contribute to the pathogenesis of some autoimmune disorders and models of allergic asthma show TH17 cells are involved in the development of allergic responses. In order to investigate the function of TH17 cells we polarized naïve CD4+ T cells from DO11.10 TCR transgenic mice by culturing in conditioned media containing OVA peptide, TGF-β, IL-6, IL-23, anti-IFN-γ and anti-IL-4. For comparisons we also generated TH0 cells (grown in IL-2 only) and polarized TH1 and TH2 cells.TH17 cells secreted > 5 ng/ml IL-17 in an antigen-recall response when stimulated with OVA peptide. Only transfer of TH17 cells into mice challenged with OVA protein resulted in increased chemokine secretion in the BAL fluid, including substantial levels of the IL-8 homolog KC whereas only transfer of Th2 cells resulted in eosinophil emigration into the lung. Both TH17 and TH2 cells resulted in increased mucous secreting cells in the airway. These data demonstrate that the polarized antigen specific T-cells result in specific lung pathologies.
Differential expression of beta2-integrins, CNS trafficking, and cytokine production between gamma/delta and alpha/beta T cells in EAE (99.4)
Authors: Smith Sherry Susan , Barnum Scott R
Published: January 2023

A critical step in the development of EAE is trafficking of leukocytes into the CNS. Several families of adhesion molecules, including the b2-integrins, contribute to leukocyte trafficking in EAE. However, the expression of these integrins on gd T cells in naïve mice or those with EAE remains poorly characterized. Using multiparameter flow cytometric analysis, we compared b2-integrin expression, kinetics of CNS trafficking, and cytokine production between gd and ab T cells in C57BL/6 mice over the acute course of MOG-induced EAE. We observed that b2-integrin expression on gd T cells significantly increased from baseline, peaked at day 10, and remained unchanged in the DLN or declined in the spleen and CNS by day 15. CD11a and CD11d expression on ab T cells fluctuated throughout disease in all tissues examined, while CD11b and CD11c expression peaked 10 days post-immunization then declined. Ab T cells infiltrated the CNS prior to gd T cells and were more abundant in the CNS. However, early in disease gd T cells infiltrated the CNS at a significantly higher rate than ab T cells. In addition, gd T cells, particularly those in the CNS, produced more IFN-g and TNF-a than ab T cells throughout the course of EAE. These results suggest unique roles for b2-integrins in the trafficking of gd versus ab T cells during EAE. Furthermore, our data suggest that gd T cells rapidly infiltrate the CNS early in EAE and produce cytokines that modulate acute disease.

This work was supported by grants from the National MS Society (RG-3437-A-6) and from the NIH (NS46032) to S. Barnum and (T32 AI07051-30) to S. Smith.
CD80 blockade suppresses inflammatory response to syngenic flora in TNBS colitis. (B100)
Authors: Srinivasan Mythily , Eri Rajaraman , Eri Rajaraman
Published: January 2023

Inflammatory bowel disease (IBD) is an immune-mediated inflammatory disorder of the gastrointestinal organ system characterized by chronic inflammation of the bowel with periods of exacerbation and remission. Considerable evidence suggests that IBD is a result of abnormal activation of CD4+ T cells to intestinal microflora. Therapies targeting T cell costimulation such as anti-CD80 (B7-1), anti-CD86(B7-2) mAb and CTLA-4 mAb have provided some insights on the control of intestinal mucosal inflammation. Recently short polyproline helical peptide designed to selectively inhibit CD80: receptor interactions has been reported. We investigated the therapeutic potential of this peptide in animal models of colitis. When administered at the time of transfer, the CD80 blocking peptide (500μg i.v.) protected the SCID mice from CD4+ CD45Rbhi mediated transfer colitis efficacy of this peptide. Significantly when administered at the time of boosting (day 6, 500μg i.v.), the CD80 binding peptide suppressed clinical and microscopic inflammation in TNBS colitis. The lamina propria lymphocytes isolated from CD80 binding peptide treated mice secreted significantly less IL-12p40 and IFN-g when stimulated with syngenic normal colon flora. Increased number of CD4+ mesenteric lymph node cells were annexin positive in CD80 binding peptide treated mice suggesting that peripheral deletion activated T cells as the mechanism of mediating protection.
Pulmonary Expression of Vasoactive Intestinal Peptide in a Murine Model of Allergic Asthma (B107)
Authors: Samarasinghe Amali Eashani , Hoselton Scott Alan , Schuh Jane Marie
Published: January 2023

