bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential and temporal immunomodulation of α4 integrin receptors on memory T cells by Bordetella pertussis and Bordetella parapertussis infection in mice (173.24)
Authors: Nguyen Tuan , Ravindra Dipti , Ferguson Ryan , Kwong Brian , Waheed Sana , Tarlton Nicole , Abramson Tzvia
Published: January 2023

The hallmarks of B. pertussis (Bp) infection are extreme lymphocytosis and delayed recovery, which are linked to pertussis toxin (PTX). We hypothesize that PTX interferes with imprinting of α4 integrins on T-cells leading to their compromised homing to respiratory mucosa. In a murine model, immunofluorescence of lung sections implies a delayed recruitment of leukocytes in Bp but not in B. parapertussis (Bpp) and B. pertussis PTX mutant (BpTOX6) on day 5 p.i; lymphocyte recruitment is restored at day 25 p.i in Bp. At day 5 p.i, in Bpp and BpTOX6 models we reveal by flow cytometry an increase of blood memory CD4+ cells (CD44high and CD45RBlow) that express α4β1, α4β7; the respective cells are reduced in Bp infection. However, their frequency is increased at day 25 p.i, confirming delayed immune response. In vitro assays reflect a PTX-mediated inhibition of blood lymphocytes α4 integrins dependent adhesion and migration toward VCAM-1. In Bp, a high percent of lung dendritic cells (DC) expressing MHC-II and CD86 persists through day 25 p.i; however, the percent of CCR7+ DC in Bp is lower than that in Bpp at day 5 p.i. Lastly, after co-culturing with isolated Bp-lung DC from day 5 p.i for 4 days, a reduced percent of allogeneic CD4+ cells expresses α4β1, α4β7 compared to the respective models with Bpp or BpTOX6. We conclude that α4 integrins play an important role in memory CD4+ cell migration in Bordetellae, and that PTX interferes with their imprinting early in Bp infection.
Vitamin E Isoforms Differentially Regulate Intercellular Adhesion Molecule-1 Activation of PKCα in Human Microvascular Endothelial Cells (173.25)
Authors: Abdala-Valencia Hiam , Berdnikovs Sergejs , Cook-Mills Joan
Published: January 2023

Leukocytes bind to ICAM-1 on endothelial cells during leukocyte transendothelial migration. ICAM-1 is reported to activate endothelial cell xanthine oxidase (XO), protein kinase C(PKC) and ERK1/2, but the PKC isoform and the pathway involving these signals is not known. PKCα can be directly regulated by vitamin E, but it is not known whether vitamin E regulates ICAM-1 activation of PKC. Therefore, we identified an ICAM-1 signaling pathway and determined whether these signals are regulated by the vitamin E isoforms d-α-tocopherol and d-γ-tocopherol. In primary cultures of TNFα-stimulated human microvascular endothelial cells, ICAM-1 crosslinking activated the PKC isoform PKCα but not PKCβ. ICAM-1-activated XO stimulated ERK1/2 activation that then induced activation of PKCα. ICAM-1 crosslinking did not induce oxidative activation of PKCα. D-α-tocopherol inhibited ICAM-1 activation of PKCα but not the upstream signal ERK1/2. The d-α-tocopherol inhibition of PKCα was ablated by the addition of d-γ-tocopherol. In summary, ICAM-1 activation of XO and ERK1/2 stimulates endothelial PKCα without oxidation of PKCα. ICAM-1 activation of PKCα is inhibited by d-α-tocopherol and this inhibition is ablated by d-γ-tocopherol. These data are consistent with PKCα antagonist and agonist functions of d-α-tocopherol and d-γ-tocopherol, respectively, during ICAM-1 signaling.
Lymph Node Trafficking of Regulatory T Cells Is Prerequisite for Immune Suppression (173.40)
Authors: Huang Miao-Tzu , Chiang Bor-Luen
Published: January 2023

Treg cells are well known to suppress the activation of immune competent cells by direct cell contacts or by production of immune modulatory cytokines. However, the anatomic sites where Treg cells exert optimal immune regulation are unclear. Trafficking of Treg cells to either the lymph nodes or the peripheral tissues was reportedly essential for their immune suppression function and a consensus regarding this aspect is elusive. To address this issue, we investigated the transmigration characteristics of Treg cells by using the shear-stress flow assay. We found that Treg cells exhibited a significantly decreased adhesion to either IL1-beta-activated endothelial monolayer or ICAM-1/ E-selectin-coated surface as compared to activated effector T cells. The less transmigration capacity of the Treg cells suggests probable preferential localization of these cells during the exertion of immune regulation. In vivo, adoptively transferred Treg cells were able to inhibit footpad inflammation; however, pretreatment of the Treg cells with anti-CCR7 or anti-CD62L blocking antibodies prevented trafficking of Treg cells into the draining lymph nodes and abrogated their immune suppression effect during footpad inflammation. Our findings demonstrate a crucial role of lymph node localization for Treg cell immune suppression and suggest a hierarchy of the anatomic sites in Treg cell-mediated immune regulation.
IL-2/15 receptor signal strength controls T lymphocyte proliferation and metabolism in a quantitatively distinct manner. (174.8)
Authors: Arneja Abhinav , Lauffenburger Douglas , White Forest
Published: January 2023

