bluuebunny/crossref_metadata_2025 huggingface dataset:
A mechanism for inheritance of active chromatin in dividing T-helper cells (P1195)
Authors: Josefowicz Steven , Li Charles , Allis C.
Published: January 2023

Immune cell lineage specification and phenotypic stability is defined by distinct transcription factor activity and cell type-specific heritable chromatin signatures, which collectively control the transcriptional program. Heritable acquisition of chromatin states is likely to be especially relevant in activated, rapidly dividing immune cells during the initial stages of the adaptive immune response. To evaluate how chromatin signatures are propagated through the cell cycle in CD4+ T cells we have combined single cell quantitative global chromatin modification analysis with cell cycle analysis and chromatin immunoprecipitation (ChIP). We have identified novel combinatorial histone H3 tail modifications that are enriched in mitotic chromatin and are selectively retained at lineage specific regulatory elements. Biochemical approaches have identified chromatin-binding proteins, including bromodomain-containing (BRD) factors whose recruitment to these motifs is being explored for a role in heritable lineage specific chromatin characteristics. Our data indicate a model in which signaling to chromatin and “writing” of a combinatorial H3 motif at open chromatin during T-helper cell activation and proliferation results in heritable maintenance of lineage specific active chromatin through the cell cycle by recruitment of specific chromatin “readers” which may act as mitotic bookmarks and subsequently facilitate transcription.
MeCP2 reinforces STAT3 signaling and effector T cell differentiation by controlling miR-124-mediated suppression of SOCS5 (P1125)
Authors: Jiang Shan , Li Chaoran , McRae Gabrielle , Lykken Erik , Wan Ying , Sevilla Jose , Li Qi-jing
Published: January 2023

Methyl-CpG binding protein 2 is an X-linked multifunctional epigenetic regulator whose heterozygous mutation causes the neurological disorder Rett Syndrome in 1:10,000 women, and has also been linked to multiple autoimmune disorders. Speculating that loss of Methyl-CpG binding protein 2 might result in mechanistically similar defects in the nervous and immune systems, we examined Methyl-CpG binding protein 2’s role in regulating CD4+ T cells’ antigen response. We showed that Methyl-CpG binding protein 2 was indispensable for Th1 and Th17 differentiation and Th1- or Th17-mediated pathology both in vitro and in vivo. Loss of Methyl-CpG binding protein 2 in CD4+ T cells resulted in impaired transcription of microRNA miR-124, and consequently relieved miR-124-mediated repression of SOCS5 translation. The accumulation of SOCS5 inhibited cytokine-induced STAT1 and STAT3 activation that was required for Th1 and Th17 differentiation. Primary neurons and astrocytes also failed to respond properly to STAT3-dependent neurotrophic factor signaling when MeCP2 was silenced. Thus, regulation of STAT3 signaling could represent a common etiology underpinning MeCP2’s importance in both the nervous and immune systems.
Epigenetic environment, transcriptional elements, and RSS sequences collectively regulate non-random VH gene usage at the mouse Igh locus. (P4394)
Authors: Choi Nancy , Verma-Gaur Jiyoti , Torkamani Ali , Feeney Ann
Published: January 2023

A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments of the immunoglobulin heavy and light chain loci. The mouse heavy chain (Igh) locus has 110 functional V gene segments that can recombine to a rearranged DJ segment. While the unequal usage of V genes has been well described, it is not understood what factors determine the frequency of gene segment utilization. To answer this question we conducted deep sequencing of 5’-RACE products amplified in an unbiased manner. The results quantitatively represented the entire expressed repertoire of Igh genes in pro-B cells. We compared this data with ChIP-seq results for several histone modifications that indicate different states of chromatin accessibility to determine whether the degree of ‘openness’ was important for differential V gene usage. RNA pol II binding and RNA-seq for both sense and antisense transcripts were examined to evaluate the role of non-coding transcription in rearrangement. ChIP-seq for transcription factors EBF and E2A were also considered, as well as insulator binding factors CTCF and Rad21, both of which have been described to influence the 3D structure and the degree of compaction at the Igh locus. The RSS sequences for each V gene family were evaluated as well. Our study describes how the collective effect of all of the above influences the decision of which V gene is recombined and emphasizes that V gene segment usage is not determined by a single factor.
A role for CD3 conformation in the preTCR/CD3 complex (P4428)
Authors: Hoffmann Michele , Parks Christopher , Gil Pages Diana
Published: January 2023

Mature T cells recognize and respond to antigens through the T cell antigen receptor (TCR). We have established that upon TCR antigenic engagement, the TCR/CD3 complex undergoes a conformational change (CD3dc) of its CD3 epsilon tails to expose a proline-rich sequence and allow downstream signaling to occur. CD3dc has been shown to be required for full TCR/CD3 signaling and function of mature T cells. Pre-TCR assembly with the CD3 complex serves as a developmental checkpoint that ensures the expression of a function variant TCRβ chain in thymocytes. Interestingly, stimulation of the preTCR complex has been found to be ligand-independent; assembly is sufficient. Knowing this, we first determined the stoichiometry of the preTCR/CD3 complex and asked the question, how does assembly impact its ability to undergo conformational change? To answer this question, we employed various novel biochemical methods developed in our laboratory including a flow-cytommetry based immunoprecipitation (IP-FCM) as well as a CD3-pull down assay (CD3-PD). We aim to study the conformation of CD3 in the preTCR/CD3 complex and establish whether or not the assembly of this receptor is enough to induce the active CD3 conformation that supports functional CD3 signaling in mature cells. With our data, we are further able to characterize previously unstudied requirements for CD3 conformational change in the preTCR/CD3 complex with the potential of using this knowledge to manipulate CD3dc in the future.
TCF-1 and LEF-1 promote CD4+ T cell lineage choice by upregulating Myb (P4439)
Authors: Steinke Farrah , Zhou Xinyuan , Yu Shuyang , Xue Hai-Hui
Published: January 2023