Chronic allergic asthma is maintained by a Th2-type immune response and is characterized by persistent inflammation of the airways, goblet cell metaplasia, smooth muscle cell hyperplasia, and airway remodeling. Among its many roles, Vasoactive Intestinal Peptide (VIP) is a cytokine known to regulate lymphocytes, macrophages, mast cells, and natural killer cells. We investigated the distribution of VIP in naïve versus allergic mice that had been sensitized and challenged with live Aspergillus fumigatus fungal spores. In situ staining revealed that, although naïve animals produced VIP in the vascular endothelium, the presence of VIP-positive cells in and around the airways is minimal. Allergic mice, however, displayed exaggerated VIP expression in the bronchial epithelium of large and small airways, Type II alveolar cells, and resident macrophages. Recruited macrophages, eosinophils, and lymphocytes were also VIP-positive. Expression of VIP in allergic mice was maximal at days one and three after antigen challenge. Airborne delivery of spores increased the VIP production in distal airways above that seen when the challenge was delivered intratracheally in liquid. The basal epithelial cell layer was conspicuously and persistently negative for VIP expression in all samples. We hypothesize that cells in this layer secrete neutral endopeptidase (NEP) which is responsible for cleaving VIP in the healthy lung.
A post-allergen challenge inhibition of PARP prevents airway eosinophilia and reduces Th2 cytokines and mucus production in animal model of asthma: a clinical potential in the treatment of asthma (B109)
Authors: Naura Amarjit Singh , Oumouna Mustapha , Zerfaoui Mourad , Hans Chetan , Suzuki Yasuhiro , Boulares Hamid
Published: January 2023

Identifying therapeutic drugs that block the release or effects of Th2 cytokines after allergen exposure is an important clinical goal for treatment of allergic inflammatory diseases such as asthma. An important portion of pre-clinical research is predominantly focused on prevention rather than treatment of symptoms associated with diseases. We recently showed, using a murine model of allergic airway inflammation, that poly(ADP-ribose) polymerase (PARP) plays an important role in the pathogenesis of asthma-related lung inflammation and that PARP inhibition, by a novel inhibitor (TIQ-A), prevented airway eosinophilia in ovalbumin-challenged mice. TIQ-A injection before ovalbumin challenge in C57BL/6 mice resulted in significant suppression of Th2 cytokines production including IL-4, IL-5, and IL-13 and secretion of mucus by airway goblet cells. In the present study, we show that a single injection of TIQ-A 1 or 6 hour after ovalbumin challenge conferred a marked reduction in ovalbumin-challenge-induced eosinophilia. The reduction in eosinohilia was accompanied with an even more pronounced suppression of IL-4, IL-5, and IL-13. Such suppression was closely associated with a more pronounced reduction in mucus production by airway goblet cells. The observed protection, as late as 6 hour after the allergen challenge, may be of a great clinical importance in our search towards novel therapeutic drugs in the treatment of asthma.

Support@ NIH-HL072889 and 1P20RR18766 to HB.
NALT CD11b+ Dendritic Cell Migration Contributes to Induction of Ag Specific Immunity (B111)
Authors: Sekine Shinichi , Kataoka Kosuke , Asanuma Hideki , Fukuiwa Tatsuya , Davydova Julia , Adachi Yasuo , Kobayashi Ryoki , Fujihashi Keiko , Shizukuishi Satoshi , Yamamoto Masato , Fujihashi Kohtaro
Published: January 2023