IL-2 and IL-15 signal through a heterotrimeric surface receptor complex (IL-2/15R) composed of an α, a β, and the common gamma (γc) chain. Signaling occurs through the shared β and γc chains, and the α-chains function as high affinity ligand capture agents. Despite sharing signaling chains and pathways, IL-2 and IL-15 have non-redundant roles in T cell biology. In vitro, although IL-2 and IL-15 function as equivalent mitogens, they greatly differ in their activities as growth factors. T cells cultured with IL-2 are larger in size, show increased protein synthesis, and increased amino-acid uptake compared to T cells cultures in IL-15. In vivo, IL-2 is critical for the initial clonal expansion and retraction of activated T cells, whereas, IL-15 is in the differentiation, and maintenance of memory T cells. The mechanism through which IL-2 and IL-15 can trigger distinct phenotypes while initiating identical signaling pathways remains elusive. Our results show that T cell proliferation and metabolism are controlled in a quantitatively distinct manner through IL-2/15R signal strength independent of the cytokine identity. Proliferation is controlled by signal strength in a digital manner, whereas cell size and metabolism are controlled in an analog fashion. These results suggest that IL-2 and IL-15 can trigger distinct phenotypes by differentially regulating the strength of IL-2/15R signal, without activating distinct biochemical signaling pathways.
JAK-STAT pathways are involved in the BAFF signaling of peripheral monocytes of patients with primary Sjögren’s syndrome. (174.9)
Authors: Yoshimoto Keiko , Tanaka Maiko , Kojima Masako , Ogata Hideko , Suzuki Katsuya , Kameda Hideto , Abe Tohru , Takeuchi Tsutomu
Published: January 2023

Background and Purpose: We have demonstrated that peripheral monocytes of patients with primary Sjögren’s syndrome (pSS) produces abnormally high amount of IL-6 in vitro upon stimulation with soluble BAFF (sBAFF). We have also found that this production is strongly suppressed by a JAK3 inhibitor. In the present study, we have further investigated a regulatory mechanism of the production of IL-6 by sBAFF-stimulated pSS monocytes. Materials and Methods: Peripheral monocytes of pSS patients and age-matched normal individuals were stimulated in vitro with sBAFF in the presence or absence of a JAK3 inhibitor. The expression levels of IL-6 and STAT protein families were analyzed by ELISA and/or quantitative RT-PCR.. Results and Discussion: The elevated production of IL-6 by sBAFF-stimulated pSS monocytes was strongly suppressed by a JAK3 inhibitor in a dose dependent manner. Quantitative PCR analysis indicated that stimulation of pSS monocytes with sBAFF remarkably induced the expression of JAK3 as compared with the control monocytes. Similarly, the expression levels of STAT3 and STAT4 in pSS monocytes were increased upon stimulation with sBAFF, while those of STAT5 and STAT6 did not change under the same conditions. Moreover, the JAK3 inhibitor suppressed the elevated expression of STAT4. Taken together, these data suggest that a JAK3-STAT4 pathway may be involved in the production of IL-6 by BAFF-stimulated peripheral monocytes.
Lunatic Fringe: A key regulator of notch ligand Dll4 mediated Th2 inflammation during viral exacerbation of allergic response. (175.4)
Authors: Mukherjee Sumanta , Berlin Aaron , Maillard Ivan , Kunkel Steven , Lukacs Nicholas
Published: January 2023

Children exposed to environmental cockroach allergen (CRA) have increased susceptibility to develop chronic pulmonary disorders such as asthma later in their life. Moreover when these children encounter a respiratory viral infection it exacerbates their existing condition. The clinical manifestations include increased mucus production and airway hyperreactivity and peribronchial Th2 inflammation, necessitating immediate hospitalization and frequent recurrent hospital visits. In the present study we examined respiratory syncytial virus (RSV) exacerbated allergic responses. Previous studies showed that notch is required for the development of Th2 responses. However the role of notch ligand-Delta like-4 (Dll4) in viral exacerbation is unknown. Here, using a clinically relevant mouse model of RSV exacerbation an abrogation of mucus production and Th2 responses when Dll4 is blocked was observed. Further, our results from CD4-T cell skewing assays suggest that Dll4 augments IL4 production in Th2 cells in an antigen specific manner. Interestingly, both in vitro and in vivo an existing Th2 environment increased lunatic fringe (L-FNG) expression, a glycosyltransferase that enhances Notch activation by ligands of the Delta-like family, in CD4 T cells. Using loss of function assays and an RNAi approach we show that Dll4 augmented IL4 production is L-FNG dependent. These studies highlight a novel regulatory mechanism involved in Dll4 mediated Th2 responses during viral exacerbation.
Structure of Nectin-2 reveals determinants of homophilic and heterophilic interactions that control cell-cell adhesion and immune regulation (176.10)
Authors: Samanta Dibyendu , Ramagopal Udupi , Vigdorovich Vladimir , Rubinstein Rotem , Almo Steven , Nathenson Stanley
Published: January 2023

Nectin comprises a family of four immunoglobulin-like molecules. Homophilic and heterophilic interactions among nectins are implicated in cell-cell adhesion, while their interactions with members of other protein families have diverse biological functions like host-pathogen interaction and immune modulation. In particular, nectin-2, found to be up-regulated on cancer cells is capable of interacting with two receptors, CD226 and TIGIT, expressed on T and NK cells. These interactions lead to the delivery of two opposing signals to both T and NK cells. This situation is reminiscent of the well-studied pathways in T cells, in which the coinhibitory receptor CTLA-4 binds the same ligand (B7) as the coactivating receptor CD28. In order to define the molecular and structural determinants underlying the homophilic and heterophilic recognitions of nectin-2, we examined the biochemical, biophysical and structural properties of human nectin-2. The structure of nectin-2 at 1.3 Å resolution reveals that the architecture of the nectin-2 homophilic dimer resembles other members of the immunoglobulin superfamily and defines the details responsible for recognition and selectivity. Of particular note, the close proximity of charged residues at the interface is a major determinant of binding affinity. Using these biochemical and structural data, we also characterized the heterophilic binding of nectin-2 with TIGIT, which is implicated in T cell and NK cell-mediated immune modulation.
Cell type specific role of B7 for T-dependnet antibody response in vivo (176.14)
Authors: Watanabe Masashi , Hodes Richard
Published: January 2023