During late stages of T cell development, positively selected CD4+CD8+ (DP) thymocytes must make a lineage choice decision to become MHC-I-restricted cytotoxic CD8+ T cells or MHC-II-restricted helper CD4+ T cells. This lineage choice process is determined by extrinsic signals such as TCR and γc cytokines and by intrinsic transcriptional factors such as Myb, Gata3, ThPOK and Runx. The canonical Wnt effector transcription factors, TCF-1 and LEF-1, are essential for the transition from CD4-CD8- (DN) to the DP stage, but their requirement beyond the DP stage is unknown. Here we show that deficiency in TCF-1 resulted in a preferential loss of CD4+ T cells and reduced CD4/CD8 ratio, and this phenotype was exacerbated upon loss of both TCF-1 and LEF-1. By crossing TCF-1-/- mice to MHC-I-deficient strain or OT-2 CD4+ TCR transgenic strain, we found that DP thymocytes selected on MHC-II were re-directed to the CD8 lineage in the absence of TCF-1 and/or LEF-1. Mechanistically, we observed that Myb expression is greatly diminished in post-select DP thymocytes lacking TCF-1 and/or LEF-1, and that activation of the Wnt signaling pathway in post-select DP thymocytes induced Myb expression. Collectively, these findings revealed a novel role of Wnt effectors TCF-1 and LEF-1 in promoting CD4+ lineage choice, which is likely mediated by Wnt-dependent upregulation of Myb.
Identification of a novel, developmental-stage-specific enhancer in the Cd4 gene (P4442)
Authors: Swan Gregory , Sarafova Sophia
Published: January 2023

CD4 helper T cells coordinate the immune response and are highly dependent on the expression of the Cd4 gene for proper development and function. The function of a promoter, proximal enhancer, and a silencer have been well documented and together explain how the Cd4 gene gets turned on in CD4 T cells and off in CD8 T cells. However once turned on, the amount and timing of Cd4 expression varies during T cell development and activation. This modulation of CD4 surface levels is essential for proper lineage specification and T cell function. Yet, how subtle changes of CD4 expression are regulated remains unclear. We have recently identified a novel positive cis-acting transcriptional regulatory element (NCE) in the Cd4 locus. Here we demonstrate that NCE enhances Cd4 promoter function in position and orientation independent manner using a transient transfection assay with an eGFP reporter construct in RLM11 murine thymoma cell line. Using cell lines at different developmental stages, we determined that NCE functions well at the intermediate, but not the DP stage of development. This formally demonstrates that NCE functions as an enhancer and may have developmental specificity. We are in the process of confirming these findings in vivo by generating BAC transgenic mice that lack the NCE in the Cd4 locus.
NKAP is required for iNKT cell development (P4450)
Authors: Thapa Puspa , Das Joy , McWilliams Douglas , Shapiro Michael , Sundsbak Rhianna , Nelson-Holte Molly , Tangen Sarah , Anderson Joshua , Desiderio Stephen , Hiebert Scott , San'tAngelo Derek , Shapiro Virginia
Published: January 2023

Invariant Natural Killer T cells (iNKT) are innate lineage of lymphocytes with distinct developmental pathway than conventional αβ T cells. The transcriptional repressor NKAP is required for early T cell development and maturation of conventional αβ T cells. Using CD4-cre NKAP cKO mice, we show that NKAP is required for positive selection of thymocytes into the iNKT cells lineage. Deletion of NKAP using CD4-cre leads to an early block in iNKT cell development at the DP stage. This cell intrinsic defect was not due to decreased DP thymocyte survival or inability of NKAP deficient thymocytes to rearrange their Vα14-Jα18 TCR. Moreover, ectopic over-expression of Vα14-Jα18 invariant TCR α chain rescued the development of iNKT cells. This suggests that NKAP plays a role in the positive selection of thymocytes into the iNKT cell lineage. Interestingly, conditional deletion of NKAP associated protein Hdac3 using CD4-cre also leads to a similar block in iNKT cell development, but not conventional T cell development. This is first evidence demonstrating for an Hdac in T cell development. Whether NKAP and Hdac3 work together functionally to regulate the positive selection of DP thymocytes into iNKT cell lineage is currently being investigated.
Ly108 enhances PLZF expression in developing thymocytes (P4452)
Authors: Kraus Zachary , Dutta Mala , Schwartzberg Pamela
Published: January 2023