Our previous study showed that targeting nasopharyngeal-associated lymphoreticular tissue (NALT) dendritic cells (DCs) with an adenovirus vector expressing Flt3 Ligand (Ad-FL) induced Ag-specific mucosal immunity that mediated by increased numbers of DCs and CD4+ Th1- and Th2-type cytokine responses. In this study, we examined the kinetics of DC subset formation and intracellular cytokine production in NALT and in cervical lymph nodes (CLNs). C57BL/6 mice were nasally immunized with Ad-FL and OVA three times at weekly intervals. As controls, mice were given Ad expressing the firefly luciferase protein (Ad-Luc). Mononuclear cells were isolated at various time points and the frequencies of DC subsets and intracellular cytokine production by CD4+ T cells were determined by FACS. An Increased frequency in CD11b+ DCs was seen in nasal passages (NPs) and submundibular glands (SMGs) at day 21. On the other hand, the frequency as well as the actual numbers of DCs in NALT was the highest 3 days after the second nasal immunization. Further, peak cell numbers of CD11b+ DCs in CLNs were seen at day 14. Of importance, intracellular cytokine FACS analysis showed highest IFN- γ and IL-4 production in CLNs at day14.

These results suggest that NALT CD11b+ DCs migrate into NPs and SMGs via the CLN and play an important role in the induction of both Th1- and Th2-type cytokine responses in mice given Ad-FL as nassal adjuvant.

Supported by DE 12242, AI 18958, AI 43197, and AG 025873.
The PD-1/PD-L costimulatory pathway plays a key role in Histoplasma capsulatum pathogenesis (B122)
Authors: Lazar-Molnar Eszter , Gacser Attila , Nosanchuk Joshua D , Freeman Gordon J , Almo Steven C , Nathenson Stanley G
Published: January 2023

The PD-1 costimulatory receptor negatively regulates T cell receptor signaling upon interacting with its ligands PDL1 and PDL2 expressed on antigen presenting cells (APCs) and has a key role in maintaining self-tolerance. In the present study we examined the role of PD-1 in Histoplasma capsulatum (Hc) infection. Hc is a human pathogenic dimorphic fungus that replicates within macrophages in vivo. To characterize immune responses in murine histoplasmosis, macrophages, as well as splenocytes were analyzed after intranasal infection with Hc. qRT-PCR and FACS analysis showed significant, IFN-γ-mediated up-regulation of PDL1 on both macrophages and splenocytes. The role of the PD-1 pathway in Hc pathogenesis is further demonstrated by the significant (100%) survival of PD-1 deficient mice infected with a lethal dose of Hc. Although initially inflammatory reactions developed in the lungs, the mice were disease free by day 10, as shown by histological analysis and cfu values. Also, when Hc infected C57BL/6 mice were treated with blocking anti-PD-1 monoclonal antibody, there was a significant increase in the survival of the animals (70%), whereas all untreated mice died. Our results show for the first time the importance of the PD-1/PDL pathway in anti-fungal immunity, and offer novel strategies for immunotherapy in clinical histoplasmosis.
Identification of an MHC class Ib-restricted CD8 T cell response to polyoma virus infection (B136)
Authors: Swanson Phillip Alan , Stroynowski Iwona T. , Jensen Peter E. , Lukacher Aron E.
Published: January 2023

While MHC class Ia-restricted CD8 T cell responses to viral infections have been extensively characterized, little is known about MHC class Ib-restricted CD8 T cells during viral infection. Mouse polyoma virus establishes a persistent low-level infection and induces tumors in MHC class Ia + class Ib deficient (i.e., b2m−/−) mice. Unexpectedly, mice selectively deficient for MHC class Ia molecules (i.e., Kb−/−Db−/−) mice efficiently control polyoma viral replication and are resistant to polyoma virus-induced tumors. Polyoma virus-specific CD8 T cell cloned lines were isolated from immune Kb−/−Db−/− mice. Using these cloned lines, we mapped their MHC restriction to H2-Q9, an MHC Class Ib molecule of the Qa-2 family. We identified the Q9-restricted viral epitope as a 9mer peptide from a viral capsid protein. In preliminary studies, we found that the magnitude of the Q9-restricted antiviral CD8 T cell response progressively inflates over the course of infection. Experiments are underway to visualize this novel virus-specific class Ib-restricted CD8 T cell response in vivo.