B7.1/B7.2 molecules are essential for eliciting immune responses through CD28-mediated T cell activation, as well as for CTLA-4-mediated negative regulation of T cell responses. Although many cell types (DC, MΦ, B cells and T cells) express B7 in vivo, the role of B7 on each cell type for immune response is not well defined. To address these questions, we generated and characterized cell type-specific B7 cKO mice. Mice expressing a B7.1flox BAC Tg on a B7.1/B7.2 DKO background (designated as B7flox) were generated and crossed to several Cre-Tg strains. To evaluate the cell type-specific role of B7 for T-dependent Ab response in vivo, B7 cKO mice were immunized with TNP-KLH/Alum, and TNP-specific Ab was measured. Ag-specific IgG1 production was B7-dependent, as evidenced by the absence of response in B7 DKO mice. Normal levels of IgG1 production were observed in B7flox mice and in B cell-specific B7 cKO (CD19-Cre x B7flox) mice. In contrast, the IgG1 response was essentially absent in DC-specific B7 cKO (CD11c-Cre x B7flox) mice. This result indicates that B7 on DC is critical for induction of IgG1 class switching, whereas B7 on B cells is not required for this response. Further studies are ongoing to elucidate cell type-specific roles of B7 for several critical steps involved in T-dependent antibody responses, including follicular helper T cell differentiation and germinal center formation, using a panel of cell type-specific B7 cKO mice.
Mast cell-macrophage interactions alter inflammatory cytokine production. (177.10)
Authors: Lama Tamang Tenchee , Pullen Nicholas , Ryan John
Published: January 2023

Increased levels of mast cells and macrophages are seen at the tumor microenvironment. Mast cells are associated with tumor progression, and macrophages have been observed to induce cancer cell motility and angiogenesis. Like mast cells, macrophages produce inflammatory cytokines such as TNF, IL-13, IL-6 and MIP-1α. The individual role of these cells are well-recognized, but very little is known about their potential interactions in this microenvironment. Our preliminary studies demonstrate that co-culturing mast cells with macrophages elevates inflammatory cytokine production. Cultures of IgE-sensitized, antigen stimulated mast cells mixed with macrophages produced higher concentration of inflammatory cytokines than either cell population alone. These data demonstrate the possibility that communication between mast cells and macrophages may alter one another’s functions, especially cytokine secretion. These studies will help us gain a better understanding of the role of mast cell-macrophage interactions and how they may contribute to both normal and pathological immunity.
Autocrine secretion of hemokinin-1 promotes the innate immunostimulatory function of mast cells. (177.21)
Authors: Sumpter Tina , Ho Chin , Tkacheva Olga , Larregina Adriana
Published: January 2023

Mast cells (MC), with pro- and anti-inflammatory functions, control innate and adaptive immune responses. Mechanisms causing such disparate functions are only starting to be elucidated. Recent advances in neuroimmune regulation suggest that MC function may be regulated, in part, by neuropeptides. MC express the neurokinin-1 receptor (NK1R), the preferred receptor for the inflammatory neuropeptides, Substance P (SP) and Hemokinin-1 (HK1). We hypothesized that agonistic signaling through the NK1R regulates MC function. We compared the ability of C57BL/6 wild type (WT) or NK1RKO bone marrow-derived MC stimulated via FcϵRI or TLR4 to secrete pro-inflammatory IL-1β, IL-6, IL-33, and TNFα, or anti-inflammatory, IL-10. NK1RKO MC secreted significantly less TNFα and IL-33 but equal levels of IL-10, IL-6 and IL-1β compared to WT-MC, results that were further confirmed with WT-MC incubated with the NK1R antagonist L760,330. As our experimental conditions lack nerve fibers and exogenous SP or HK1 did not increase cytokine secretion we postulated that the observed effects were caused by autocrine secretion of NK1R agonists by MC. Using quantitative RT-PCR, we found high expression of the tac4 gene encoding for HK1 which was further increased following MC stimulation while the levels of SP mRNA remained significantly lower in spite of MC simulation. Together our results demonstrate that autocrine HK1 and NK1R signaling are potent stimulators of the pro-inflammatory function of MC.
ADAP Regulates the Stability and Movement of SLP-76 Microclusters (178.10)
Authors: Lewis Juliana , Nguyen Ken , Bunnell Stephen
Published: January 2023

The adaptor molecule ADAP and its paralog, PRAM-1, are expressed in mononuclear cells, lymphocytes, and granulocytes. ADAP plays a crucial role in T cell receptor-induced integrin activation, conjugate formation, NF-kB activation, IL-2 production, and T cell proliferation. However, it is not clear how ADAP exerts its profound effects on T cell activation. Here, we demonstrate that endogenous ADAP is an integral component of TCR-induced SLP-76 microclusters, and is essential for their persistence and movement. Our live cell imaging studies show that when we break the ADAP-SLP-76 interaction ADAP is not recruited into SLP-76 MC and SLP-76 MC are immobile and non-persistent. Consequently, T cell adhesion via the TCR is severely impaired, indicating a novel role for ADAP in adhesion to TCR ligands. Phosphorylation of highly conserved, SLP-76-interacting tyrosines within the ADAP C-terminus is necessary for ADAP to enter SLP-76 MC, and associations mediated by the N-terminus are required to stabilize ADAP within SLP-76 MC. These data demonstrate that ADAP could participate in TCR-mediated adhesion to antigen in vivo. Consequently, ADAP and PRAM-1 could be therapeutically relevant targets for modulating TCR- and FcR-dependent signals in clinical settings.
High-throughput sequencing of Memory and Naïve T-cell receptor repertoires at the RNA and DNA levels reveals differences in relative expression of expanded TCR clones (178.12)
Authors: Desmarais Cindy , Robins Harlan
Published: January 2023