Promyelocytic zinc finger transcription factor (PLZF) is an important transcription factor expressed by natural killer T (NKT) cells and a variety of other innate T cells. Development of these PLZF positive cells require the adaptor molecule SLAM- associated protein (SAP) and the interaction of the signaling lymphocyte activation molecule (SLAM) family member receptors in addition to TCR stimulation. However, it remains unclear how these interactions influence the development of innate thymocytes. We have found that costimulation of pre-selection double positive (PS-DP) thymocytes with an agonist to the SLAM family receptor Ly108 greatly enhances PLZF expression compared to TCR stimulation alone. Furthermore, PLZF expressing cells were reduced in Ly108 deficient mice and increased in transgenic mice expressing a hyperactive Ly108 isoform. Costimulaiton with Ly108 also increased the expression of Egr-2 and the binding of Egr-2 to the PLZF (Zbtb16) promoter. Surprisingly costimulation with CD28 was unable to enhance Egr-2 nor PLZF expression in vitro suggesting that Ly108 has a unique role in PLZF expression. This work provides a novel mechanism by which innate thymocyte development is regulated via Ly108 and possibly other SLAM family receptor interactions.
Differential education of human fetal natural killer cells provides a mechanism for fetal-maternal tolerance. (P4458)
Authors: Ivarsson Martin , Loh Liyen , Marquardt Nicole , Björkström Niklas , Westgren Magnus , Nixon Douglas , Michaelsson Jakob
Published: January 2023

Little is known about the human fetal immune system, but it has for long been considered as immature and unresponsive. Here we have investigated the development and function of human fetal natural killer (NK) cells. Using advanced flow cytometry, the phenotype and function of fetal NK cells was examined. In contrast to the notion that the fetal immune system is immature, we found that human fetal NK cells differentiate as early as gestational week 15 in utero, evidenced by the presence of NK cells with decrease in CD94/NKG2A and increase in CD16 and KIR expression. NK cells with varying degrees of differentiation were found in the fetal lung, liver, spleen, lymph nodes and bone marrow. Moreover, we show that fetal NK cells are highly responsive to cytokine stimulation and are capable of killing target cells both via natural cytotoxicity and by antibody-dependent cellular cytotoxicity. Strikingly, and in contrast to the education of adult NK cells, we demonstrate that while education via CD94/NKG2A was established in fetal NK cells, both self- and non-self KIR expression rendered fetal NK cells hyporesponsive, thus potentially blunting the response to maternal allogeneic cells. Fetal NK cells were also found to be highly sensitive to TGF-β-mediated inhibition as compared to adult NK cells. Taken together, our data indicate a fundamental difference in the education and regulation of fetal compared to adult NK cells, which have implications for fetal-maternal tolerance in utero.
Critical role of histone deacetylase 11 in lineage differentiation of immune cells (P4378)
Authors: Sahakian Eva , Powers John , Rock-Klotz Jennifer , Deng Susan , Woods David , Woan Karrune , Nguyen Michael , Gabrilovich Dmitry , Sotomayor Eduardo , Pinilla Javier
Published: January 2023

Previously our group reported HDAC11 as a regulator of IL-10 expression in immune cells; however the functions of HDAC11 still remain largely unknown. Here we reveal the novel role of HDAC11 as an epigenetic regulator of lineage differentiation in leukocytes. To “visualize” dynamic changes in HDAC11 transcriptional activation, we utilized HDAC11 promoter-driven eGFP reporter mice, where eGFP expression is indicative of HDAC11 transcription. Using this system, we found that hematopoietic stem cells are devoid of eGFP expression. However, eGFP expression increases from CD4-/CD8- T-cells to single positive CD4+ and CD8+, and decreases in effector compared to naïve T-cells. In the bone marrow, expression of eGFP moderately increases in the transition from Pro- to Pre-B-cells, however in the periphery eGFP significantly increases in the B-1 stage of differentiation. Remarkably, in plasma cells eGFP expression appears to be at its highest. Moreover, less plasma cells and more B-1 cells were observed when HDAC11KO were compared to wild-type mice. Expression of eGFP in myeloblasts appears to be absent, however it increases to 50% in the promyelocytes and to 98% in the granulocytes. Strikingly, monocytes showed no eGFP expression. These results unveiled a previously unknown role of HDAC11 in hematopoiesis, regulating leukocyte lineage differentiation and expansion. These key findings will potentially lead to targeted epigenetic therapies to influence immune cell differentiation.
Human T cell responses to IL-6 and IL-27 are inhibited during cancer and chronic inflammation (P2019)
Authors: Miyahira Andrea , Simons Diana , Murad John , Lee Peter
Published: January 2023