This work was supported by R01CA71971.
Vaccination with recombinant adenoviruses and dendritic cells expressing prostate-specific antigens is effective in eliciting CTL and suppresses tumor growth in the experimental prostate cancer (B153)
Authors: Kim Sol , Lee Jee-Boong , Chang Jun
Published: January 2023

Prostate cancer is currently the most commonly diagnosed cancer in men and the second leading cause of cancer-related death in men in the US. Immunological approaches may provide an alternative option for prevention and treatment of prostate cancer, since the malignant cells overexpress many unique antigens that can be targeted by immune cells. To develop vaccine candidate against prostate cancer using mouse model, we constructed three recombinant adenoviruses expressing prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA) and six-transmembrane epithelial antigen of the prostate (STEAP), that were specifically upregulated in the TRAMP-C1 cancer cell line as well as the orthotopic murine prostate cancer. Male C57BL/6 mice were immunized by intravenous injection of these recombinant adenoviruses and subsequently by subcutaneous injection of dendritic cells pulsed with TRAMP-C1 cell lysate. After subcutaneous challenge with TRAMP-C1 cells, tumor growth was significantly delayed in the immunized mice compared to the control group. Surprisingly, high levels of STEAP-specific CD8 T cell responses were detected using MHC I tetramers in the peripheral blood and the spleen, which is inversely correlated with tumor growth in tumor-challenged mice. In addition, the therapeutic effect of our vaccine regimen was also accessed and induced significant delay in growth of established tumors. Together, our results suggest that STEAP is a good immunologic target against prostate cancer and our vaccine regimen successfully elicits effective tumor-specific CD8 T cell responses and suppresses tumor growth. Further study will improve the efficacy of our vaccine candidate and could be applied for the human disease in the future.
Memory and protective cellular immunity induced by the murine orthologue of metastasis associated protein TPD52 (B155)
Authors: Bright Robert K , Lewis Jennifer D , Payton Laura A
Published: January 2023

Tumor protein D52 (TPD52) is involved in tumor cell proliferation, trafficking and metastasis. TPD52 over expression has been demonstrated in prostate, breast, lung and colon carcinomas. Murine TPD52 (mD52) has been shown to induce transformation and metastasis, and mirrors the known function and expression patterns of the human orthologue. We believe TPD52 represents a self, non-mutated tumor associated antigen (TAA) with direct relevance to human cancers. The Transgenic Adeno-carcinoma of the Mouse Prostate (TRAMP) model was employed to study mD52 as a vaccine antigen. Naïve mice were immunized with plasmid DNA encoding the full length cDNA of mD52. Following the final immunization, mice were challenged s.c. with a tumorigenic dose of mD52 positive, autochthonous TRAMP-C tumor cells. Greater than 60% of mice were tumor free 85 days post challenge with TRAMP-C1 and 40% were tumor free post challenge with TRAMP-C2 tumor cells, without induction of observable autoimmunity. Survivors of TRAMP-C2 initial tumor challenge rejected a second TRAMP-C2 tumor challenge 73 days later and remained tumor free greater than 90 days. The T cell cytokine secretion patterns from tumor challenge survivors indicated that a TH1 cellular immune response was involved in tumor rejection. These data suggest that mD52 vaccination induced a memory immune response that resulted in rejection of mD52+ tumors without inducing autoimmunity.
4-1BB and 4-1BB ligand interaction increases IL-10 during acute inflammation of mice (B162)
Authors: Shin HH , Lee JE , Kyung TW , Oh SR , Choi HS
Published: January 2023