Recent advances in high throughput sequencing technologies have enabled the profiling of TCR repertoires at an unprecedented level of resolution. Adaptive Biotechnologies has developed sequencing methods that are able to sequence TCR rearrangements by sequencing either using cDNA isolated from RNA, or using genomic DNA directly. We used Adaptive’s immunoSEQ technology to profile CD8+ Memory, CD8+ Naïve, and unsorted PBMC repertoires from 2 individuals using cDNA and genomic DNA isolated from the same starting population of cells. Using this method, it is possible to identify instances of either differential over- or under-expression of particular rearrangements, and to study the overall impact of starting material on sequencing results. Understanding the implications of assaying TCR repertoires at the genomic versus RNA level is important for the interpretation of experimental results, as well as for informing the design of immune profiling experiments that use high-throughput sequencing methodologies.
The B7-CD28 paradigm revisited--- a systemic receptor array approach (178.19)
Authors: Yao Sheng , Zhu Yuwen , Zhu Gefeng , Augustine Mathew , Goode Diana , Wang Jun , Zheng Linghua , Han Xue , Luo Liqun , Broadwater Megan , Ruff William , Chen Lieping
Published: January 2023

T cell cosignaling pathways regulate T cell activation and control the fate of T cell responses. They consist of complex receptor-ligand interactions on cell surface between T cell and antigen presenting cell. The effort to identify co-signaling interactions has been hampered by low expression levels and weak affinities. We generated a comprehensive receptor array system in which over 2,500 human transmembrane proteins of hematopoietic system are expressed individually at high level and could be screened in a high throughput format to determine the binding partner of a fluorescence-labeled target protein. We report here the identification and characterization of two new interactions in the B7-CD28 family. Firstly, B7-H2, formerly known as the ligand for ICOS, is identified as the third ligand for CD28 and CTLA-4 in human. B7-H2 interacts with CD28 through a non-overlapping domain as B7-1 and B7-2. B7-H2-CD28 interaction is essential for the costimulation of human T cells’ primary responses to allogeneic antigens and memory recall responses. B7-H2-CD28 costimulation upregulates survival factor Bcl-xL, suppresses cell cycle inhibitor p27kip1 and induces signaling cascade of ERK and AKT kinases-dependent pathways. Secondly, B7-1 is identified as an inhibitory counter-receptor on T cell for B7-H1. Our study reveals new cosignaling interactions as potential targets for immune-modulation and provides a platform to rapidly discover receptor-ligand interactions in a large scale.
T lymphocytes are sensitive to LIF and possess (i) a LIF/IL6 axis and (ii) a LIF/NANOG axis (178.2)
Authors: Metcalfe Su
Published: January 2023

Subtractive genome-wide analysis of transplantation tolerance has revealed a novel stem cell gene - MARCH-7 - as one of only 8 genes specifically associated with tolerance in vivo. Further studies revealed (i) MARCH-7 as an E3 ligase required for degradation of leukaemia inhibitory factor receptor (LIF-R); and (ii) LIF itself is linked to T cell lineage determination and tolerance. Further, LIF proved to be functionally linked to IL6, each generating opposing feed forward loops during T cell differentiation and leading respectively to either Treg (LIF), or TH17 (IL6). Modelling this LIF/IL6 axis would place MARCH-7 as pivotal: indeed, experimental evidence supports this notion with low MARCH-7 correlating with high LIF-R expression and high responsiveness to LIF. Conversely, high MARCH-7 reduced LIF responsiveness. In stem cells LIF induces NANOG, critical for pluripotency. Given the various links between (i) MARCH-7 and stem cells; (ii) MARCH-7 and regulation of endogenous LIF signalling; and (iii) LIF and NANOG expression in stem cells, we asked, in MARCH-7 null T cells, (a) is NANOG expressed ? (b) is NANOG expressed in response to exogenous LIF ? (c) does MARCH-7 gene-dose influence expression of short non-coding RNAs (miR) ? Each question was positively confirmed, unmasking a novel LIF/NANOG axis linked to MARCH-7 in the T cell lineage. A model is proposed where Treg and TH17 are downstream of a common precursor lineage sensitive to “stemness”-type signals.
IGSF4 is a novel TCR ζ-chain-interacting protein that enhances TCR-mediated signaling (178.5)
Authors: Kim Hye-Ran , Jun Chang-Duk
Published: January 2023

Immunoglobulin superfamily member 4 (IGSF4) is a known ligand of CRTAM, a receptor expressed in activated NKT and CD8+ T cells, but its function in T cell immunity has not been elucidated. Here, we show that IGSF4 directly interacts with the TCR ζ-chain and enhances TCR signaling by enhancing ζ-chain phosphorylation. Ectopic overexpression of IGSF4 enhances TCR-mediated T cell activation. In contrast, IGSF4 knockdown shows a dramatic decrease in markers associated with T cell activation compared with those in control siRNA. The transmembrane domain is essential for TCR ζ-chain association and clustering to the immunological synapse, and the ectodomain is associated with T cell interaction with APCs. IGSF4-deficient mice have impaired TCR-mediated thymocyte selection and maturation. Further, these mice reveal attenuated effector T cell functions accompanied by defective TCR signaling. Collectively, the results indicate that IGSF4 plays a central role in T cell functioning by dual independent mechanisms, control of TCR signaling and control of T cell-APC interaction.
A mixed Th1/Th2 response induces optimal resistance to Leishmania braziliensis (43.21)
Authors: Novais Fernanda , Lee James , de Oliveira Camila , Scott Phillip
Published: January 2023