In the complex interplay between an evolving tumor and its host, the functionality of the immune system is a key determinant in patient outcome. Optimal immune responses eliminate tumors while immune dysfunction allows disease progression. Cytokines play key roles in many immune functions known to be abrogated in cancer patients. We have previously shown that interferon responses are impaired in cancer patient lymphocytes. We hypothesized that additional alterations in cytokine signaling pathways occur in cancer patients and contribute to immune dysfunction. We used phospho-flow cytometry to assess if STAT phosphorylation in response to additional cytokines is impaired in peripheral blood immune cells from patients with various carcinomas or psoriasis, a chronic inflammatory skin condition. Compared with healthy controls, T cells from cancer and psoriasis patients exhibited significantly defective STAT1 and STAT3 responses to the gp130 cytokines IL-6 and IL-27, and correlating decreased expression of the gp130 and IL-6Rα receptors. Chronic exposure of healthy PBMCs to IL-6 led to down-regulated receptor expression and hyporesponsiveness to IL-6 and IL-27. Our studies indicate that that chronic inflammation which occurs in the settings of cancer and psoriasis contributes to T cell IL-6 and IL-27 hypo-responsiveness via exposure to well known systemically elevated IL-6 levels. We are exploring the functional consequences of these defects on T cell function in cancer.
Protective effects of orally administered fucoidan mix on immunosuppression by an experimental cancer chemotherapy (P2045)
Authors: Miyazaki Yoshiyuki , Nakamizo Masaji , Shibasaki Tetsuya , Kirino Tomomi , Saito Yuzu , Kawahara Kazuhiro , Otsuka Ken , Tachibana Hirofumi , Yamada Koji , Tachikawa Daisuke
Published: January 2023

Fucoidan are sulfated polysaccharides derived from brown seaweeds that mainly consist of L-fucose. Peoples in Asian countries eat seaweeds willingly as healthy foods. In previous studies, we have demonstrated that fucoidan augment anti-tumor immunity in an experimental mouse model and in healthy human subjects. However, availability of fucoidan to protect immune system from hazardous side effects of cancer chemotherapy has not been verified enough. Therefore, we performed a mouse model experiment in which immunosuppression was induced by subcutaneous 5-fluorouracil (5-FU) injection (1 mg/mouse/day, 5 times on alternate days) and evaluated protective effects of orally administered fucoidan mix (40 mg/mouse/day). The immunosuppression by 5-FU s.c. was confirmed with the loss in spleen weight of 5-FU-injected mice. NK cell activity was lower in 5-FU-injected mice than sham-treated control mice, but the decline was recovered by the fucoidan mix feeding. The intake of fucoidan mix significantly alleviated the reduction in interferon (IFN)-gamma production of splenocytes from 5-FU-injected mice. Furthermore, flow cytometric analyses revealed that specific population such as Th1 and CD11b+ cells were supported by the fucoidan mix feeding even in the 5-FU-injected mice. These results suggested that oral administration of fucoidan mix is effective to maintain tumor immunity during immunosuppressive cancer chemotherapy.
Deficiency in IL-4Rα signaling is associated with impaired tumor immunity (P2057)
Authors: Manzar Gohar , Kim Eun-Mi , Zavazava Nicholas
Published: January 2023

We recently made a seminal observation that Ym1, a chitinase-like lectin, activates NK cells, thereby promoting robust anti-tumor responses in vivo. This activation requires dendritic cells. We next sought to characterize the significance of endogenously expressed Ym1 in tumor immunity. In previous studies, Ym1 has been described as a marker for alternatively activated macrophages. Furthermore, the Th2-associated cytokines IL-4 and IL-13 were shown to induce Ym1 expression in macrophages. From these data, we hypothesized that IL-4Rα-/- mice are deficient in Ym1 and therefore more susceptible to tumor metastasis and growth. Indeed, IL-4Rα-/- mice exhibited reduced serum levels of Ym1. When WT or IL-4Rα-/- mice were i.v. challenged with the allogeneic murine melanoma cell line B16-F1, we observed a 12-fold increase in tumor burden in the lungs of IL-4Rα-/- mice compared to WT mice. Next, we generated bone marrow chimeras in which IL-4Rα-/- mice were adoptively transferred with either WT or IL-4Rα-/- bone marrow cells. Subsequently, the mice were challenged with B16-F1 cells and tumor burden evaluated after two weeks. Mice that received WT bone marrow exhibited significantly fewer tumor nodules in the lungs compared to control mice. In addition, their serum levels of Ym1 had been restored by the introduction of WT bone marrow cells. Altogether, these data indicate that IL-4 is important for tumor immunity via the induction of Ym1.
Immunogenicity assessment of in silico-selected T cell epitopes for a burkholderia biodefense vaccine (P4282)
Authors: Liu Rui , Desrosiers Joe , Martin Barbara , Moise Lenny , Groot Annie
Published: January 2023

Background: Burkholderia pseudomallei (BPM) and Burkholderia mallei (BM), the causative agents of Melioidosis and Glanders respectively, are Category B pathogens. Burkholderia cepacia (BC) causes severe disease in Cystic Fibrosis patients. No vaccines are available for these pathogens. We are developing a single Burkholderia vaccine that is effective for all three pathogens. Previously, we used informatics tools and HLA binding assays to select 70 promiscuous Class II T epitopes that are highly conserved in BPM, BM and/or BC. Here, an epitope-based vaccine is produced and its immunogenicity is evaluated in a mouse model. Methods: Epitopes were concatenated to form 3 multi-epitope gene constructs (PB1, PB2 and PB3) to produce DNA and protein vaccines. The VaxCAD algorithm ordered epitopes to minimize potential immunogenicity at epitope junctions. HLA DR3 transgenic mice were immunized by a DNA-prime/peptide- or protein-boost strategy. Immunogenicity was determined by IFNγ production to individual peptides by ELISpot assay. Results and Conclusion: Following DNA/peptide immunization, significant IFNγ production (p<0.01) was found in PB3- but not PB1-and PB2-immunized mice. Notably, only PB3 concatameric protein successfully expressed. DNA/PB3 protein immunization studies are ongoing. We describe the step-wise process of developing genome-derived vaccines and identify solutions to the problem of engineering epitope-rich constructs for a broadly reactive Burkholderia vaccine.
A novel multi-stage subunit vaccine against paratuberculosis induces significant immunity and reduces bacterial burden in tissues (P4304)
Authors: Thakur Aneesh , Aagaard Claus , Riber Ulla , Skovgaard Kerstin , Andersen Peter , Jungersen Gregers
Published: January 2023