The cell influx in the peritoneal cavity after the inflammatory stimulus lipopolysaccharide (LPS) was analyzed from wild-type and 4-1BB-deficient mice. The peritoneal injection of LPS resulted in an increase of influx of polymorphonuclear leukocytes (PMN) and macrophages in the peritoneal cavity with significantly higher number of macrophages in 4-1BB-deficient mice. Anti-4-1BBL Ab, which blocks the interaction between 4-1BB and 4-1BBL, resulted in higher number of cell influx, suggesting that blocking of 4-1BB and 4-1BBL interaction can increase PMN and macrophages influx after acute inflammation. The level of IL-10 production in wild type mice was significantly higher than that of 4-1BB-deficient mice. The peritoneal macrophages of 4-1BB-deficient mice expressed lower level of IL-10 at the mRNA and protein levels than did those of wild-type ones after LPS treatment, suggesting that peritoneal macrophages are main source of IL-10 in response to LPS. Significantly lower numbers of macrophages and PMN were recruited in PEC of 4-1BB-deficient mice after IL-10 injection, comparing with after PBS injection.

This work was supported by SRC fund to IRC, University of Ulsan from KOSEF and Ministry of Korea Sciences and Technology.
Functions of Notch signaling-dependent Thy1-expressing dendritic cells in immune response (B164)
Authors: Ishifune Chieko , Maekawa Yoichi , Yasutomo Koji
Published: January 2023

Dendritic cells (DCs) are one of key regulators in immune response through antigen presentation and producing several cytokines. Although recent studies have shown that several DC subsets have different phenotypes and functions, it is still unclear how differentiation of various DC subsets is regulated and determined. We here investigated whether Notch signaling contributes to DCs development and their functions. Thy1-expressing DCs (Thy1-DCs) dominantly differentiated by co-culture of bone marrow cells and OP9 cells overexpressing Delta1 in the presence of GM-CSF. Thy1-DCs had different phenotypes from those of reported DCs and developed in Notch signaling dependent manner. The Thy1 expressing DCs were also found in vivo and the proportion of Thy1-DCs was markedly increased in both spleen and liver of mice infected with Trypanosoma cruzi (T.cruzi). The Thy1-DCs from mice infected with T. cruzi were capable of producing high amount of IFN-gamma. Considering that IFN-gamma is a crucial cytokine for excluding T.cruzi, Notch signaling might have an important role in controlling T.cruzi infection by expanding Thy1-DCs.
CD1d-dependent activation of NKT cells by Cryptococcus neoformans-pulsed dendritic cells (B172)
Authors: Aoyagi Tetsuji , Nakamura Kiwamu , Miyazato Akiko , Nakagawa Kiyotaka , Miyazawa Teruo , Kinjyo Yuki , Kronenberg Mitchell , Kaku Mitsuo , Kawakami Kazuyoshi
Published: January 2023

Previously, we reported that NKT cells play an important role in the development of Th1 responses and host resistance to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS. In the present study, we addressed the role of dendritic cells (DCs) to activate mouse liver mononuclear cells (LMNCs), known as a rich source of NKT cells. LMNC were activated in response to C. neoformans-pulsed mouse bone marrow-derived dendritic cells (BMDCs) and produced IFN-γ, which was significantly suppressed by anti-CD1d antibody. This response was abrogated or reduced when BMDCs from CD1dKO or LMNCs from Jα18KO mice were used. In addition, NKT hybridoma cells were cultured on recombinant CD1d protein-coated plates with lipid from this fungal pathogen and IL-2 synthesis was measured. The total lipid led to their activation, although not profound in magnitude. These results suggest that DCs present some lipid antigen from C. neoformans in a CD1d-dependent manner for the activation of NKT cells.
Suppression of Toll-like receptor-mediated signaling by Cryptococcus neoformans (B177)
Authors: Inden Ken , Xiao Gang , Miyazato Akiko , Nakamura Kiwamu , Kaku Mitsuo , Kawakami Kazuyoshi
Published: January 2023

Cryptococcus neoformans is an intracellular fungal pathogen, host defense to which is mediated by cellular immunity and Th1 immune responses. This microorganism is eliminated by nitric oxide (NO) produced from activated macrophages. In the current study, we elucidate the influence of C. neoformans on NO synthesis by macrophages activated via Toll-like receptors (TLR).