Leishmania braziliensis is a protozoan that causes cutaneous lesions that heal spontaneously or induces a disease where the parasites metastasize to the nasopharyngeal mucosa. The factors responsible for the severe forms of the disease remain poorly understood, but an exaggerated Th1 response is associated with the immunopathology observed in these patients. In the absence of IFN-γ or iNOS mice are extremely susceptible to L. braziliensis, indicating that Th1 responses are essential to control these parasites. However, we hypothesized that a more moderated response might be associated with less pathology. Since we previously found that CD8 T cells inhibit a Th2 response in leishmaniasis, we tested whether depletion of CD8 T cells might promote a mixed Th1/Th2 response and less pathology. In fact, BALB/c mice infected with L. braziliensis and depleted of CD8 T cells developed smaller lesions, and unexpectedly were better able to control the parasites. This enhanced protection was accompanied by an increase in IL-4 and IL-5 levels, and an infiltration of neutrophils and eosinophils into the lesions. Eosinophils were essential for the increased resistance, since the protective effect of anti-CD8 treatment was reversed when mice were depleted of eosinophils by anti-CCR3 or anti-IL-5 monoclonal antibodies. Taken together, these data demonstrate that a mixed Th1/Th2 response induces optimal control of a L. braziliensis infection, and eosinophils are required for this outcome.
Polyfunctional T cell responses to ESAT-6 and PPD after treatment in multidrugresistant tuberculosis patients. (43.26)
Authors: Sada Isabel , Carrillo-Alduenda José Luis , Chávez-Galán Leslie , Sköld Markus , Salazar-Lezama Miguel Angel
Published: January 2023

Background: Treatment failure against Mycobacterium tuberculosis (Mtb) infection has contributed to the development of multidrug resistant (MDR) tuberculosis. Polyfunctional (PF)-T cells secrete IFN-γ+IL-2+TNF-α+ and mediate protective immunity during chronic infections. Objective: characterize and compared the frequency of PF T cells responding to ESAT-6 and PPD antigens. Methods: We followed 10 MDR TB patients longitudinally 3 months after anti-MDR TB treatment. All samples were obtained in accordance with the Institutional Review Board’s protocol. Peripheral blood mononuclear cells (PBMCs) were stimulated with ESAT-6 or PPD and prepared for FACS analysis of CD3, CD4, CD8, IFN-γ, TNF-α and IL-2. Results: Basal samples from all patients showed a positive response to ESAT-6 and PPD antigens; however, the frequency of CD4+ and CD8+ T cells producing any combination of cytokines was not different. The proportion of IFN-γ+TNF-α+IL-2+, IFN-γ+ and TNF-α ESAT-6 CD4+ T cells increased significantly (p=0.02) when compared with pretreatment values. When cells were stimulated with PPD, IFN-γ+TNF-α+, IFN-γ+ and TNF-α+ CD4+ T cells also increased. The frequency of triple positive ESAT-6 CD8+ T cells was not different. For both, ESAT-6 and PPD antigens IFN-γ+TNF-α+, IFN-γ+ and TNF-α+ CD8+ T cells increased significantly (p<0.001). Conclusions: Our results support that PF T cell responses are actually recovered during the first 3 months of therapy.
IL-18-primed ‘helper’ natural killer cells mediate the attraction and activation of dendritic cells, promoting the accumulation of type-1-polarized T cells at effector sites (45.6)
Authors: Wong Jeffrey , Muthuswamy Ravikumar , Kalinski Pawel
Published: January 2023

The chemokine-driven interaction of immune cells is essential for effective immunity against infection and cancer. We have previously shown that natural killer (NK) cells can be primed by IL-18 for unique ‘helper’ activity, promoting dendritic cell (DC) activation and DC-mediated induction of type-1 immune responses. Here, we further demonstrate that such IL-18-treated ‘helper’ NK cells are selectively primed for high expression of immature DC (iDC)-attracting chemokines, including CCL3 and CCL4, upon subsequent exposure to type I interferons, tumor cells, or other secondary signals. These ‘helper’ NK cells potently attract iDCs in a CCR5-dependent mechanism, situating them for efficient NK cell-mediated activation. We further describe the unique ability of these ‘helper’ NK cells to induce high DC production of CXCR3 and CCR5 ligands, including CXCL9, CXCL10, and CCL5, facilitating the recruitment of type-1 effector T (Teff) cells to sites of infection or tumor. Furthermore, using cells from the ascites of patients with advanced ovarian cancer, we demonstrate the therapeutic potential for using helper NK cell-inducing stimulatory factors to enhance Teff cell-attracting chemokines directly within the human tumor environment. This study demonstrates for the first time the unique chemokine regulation of ‘helper’ NK cell differentiation, and supports the therapeutic use of properly-activated NK cells in promoting type-1 immune responses against infection and cancer.
Role of Dectin-2 in the host defense against Streptococcus pneumoniae infection (45.8)
Authors: Ishii Keiko , Akahori Yukiko , Toyama Masahiko , Aoyagi Tetsuji , Yamamoto Natsuo , Saijo Shinobu , Iwakura Yoichiro , Yamasaki Sho , Kawakami Kazuyoshi
Published: January 2023