Effective control of paratuberculosis is hindered by lack of a vaccine preventing infection, transmission and without diagnostic interference with tuberculosis. We have developed a novel multi-stage recombinant subunit vaccine in which a fusion of four early expressed MAP antigens is combined with a MAP protein expressed in latent infection (FET11 vaccine). FET11 vaccine proteins were formulated with CAF01 adjuvant and injected to MAP challenged calves at two different ages. 28 calves divided into two FET11 vaccine groups, a commercial vaccine and a control group were used in the study and followed for a year. The FET11 vaccine induced a significant T cell response against constituent vaccine proteins characterized by a high percentage of CD4+ T cells and participation of polyfunctional CD4+ T cells. Of the two different age groups, late FET11 vaccination conferred protective immunity characterized by a significant containment of bacterial burden in gut tissues compared to non-vaccinated animals. There was no cross-reaction with bovine tuberculosis in vaccinated animals. This novel multi-stage vaccine has the potential to become a marker vaccine for paratuberculosis.
Antigen-specific B cells responding to pneumococcal polysaccharide vaccination in newly diagnosed HIV infected individuals. (P4264)
Authors: Leggat David , Khaskhely Noor , Iyer Anita , Westerink M.A. Julie
Published: January 2023

The peripheral blood of newly diagnosed HIV infected individuals is subjected to high uncontrolled viral titers. The decimation of both T and B cells thus leaves these individuals highly susceptible to opportunistic infections, including Streptococcus pneumoniae, despite the availability of vaccines. We have previously shown using directly labeled fluorescent pneumococcal polysaccharides that the majority of pneumococcal polysaccharide-specific B cells in healthy individuals were IgM+ memory B cells. Using this method, we identified specific deficiencies in B cell populations responding to pneumococcal polysaccharide vaccination in HIV infected individuals. Namely, a significant decrease in pneumococcal polysaccharide-specific IgM+ memory B cells compared to healthy individuals. Consequently, HIV infected individuals showed reduced antibody response and opsonophagocytic titers compared to healthy individuals. Furthermore, we correlated changes in pneumococcal polysaccharide-specific B cell populations post-vaccination with individual CD4 T cell counts. We observed a decreasing trend in pneumococcal polysaccharide-specific IgM+ memory B cells with lower CD4 T cell counts. These data indicate that the loss of pneumococcal polysaccharide-specific IgM+ memory B cells in HIV infected individuals correlates with CD4 counts and results in poor responsiveness to pneumococcal vaccination.
Recombinant Ehrlichia P29 protein, but not P13 protein, induces a protective immune response in a mouse model of ehrlichiosis (P4280)
Authors: Thirumalapura Nagaraja , Crocquet-Valdes Patricia , Saito Tais , Dadfarnia Tahereh , DiCicco Beau , Walker David
Published: January 2023

Ehrlichioses are emerging tick-borne bacterial diseases of humans and animals. The diseases are caused by obligately intracelluar bacteria belonging to the genus Ehrlichia. Currently, no vaccine is available against any ehrlichial diseases. Several immunoreactive proteins of ehrlichiae identified using immune sera from human patients and animals that include the major outer membrane proteins, ankyrin-repeat proteins and tandem repeat proteins (TRPs). In the present study, we evaluated E. muris P13 and P29 proteins, which are orthologs of E. chaffeensis TRP32 and TRP47, respectively, as subunit vaccines in a mouse model of ehrlichiosis. Immunization of mice with recombinant P29, but not P13, induced significant protection against a challenge infection. The protection induced by recombinant P29 was associated with the induction of a strong IgG antibody response. In contrast to development of IgG antibodies to P13 in all E. muris-infected mice, fewer animals infected with E. muris had IgG antibodies to P29. We hypothesize that polyclonal activation of B cells by CD4+ Th1 cells and subsequent development of hypergamaglobluinemia during persistent E. muris infection impairs formation of antibodies to antigens, such as P29, with low B cell precursor frequencies. In conclusion, our study indicated that Ehrlichia P29, but not P13, induces protective immune response, and induction of antigen-specific antibody responses impaired during persistent ehrlichial infections.
MicroRNAs induced during Mycobacterium leprae infection and their effects on immune response (P3019)
Authors: Mehta Manali , Liu Philip , Modlin Robert
Published: January 2023