A mouse alveolar macrophage cell line, MH-S, was activated by TLR ligands such as peptidoglycan (PGN), lipopolysaccharide (LPS) and CpG-oligo DNA (ODN) in the presence of IFN-ƒ×. These treatments caused NO synthesis measured by Griess method and expression of iNOS mRNA detected by RT-PCR. C. neoformans encapsulated strains, YC-11 and B3501, and an acapsular strain, Cap67, strongly inhibited these activities. For this suppression, physical interaction between MH-S and C. neoformans was requied, and heat-killed organism was not effective. Lipid fractions derived from C. neoformans did not show such inhibitory activity. This fungal microorganism inhibited the activation of NFƒÛB in TLR-transfected HEK293 cells using a luciferase reporter assay. These results suggest that C. neoformans may suppress the NO-mediated killing by alveolar macrophages activated via a TLR pathway.
Age-associated decreases in influenza-specific CD8+ T cell immunity and the effect of androgen removal (B18)
Authors: Reiseger Jessica , Fletcher Anne , Boyd Richard , Turner Stephen
Published: January 2023

Age-associated decreases in the CD8+ T cell primary and secondary responses of mice to influenza A virus infection were examined by comparing young (2 months) and aged (22–24 months) C57BL/6 mice. A significant age-associated decrease was seen in both the percentage and number of virus-specific CD8+ T cells following both primary and secondary infection. In both age groups there was a strong correlation between influenza peptide-specific tetramer+ and IFNg+ cells. Clearance of virus from the lungs, following primary infection corresponded with the time point at which maximal levels of both percentages and numbers of virus-specific CD8+ T cells were seen for both age groups. There was a delay in the maximal expansion of virus-specific CD8+ T cells in the aged mice compared to young mice following primary infection and a corresponding delay in viral clearance. A significant age-associated decrease in the cytotoxic activity was seen. Cytotoxic activity was closely correlated with the numbers of virus-specific CD8+ T cells suggesting that the age-associated problems with CD8+ T cell activity against influenza are due to decreased numbers of cells able to respond initially or a defect in expansion of these cells once exposed to the virus, rather than effector activity on a per cell basis.

This was further supported by the fact that following thymic regeneration, caused by castration, there was an increase in the numbers of naïve T cells in the periphery of aged-mice with a consequent increase in the numbers of virus-specific cells and an increased ability to clear virus.
PKCζ is a novel signal transducer in TLR4-mediated NF-κB activation (B186)
Authors: Huang Xuesong , Pan Zhixing Kevin
Published: January 2023

NF-κB is a key transcription factor mediating a wide spectrum of innate immune responses induced by bacterial lipopolysaccharide (LPS). Although LPS activating NF-κB through TLR4-TRAF6-IKK signaling cascade has been extensively studied, the detailed signal transduction pathways induced by LPS that lead to NF-κB activation, still need to be defined. Recently we found that atypical PKCζ is involved in LPS-induced activation of NF-κB in macrophages. LPS induces time-dependent activation of PKCζ, and blocking PKCζ activity by PKCζ pseudosubstrate inhibitor or siRNA effectively inhibits LPS-induced NF-kB activation. Co-immunoprecipitation with anti-PKCζ antibody indicates association between PKCζ, and TRAF6 or TAK1. PKCζ activity is TRAF6-dependent because inhibition of TRAF6 by transfection with siRNA and dominant-negative mutant attenuates LPS-induced PKCζ activation. PKCζ activating NF-κB is mediated by TAK1, which is supported by the observation that inhibition of PKCζ impairs activation of TAK1 and knock-down of TAK1 attenuates the activation of NF-κB induced by constitutively active PKCζ. The results demonstrate a signaling pathway involving PKCζ, presents in TLR4-mediated NF-κB activation, and the function of PKCζ is TRAF6-dependent and via TAK1.
Virulent Mycobacterium tuberculosis H37Rv suppresses cell membrane repair of host macrophages (B187)
Authors: Chen Minjian , Remold Heinz G.
Published: January 2023