Streptococcus pneumoniae possesses a thick polysaccharide capsule. Host defense to this bacterium is mediated by neutrophilic inflammatory responses, which is critically regulated by innate immune mechanism. S. pneumoniae has been reported to be sensed by pattern-recognition receptors such as TLR2, TLR4 and SIGN-R1. In this study, we examined the role of Dectin-2 in the host defense to S. pneumoniae infection. When bone marrow-derived dendritic cells from wild-type (WT) and Dectin-2KO mice were stimulated with culture supernatants (Sup) of a strain of S. pneumoniae (URF918) or its ConA-bound fraction, IL-12p40 production was abrogated in Dectin-2KO mice. In a NFAT-GFP reporter assay, Sup provided a positive signal in Dectin-2 gene-transfected reporter cells. These results suggest that Dectin-2 senses mannose-containing polysaccharides in S. pneumoniae capsule. Dectin-2KO mice infected intratracheally with S. pneumoniae showed shorter survival and larger bacterial burden and lower IFN-γ production in lungs than WT mice. Although the neutrophilic infiltration in lungs was equivalent in both mice, the ratio of engulfed/un-engulfed neutrophils was lower in KO mice than in WT mice. The levels of anti-pneumococcal polysaccharide Ab in bronchoalveolar lavage fluid and serum were lower in KO mice than WT mice. These results suggest that Dectin-2 plays critical roles in the host defense to S. pneumoniae infection through promoting the Ab-mediated phagocytosis by neutrophils.
MHC II retrograde signaling in MDSC (46.15)
Authors: Nagaraj Srinivas , Nelson Allison , Nair Sajitha , Mcmillan Christina
Published: January 2023

It is now evident that inadequate function of the host immune system is due to suppressive factors such as MDSC. A number of studies demonstrated that MDSC induced antigen-specific tolerance of CD8+, but not CD4+ T cells. In our recent study we found that the ability of MDSC to induce antigen-specific CD4+ T-cell tolerance in vivo was dependent on the expression of MHC class II. We also found that MDSC in tumor-bearing mice can induce this suppression, as long as MDSC express a sufficient level of MHC class II molecules. Antigen-specific CD4+ T cells, but not CD8+ T cells, were able to convert MDSC to non-specific suppressor cells in vitro and in vivo and this effect was dependent on MHC class II. Our data have demonstrated that this effect required MHC class II cross-linking, which leads to up-regulation of cox2 and PGE2, which were previously implicated in MDSC mediated immune suppression. These functions of MHC class II was linked to increase in intracellular Ca2 mobilization, and activation of PKC and the mitogen activated kinases p38 and Erk. Cross-linking of IAb resulted in up-regulation of Ets-1 in MDSC. Down-regulation of Ets-1 abrogated increased cox2 expression and PGE2 production caused by IAb ligation. Our data suggests that CD4+ T cell / ETS-1/ COX2 play a major role in retrograde MHC class II signaling in MDSC and results in PGE2 synthesis. Our study may provide an understanding of the initiation and maintenance of immune suppression in cancers.
Phenotyping of tumor-infiltrating lymphocytes in melanoma using tissue cytometry (46.28)
Authors: Mansfield James , Lane Kristin , van der Loos Chris , Hoyt Clifford
Published: January 2023

In many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor immunogenicity and are a strong predictor of survival. An understanding of the phenotype and spatial distribution of TILs within tumor regions would be advantageous for characterizing host response. However, visual TIL assessment is prone to error and multimarker quantitation is difficult with standard methods. Here we present a multi-marker, computer-aided event-counting method for the determining the phenotypes of lymphocytes in melanoma sections using a novel multispectral imaging (MSI) approach. A section of a tissue microarray containing 120 melanoma cores was stained for CD3, S100, Foxp3 and hematoxylin. This was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. The images were analyzed using software which had been trained to recognize the tumor area based on the S100 staining pattern. Then the Foxp3 status of each CD3+ TIL was then determined. Results indicate that machine-learning software can be trained to accurately recognize tumor regions within each core. MSI enabled the accurate quantitation of three immunostains in the sample without crosstalk. Within the tumor region of each core it was possible to count the CD3+ TILs and then determine the Foxp3 status of each. This multimarker phenotyping and counting approach shows the potential for broad applicability in the assessment of TILs in many solid tumors.
An essential role for SREBP signaling in T cell blastogenesis (47.17)
Authors: Bensinger Steven , Kidani Yoko , Hock M. Benjamin , Argus Joseph , Komisopoulou Evangelia , Graeber Thomas
Published: January 2023

Accumulating evidence indicates that cellular metabolism is an important regulator of T cell immunity. We and others have shown that activation of T cell rapidly initiates a robust genetic and biochemical program that drives the biosynthesis of lipids. Surprisingly, inhibiting lipid synthesis markedly decreases DNA synthesis and lymphocyte proliferation, suggesting a fundamental link between lipid anabolism and cell cycle progression. To date, the molecular mechanisms underlying these intriguing observations are largely undefined. The Sterol Regulatory Element Binding Proteins (SREBP) are b-HLH transcription factors with a well-placed role in lipid homeostasis. The impact of SREBPs on T cell development and function has not been evaluated. Herein we demonstrate an essential role for SREBPs in CD8 T cell blastogenesis. Gene expression and ChIP studies coupled with metabolic flux analysis revealed an essential role for SREBP signaling in the acquisition of a lipid anabolic program by blasting T cells. In the absence of SREBP activity, mitogen-stimulated T cells do not efficiently enlarge and arrest in G0/G1 of cell cycle before undergoing apoptosis-independent cell death. Homeostatic proliferation and antigen-specific viral immunity is also compromised in the absence of SREBP activity. Taken together, these data delineate a critical role for SREBPs in T cell growth and provide a mechanistic understanding of how SREBPs regulate the acquisition of anabolic metabolism in T cells.
Visualization of bacterial dissemination and antigen presentation in gut mucosa following oral infection with murinized Listeria monocytogenes (49.15)
Authors: Xu Daqi , Sheridan Brian , Freitag Nancy , Schubert Wolf-Dieter , Lefrançois Leo
Published: January 2023