MicroRNAs have been shown to be crucial in regulation of immune cell development and cancer progression. Recently, it has become increasingly clear that their involvement in regulation of immune responses to infection is also important. Here we use Mycobacterium leprae, the causative agent of leprosy, as a model to determine the potential roles of microRNAs in infectious disease. The immune response to M. leprae has been well-characterized as presenting as a spectrum, ranging from a contained infection characterized by a Th1 response in tuberculoid (T-lep) patients to a disseminated infection reflecting a Th2 response in lepromatous (L-lep) patients. We performed mRNA and microRNA microarray analysis on 6 T-lep and 4 L-lep lesions and subsequently integrated this data using Ingenuity. Preliminary gene network analysis of differentially expressed microRNAs in L-lep versus T-lep yielded multiple networks potentially targeted, including antigen presentation. A supervised analysis of Th1 and Th2 gene targeting indicates that microRNAs more highly expressed in L-lep lesions, such as miR-21 and miR-146a, may preferentially target proinflammatory genes required for a Th1 immune response. Further analysis to determine the functional effects of these microRNAs and their involvement in skewing towards a Th2 immune response to infection could explain the bias in disease outcome.
Activated CD8+ T cells exhibit blunted effector functions in early HIV-1 disease (P3035)
Authors: Crawford Timothy , Hecht Frederick , Ndhlovu Lishomwa , Barbour Jason
Published: January 2023

Elevated, persistent CD8+ T cell activation is a hallmark of HIV-1 infection. CD8+ T cell activation is measured by expression of CD38 and HLA-DR antigens on CD8+ T cells from the blood. How CD8+ T cell activation state may define impairments to CD8+ T cell function remains unclear. ERK1/2 signaling is critical for multiple T cell functions, and interruption of this pathway due to immune activation and inflammation may blunt CD8+ T cell function. We previously demonstrated that a major proportion of activated (CD38+HLA-DR+) CD8+ T cells from recently HIV-1-infected antiretroviral-treatment-naïve adults are refractory to phosphorylation of the ERK1/2 kinases (p-ERK1/2-refractory). Here we hypothesized that p-ERK1/2-refractory CD8+ T cells would exhibit reduced effector function compared to CD8+ p-ERK1/2-responsive cells. We combined single-cell phospho-kinase flow cytometry with intracellular cytokine staining into a single assay (PFICS), to examine IFN-γ and perforin responses in CD8+ T cells by ERK1/2 signaling ability. On a per cell basis, p-ERK1/2-refractory cells produced less IFN-γ in response to polyclonal or HIV-1 Gag peptide stimulation (% IFN-γ+ cells was 5.5-fold lower, p=0.006 for polyclonal and 1.5-fold lower, p=0.048 for Gag) and expressed reduced levels of perforin (2.1 and 1.6-fold lower perforin MFI). Blunted CD8+ T effector functions, secondary to ERK1/2 signaling deficits concentrated within activated CD8+ T cells, may contribute to HIV immunodeficiency.
Higher expression of several interferon-stimulated genes in HIV-1-infected females after adjusting for the level of viral replication (P3039)
Authors: Chang J. Judy , Woods Matt , Lindsay Robert , Doyle Erin , Griesbeck Morgane , Chan Ellen , Robbins Gregory , Bosch Ronald , Altfeld Marcus
Published: January 2023

Background. Clinical studies have shown faster disease progression and stronger immune activation in HIV-1-infected females as compared to males for the same level of HIV-1 replication. This study assesses whether the elevated levels of HIV-1-induced IFN-α production observed in females were associated with higher interferon-stimulated gene expression levels in T cells, and hence suggesting type-I-IFN as a mechanism for the higher HIV-1-associated immune activation observed. Methods. Fluorescence activated cell sorting was used to isolate T cells and dendritic cells from PBMCs of treatment-naïve chronically HIV-1-infected individuals enrolled in ACTG 384. The expression of 98 genes involved in toll-like receptor and type-I-IFN signaling pathways were quantified using Nanostring technology on sorted cell populations. Results. Several interferon-stimulated genes were significantly correlated with HIV-1 viral load and/or CD4+ T cell count. Higher expression levels of a subset of these interferon-stimulated genes were observed in cells derived from females compared to males after adjusting for viral load, and were correlated to higher levels of T cell activation. Conclusion. These data show that higher IFN-α production and resulting higher ex vivo expression of several interferon-stimulated genes in females can contribute to higher levels of immune activation, and subsequently the observed faster HIV-1 disease progression in females for a given level of viral replication.
B cell exhaustion in sickle cell disease patients is associated with inhibited memory generation after influenza vaccination (P3009)
Authors: Szczepanek Steven , Adami Alex , Louis Deirdre , Farrington Narye , Bracken Sonali , Natarajan Prabitha , Secor Eric , Jellison Evan , Thrall Roger , Andemariam Biree
Published: January 2023