Virulent Mycobacterium tuberculosis (Mtb) H37Rv induces necrosis of host macrophages (Mφ), initiated by mitochondrial inner membrane perturbation and plasma membrane disruption, which serves as a microbial virulence mechanism allowing dissemination of the pathogen. Infection of Mφ with Mtb creates a local disruption of the plasma membrane, which can be repaired in a Ca2+dependent manner by trafficking of intracellular vesicles to the plasma membrane as membrane donors. We demonstrate that attenuated H37Ra induce significant exocytosis of LAMP1, a lysosome marker, and mannosidase 2, a Golgi apparatus marker, to the plasma membrane surface leading to cell membrane repair. Successful membrane repair prevents fluorescent dextran (FDx) diffusion into the cytosol. In contrast, Mφ infected with virulent H37Rv show much less LAMP1 and mannosidase 2 exocytosis and more FDx diffusion. Confocal analysis indicates that plasma membrane LAMP1 is partially colocalized with synaptotagmin7 (Syt 7), an essential regulator of exocytosis. Silencing of Syt 7 leads to less LAMP1 exocytosis and increases FDx diffusion and necrosis. Suppression of mannosidase 2 exocytosis by Brefeldin A implies the endoplasmic reticulum indirectly as a contributor to membrane repair. These findings reveal a virulence mechanism of H37Rv that blocks plasma membrane repair and leads to necrosis

(Supported by NIH grant AI50216).
The Histamine H4 Receptor (H4R) Mediates Inflammatory Responses in a Th2-dependent, FITC-induced Atopic Dermatitis Model (B199)
Authors: Zhang Mai , Cowden Jeffery M. , Dunford Paul J. , Thurmond Robin L.
Published: January 2023

Histamine is an important inflammatory mediator in atopic dermatitis. The histamine H4 receptor (H4R), primarily expressed on immune cells, affects Th2 responses, dendritic cell functions, eosinophil and mast cell chemotaxis. Recent studies also demonstrate its role in experimental pruritus. In this study, we investigated the function of H4R in a Th2-dependent mouse model of atopic dermatitis. Mice were sensitized with Fluorescein isothiocyanate (FITC) on the abdominal skin, and later challenged with FITC on the ear. Ear edema and pruritic response were then evaluated. The experimental group that received JNJ7777120, a potent and specific H4R antagonist, developed less edema (28%, P <0.01) and pruritic response (68%, P <0.05). JNJ7777120 administration also inhibits cytokine and chemokine production measured from homogenates of inflamed ear. Taken together, these results suggest that the H4R is involved in inflammatory responses in this Th2-dependent atopic dermatitis model. Therefore, antagonists to the H4R may be an effective treatment for atopic dermatitis, given their dual anti-inflammatory and anti-pruritic effects.
Primary and memory B cells occupy independent homeostatic niches (B20)
Authors: Crowley Jenni Eileen , Scholz Jean L. , Migone Thi-Sau , Cancro Michael P.
Published: January 2023

The cytokines BLyS (B lymphocyte stimulator) and APRIL (a proliferation-inducing ligand) interact with three receptors – BR3, TACI, and BCMA – to regulate peripheral B cell homeostasis. Experimental evidence indicates that the BLyS-BR3 interaction governs the size and composition of primary B cell subsets by controlling the proportion of transitional (TR) cells that complete differentiation and determining the lifespan of follicular (FO) and marginal zone (MZ) B cells. The role of the BLyS family in the regulation and maintenance of memory B cell subsets remains less understood. Accordingly, we have directly tested the role of BLyS in the maintenance of memory B cells in vivo using a neutralizing anti-BLyS antibody. Mice injected with anti-BLyS show profoundly reduced numbers of TR, FO, and MZ B cells within two weeks following treatment, as well as significantly reduced serum BLyS levels. However, for mice previously immunized with NP-CGG and injected with anti-BLyS, this treatment, while similarly reducing primary B cell pools, has no effect on NP-specific memory B cell numbers. Further, upon rechallenge, the expansion of NP specific memory B cells and serum anti-NP antibody levels are similar to controls that did not receive anti-BLyS. Together, these findings indicate that memory B cells are largely independent of BLyS availability, and therefore occupy a separate homeostatic niche from primary B cells.

Supported by USPHS AI054488 to MPC and T32-AI-055428 to JEC