Listeria monocytogenes (Lm) crosses the intestinal barrier via Peyer’s patches (PP) and via intestinal epithelial cell (IEC) invasion. Most previous studies examining antigen (Ag) presentation and T cell priming with Lm infection were accomplished using systemic infection. However, Lm dissemination in the gut mucosa and its association with Ag presentation following a natural infection route is not well understood. To understand the mechanisms orchestrating intestinal T cell responses, we analyzed the kinetics and localization of Lm dissemination and Ag presentation among different mucosal tissues following oral infection with a murinized recombinant Lm expressing a modified internalin A protein that allows IEC entry. One day after infection, Lm were located in the subepithelial dome of the PP but were not visualized in the mesenteric lymph nodes (MLN). However, Ag in MLN could be as well detected as in PP by T cell proliferation assay. By 48 hours after infection, Lm had disseminated to the cortex of the MLN, apparently associated with cortical lymphatic sinuses where they co-localized with CD11b+Ly6g+ neutrophils. With time, Lm accumulated in the medulla of the MLN and were present in both PP and MLN for at least 5 days after infection. CD103+ dendritic cells (DC) surrounded the Lm and neutrophil clusters. Ongoing studies will visualize the location of Ag presentation and identify the relationship between DC subsets, bacterial dissemination and Ag presentation.
Non-depleting down-modulation of human B-cells through inhibitory coupling of FcγRIIB (CD32B) and the BCR with bi-specific DART molecules with extended pharmacokinetics. (51.8)
Authors: Chen Wei , Burke Steve , Huang Ling , Ciccarone Valentina , Zhang Tengfei , Alderson Ralph , Barat Bhaswati , Hotaling Tim , Wildjaja Lusiana , Gorlatov Sergey , Li Hua , Koenig Scott , Bonvini Ezio , Johnson Syd , Moore Paul
Published: January 2023

We have previously demonstrated the ability of bi-specific Dual-Affinity ReTargeting (DART) molecules to coligate the inhibitory FcγRIIb (CD32B) receptor and the BCR component, CD79B, to inhibit B-cell activation and dampen autoimmunity (Veri et al, Arthritis. Rheum. 2010). Here we describe development and characterization of CD32BxCD79B DARTs with enhanced pharmacokinetic (PK) properties through incorporation of an albumin binding domain (ABD) or an Fc region. These second generation CD32BxCD79B DARTs, expressed in CHO cells and purified to homogeneity, demonstrated anticipated multi-specific binding properties and effectively reduced naïve and memory B-cell responses in both healthy subjects and SLE patients. Both the ABD and Fc- bearing DARTs displayed increased serum half-life resulting in prolonged ability to functionally block human B-cell responses resulting in reduced circulating IgM and IgG levels in mice engrafted with human PBMC. In contrast to rituximab, CD32BxCD79B DARTs did not induce B-cell depletion in a human fetal liver CD34+ HSC reconstituted mouse model. In summary, CD32BxCD79B DARTs with extended PK properties have been developed that inhibit human B-cell activation in the absence of depletion, forming the basis for an alternate B-cell intervention strategy for controlling autoimmune disease through convenient dosing, while avoiding the more general immune suppression associated with existing B-cell targeted strategies.
Cellular sentinels for Heat Shock Protein-peptide complexes in vivo. (52.15)
Authors: Messmer Michelle , Binder Robert
Published: January 2023

Heat shock proteins (HSPs), such as gp96, harvested from cancer cells elicit potent anti-tumor immunity in mice and in patients with cancer. HSPs in the extracellular environment, released by cells or introduced through vaccination, are taken up by antigen presenting cells of the immune system, which allows for cross-presentation of the HSP-chaperoned peptides to T-cells. The goal of this study is to identify the subset(s) of cells that sample HSPs in vivo prior to initiating T cell responses. By vaccinating mice with fluorescent-labeled gp96, cells in the draining lymph node (dLN) that engage extracellular HSPs have been explored in a time and dose dependent manner. Our results show that CD4+CD11b+ cells are the predominant cells that internalized the HSP in the subcapsular region of the dLN while total CD11c+ cells internalized minor amounts of HSP. Upon further subtyping, CD8+CD11c+ cells, which have been shown by other groups to be the major cross-priming dendritic cells, did not significantly internalize the HSP. The ability of the CD4+CD11b+ and CD11c+ cells to prime T cells is examined to demonstrate cross-presenting function following internalization. These studies illustrate how HSPs act to alert the immune system of cellular damage and will be useful for optimizing HSP-based vaccination regimes by identifying possible cellular targets.
Establishment of T cell stimulation by flucloxacillin in HLA-B*5701+ flucloxacillin-naïve individuals (52.21)
Authors: Wuillemin Natascha , Adam Jacqueline , Fontana Stefano , Pichler Werner , Yerly Daniel
Published: January 2023