Dysregulated immunity is a hallmark of Sickle Cell Disease (SCD), and clinical evidence indicates that SCD patients mount adequate initial antigen-specific antibody responses only to have significant drops in titer levels 1-2 years post-vaccination. We thus hypothesized that SCD patients do not generate sufficient B-cell memory after vaccination. We conducted a translational study (IRB #11-026-1) using flow cytometric analysis of memory B-cells from frozen PBMCs taken from 11 SCD patients and 9 healthy controls before and 4-6 weeks after receiving the influenza vaccine. Percentages of CD19+ B-cells were similar between groups before vaccination, but only SCD patients showed a significant increase in B-cells after vaccination (P=0.04). However, control subjects generated approximately 2-fold more CD27+ memory B-cells after vaccination (P=0.03) while almost no change was observed in the SCD patients. This indicates that SCD patients generate naïve, but little to no memory B-cells after vaccination, thereby explaining the diminishing antibody titers observed by clinicians. Further analysis demonstrated that SCD patients have a significantly higher proportion of FcRL4+ exhausted B-cells at baseline when compared to controls (33% more; P=0.02). Given that FcRL4 down-regulates BCR signaling and is associated with reduced memory B-cell generation in people with chronic infections, we propose that chronic inflammation in SCD leads to B-cell exhaustion through BCR signal dampening.
Pro-inflammatory response of human bronchial epithelial cells following Burkholderia cenocepacia infection: focus on IL-1β. (P1265)
Authors: Gillette Devyn , Shah Prexy , Cremer Thomas , Gavrilin Mikhail , Besecker Beth , Sarkar Anasuya , Knoell Daren , Cormet-Boyaka Estelle , Wewers Mark , Butchar Jonathan , Tridandapani Susheela
Published: January 2023

Burkholderia cenocepacia is the causative agent of the fatal lung disease, Cepacia syndrome. Epithelial cells are the first cells challenged in airway infections, yet, their pro-inflammatory response to B. cenocepacia remains under investigated. Here, we aimed to determine pro-inflammatory mechanisms initiated by airway epithelial cells following B. cenocepacia infection. We first examined signaling pathways activated post B.cenocepacia infection, and found that phosphorylation of Erk, Akt, Iκk, and NF-κB occurred. Cytokine profiles showed that while epithelial cells secreted TNFα, IL-6, and IL-8, they did not secrete IL-1β. Strikingly, qRT-PCR showed significantly elevated mRNA levels of IL1b. Examination of IL-1β within cell lysates revealed pro-IL-1β was present at significant amounts, but the mature form failed to be secreted. This inability to secrete IL-1β was not specific to B. cenocepacia, as LPS stimulation showed similiar results. Caspase-1 is required for pro-IL-1β processing and mature IL-1β release. Interestingly, we found that caspase-1 was expressed at minimal levels in both mRNA and protein forms within epithelial cells. Transfection experiments showed that restoration of caspase-1 expression in epithelial cells permits IL-1β secretion. Our data show, for the first time, that deficiency of fundamental inflammasome components, particularly caspase-1, may be responsible for the lack of IL-1β secretion by infected epithelial cells.
Glutathione reductase is essential for host defense against bacterial infection (P1239)
Authors: Liu Yusen , Yan Jing , Ralston Melissa , Meng Xiaomei , Bongiovanni Kathleen , Jones Amanda , Nelin Leif , Rogers Lynette , Smith Charles
Published: January 2023

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione. We have recently shown that Gsr is essential for host defense against Escherichia coli in a mouse model of sepsis. While we have demonstrated that Gsr is required for the oxidative burst and the development of NETs, the role of Gsr in other phagocytic functions remains unclear. It is also unclear whether Gsr-null mice exhibit host defense defects against Gram-positive bacteria. Here we characterized the effects of Gsr deficiency on the innate immune responses to group B Streptococcus and LPS. Like E. coli, group B Streptococcus also resulted in a substantially more robust cytokine response, a markedly higher morbidity and mortality, and greater bacterial burden in Gsr-null mice than in wildtype mice. Interestingly, Gsr-deficient mice did not exhibit a greater sensitivity to LPS than did wildtype mice. Analysis of Gsr-null neutrophils revealed an impaired phagocytosis. In response to thioglycollate stimulation, Gsr-null mice mobilized far fewer phagocytes, including neutrophils, macrophages, and eosinophils, into their peritoneal cavities than did wildtype mice. The defective phagocyte mobilization is associated with heavy oxidation and aggregation of ascitic proteins, particularly albumin. Our results indicate that the oxidative defense mechanism mediated by Gsr is required for an effective innate immune response against bacteria, likely by preventing phagocyte dysfunction due to oxidative damage.
Mice lacking tissue factor in myeloid cells are susceptible to Mycobacterium tuberculosis infection (P1243)
Authors: Venkatasubramanian Sambasivan , Paidipally Padmaja , Barnes Peter , Mackman Nigel , Vankayalapati Ramakrishna
Published: January 2023

Tissue Factor (TF) is a trans membrane glycoprotein important for initiation of the coagulation cascade. Recent observations indicate that TF is expressed in cancer, metabolic diseases and infections. In the current study, we determined the role of TF in M. tuberculosis (M. tb) infection using mice lacking the TF gene in myeloid cells (Cre+). One month after infection with the M. tb virulent laboratory strain, H37Rv, the lung bacterial burden was higher in Cre+ than Cre- mice (3.7 ± 0.2 X 106 vs 1.9 ± 0.1 X 106 CFU, p=0.01). M.tb infected Cre+ mice had more CD4+ and CD8+ lymphocytes and expressed 33 and 38 fold less of IFN-γ and IL-17 mRNA in the lungs, compared to Cre- mice, as measured by real time PCR. In contrast, lung IL-10 mRNA levels were higher in M. tb-infected Cre+ mice than Cre- mice (16.04 ± 3.3 vs 4.4 ± 1 fold, p=0.03). Immunohistochemical analysis indicated that M. tb-infected Cre+ mice had more foamy macrophages in the lungs than Cre- mice. M. tbH37Rv grew more rapidly in peritoneal exudate macrophages of Cre+ mice, compared to Cre- macrophages (6.1 ± 1 X 106 vs 1.5 ± 0.6 X 106 CFU, p=0.02). Further studies indicated that M. tb-infected Cre+ macrophages undergo less mitochondrial apoptosis than Cre- macrophages (16.2 ± 2.1vs 27.9 ± 1.4 %, p=0.01). Studies are underway to determine the mechanism/s involved in increased IL-10 production, reduced mitochondrial apoptosis and increased bacterial burden of M tb infected Cre+ mice.
Monocyte-derived IL-1α enables host resistance to M. tuberculosis by activating stroma-derived TNF-α and stalling pathogen replication in vivo. (P1247)
Authors: Shayakhmetov Dmitry , Di Paolo Nelson , Shafiani Shahin , Day Tracey , Papayannopoulou Thalia , Russell David , Iwakura Yoichiro , Sherman David , Urdahl Kevin
Published: January 2023