Rational: Drug induced liver injury (DILI) is a main cause of drug withdrawal. A particularly interesting example is flucloxacillin (FLUX)-DILI, which is associated with the HLA-B*5701 allele. To better understand this side effect, we established an in vitro model for FLUX induced T cell activation. Methods: Peripheral blood mononuclear cells from FLUX-naïve, HLA-B*5701+ healthy donors were induced by iterative in vitro restimulations with FLUX. FLUX-specific T cells (FLUX-Tc) were enriched by positive selection of CD107a expressing cells and characterized by flow cytometry and 51Cr-release assay. Results: FLUX-Tc are mainly CD8+. They react by IFNγ secretion and CD107a upregulation upon drug stimulation and are cytotoxic against autologous antigen presenting cells (APC). Cytotoxicity and reactivity was observed only if free FLUX was added to the system. FLUX-pulsed APC were not able to elicit a FLUX specific T cell response if FLUX was not additionally added. The direct addition of FLUX to FLUX-Tc resulted in their activation even in the absence of APC. This self-presentation and reactivation was still observed in the presence of the proteasome inhibitors lactacystin and bortezomib. Conclusion: The APC independent T cell reactivity and the non-involvement of the proteasome is best explained by a direct interaction of FLUX with the restricting HLA-B*5701 molecule. How and why this activation leads to a preferential damage of liver cells needs further investigation.
Specialized proresolution mediators enhance human B cell differentiation to antibody secreting cells (53.10)
Authors: Ramon Sesquile , Serhan Charles , Phipps Richard
Published: January 2023

The resolution of inflammation is an active and dynamic process critical in maintaining homeostasis. Newly identified lipid-derived mediators are key players during the process of inflammation resolution. These specialized proresolution mediators (SPM) constitute separate classes of compounds, which include lipoxins, resolvins, protectins and maresins, all derived from essential fatty acids. SPM regulate aspects of the immune response, including inhibition of neutrophil infiltration, reduction of T cell cytokine production and stimulation of macrophage phagocytic activity. However, their effects on B lymphocytes are unknown. Our study shows that the novel SPM 17-hydroxydosahexaenoic acid (17-HDHA), resolvin D1 (RvD1) but not protectin D1 (PD1), strongly increase the ability of normal human B cells to produce IgM and IgG. The increased antibody levels are due to an increased number of antibody-secreting cells. Furthermore, SPM regulate the expression of the transcription factors Blimp-1, Xbp-1 and Pax-5, and promote B cell differentiation towards a CD27+CD38+ antibody secreting cell phenotype. None of the SPM affect proliferation and are non-toxic. The increase in plasma cell differentiation and antibody production coincides with the known involvement of SPM during the late stages of inflammation and pathogen clearance. These new findings highlight the potential applications of SPM as endogenous and non-toxic adjuvants and anti-inflammatory therapeutic molecules.
In vitro and in vivo characterization of nanolipoproteins (NLPs) conjugated with innate immune agonists: implications for host-based therapeutics (53.12)
Authors: Weilhammer Dina , Blanchette Craig , Fischer Nicholas , El-Etr Sahar , Loots Gabriela , Corzett Michele , Hwang Mona , Thomas Cindy , Lychak Cheri , Mohammadi Sanaz , Rasley Amy
Published: January 2023

Modulation of the innate immune system has the potential to provide transient, non-specific protection from a variety of infectious organisms. Activation of innate immunity is rapid and short-lived, therefore methods aimed at enhancing and prolonging the response will increase the effectiveness of innate immune targeting as a therapeutic measure. In order to enhance the stimulatory properties of innate immune agonists, we have employed a nanolipoprotein (NLP) platform. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins, analogous to reconstituted HDLs. NLPs have been successfully used in numerous biotechnology applications, including drug delivery and diagnostic imaging. Here, we show that two synthetic TLR ligands: monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODNs) can be readily incorporated into NLPs. In addition, the delivery of agonist-conjugated NLPs significantly enhances immunostimulatory effects relative to agonists alone, at low doses, both in vitro and in vivo. Our data demonstrate that conjugation to NLPs promotes agonist trafficking to secondary lymphoid organs, increases cytokine production, and induces upregulation of immunoregulatory genes in peripheral organs. These data suggest that treatment with NLP:agonist conjugates may provide transient protection against lethal pathogen challenge, and experiments to determine their effectiveness at countering infection are ongoing.
Retinoids, Stilbenes and Vitis Vinifera modulate VDR-RXR cross-talk in HT22 mouse hippocampus neuron: an approach towards prevention of neuronal dysfunction in brain inflammation (54.20)
Authors: Dasgupta Subhajit , Bandyopadhyay Mausumi , Bhatt Aruna
Published: January 2023

The vitamin D receptor (VDR) belongs to a member of nuclear hormone receptor superfamily. The present study delineated a mechanism of VDR mode of action in prevention of neuronal dysfunction and damage in central nervous system. The in vitro treatment of HT 22 mouse hippocampus neuron with RXR agonist retinoic acid was found to modulate VDR. The stilbenes and vitis vinifera were also found to influence VDR in HT22 cells. Moreover, treatment of HT22 cells with retinoic acid rich carrot extract was found to attenuate paraquat induced damage of HT 22 cells. A study has been initiated to determine mode of action of the compounds and to evaluate mechanism of VDR-RXR interactive signals in attenuation of neuronal dysfunction in hippocampus in dementia and seizures. Acknowledgement. I (SDG) acknowledge Eileen McFadden and Christopher Riley for their support in research. The HT 22 cells were provided by Dr Mark S Kindy (MUSC). I am thankful to Dr Sebastiano Gattoni- Celli (MD) for his suggestions.We are thankful to the MUSC research endeavors.