Mycobacterium tuberculosis (M.tb) is one of the most prevalent and deadly human pathogens. The molecular mechanisms determining the outcomes of infection with M.tb remain incompletely understood. Here we show that IL-1α-mediated signaling from infected macrophages is absolutely required for effective pathogen control in a model of pulmonary M.tb infection in mice. We further show that M.tb replication and survival of infected cells in vivo is controlled through an IL-1α-driven signaling to stroma cells that secrete in response TNF-α and enable cell-intrinsic resistance to the pathogen by limiting its replication at a single cell level. The lack of IL-1α leads to profound non-resolving inflammation that contributes to pathology, rather than to pathogen control. Restoration of IL-1α expression in CD11c-positive lung leukocytes in IL-1alpha-deficient mice alleviates inflammation and improves survival of animals after M.tb infection. Our study implicates IL-1α-driven leukocyte-stroma cell crosstalk as a key step in triggering M.tb control mechanisms that are critical for host survival. These data reveal fundamental role of innate immunity in M.tb pathogenesis and define novel molecular targets for the development of drugs to control tuberculosis epidemics.
Biphasic STAT1 phosphorylation in response to double-stranded RNA (P1397)
Authors: Matsumiya Tomoh , Dempoya Junich , Xing Fei , Hayakari Ryo , Imaizumi Tadaatsu , Yoshida Hidemi , Satoh Kei
Published: January 2023

Viral nucleic acids are sensed by pattern recognition receptors, leads to produce of type I IFN to exert antiviral activities. Type I IFN binds to its cognate receptor, IFNAR, resulting in the activation of STAT1. Thus it has long been thought dsRNA-introduced STAT1 phosphorylation is through transactivation of type I IFN signaling. Foreign RNA entered into cell such as viral RNA is sensed by cytoplasmic sensors retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene-5 (MDA-5). In this study, we explored the molecular mechanism of how STAT1 phosphorylation is regulated by dsRNA through the cytosolic RNA sensors. A synthetic dsRNA polyinosinic-polycytidylic acid (polyI:C) induces STAT1 phosphorylation. We found the polyI:C-induced initial STAT1 phosphorylation is dependent on RIG-I pathway and MDA-5 is not involved in the STAT1 phosphorylation. Neither the pathway for RIG-I nor type I IFN is involved in the late phosphorylation of STAT1. polyI:C were able to stimulate STAT1 phosphorylation in type I IFN receptor-deficient U5A cells, suggesting this initial phosphorylation is predominantly type I IFN-dependent; however, studies using IFN receptor neutralizing antibody indicated the presence of IFN independent- and RIG-I-dependent pathway for STAT1 activation. Taken together, our results showed comprehensive regulation in which dsRNA induces STAT1 phosphorylation, indicating importance of STAT1 with such a tight regulation in innate immune system.
Exome sequencing identifies novel mutations in the Toll-like receptor 3 pathway contributing to herpes simplex encephalitis susceptibility (P1403)
Authors: Sancho Shimizu Vanessa , Mashbat Bayarchimeg , Itan Yuval , Lorenzo Lazaro , Zhang Shen-Ying , Puel Anne , Levin Michael , Casanova Jean Laurent
Published: January 2023

Herpes simplex encephalitis is a rare manifestation of herpes simplex virus-1 infection. Mutations in genes of the toll-like receptor 3-interferon pathway have been shown to predispose to childhood herpes simplex encephalitis, including UNC93B1, TLR3, TRIF, TRAF3 and TBK1. We investigated herpes simplex encephalitis patients with impaired toll-like receptor 3 signaling for whom the involvement of the known genetic etiologies have been excluded. Exome sequencing was used to identify other mutations underlying herpes simplex encephalitis, paying particular attention to the toll-like receptor 3-interferon pathway by employing a candidate gene based ‘connectome’ approach. One patient in particular carried a novel heterozygous missense mutation in a known gene in the nuclear factor-κB / toll-like receptor 3 pathway. The patient’s cells showed a decrease in interferon regulatory factor-3 dimerization and reduced nuclear translocation of nuclear factor-κB consistent with the lack of type I interferon and IL6 production following toll-like receptor 3 stimulation. The patients’ cells were also more susceptible upon viral infections. Further characterization of the mutation and its impact on type I interferon signaling in the context of herpes simplex virus-1 infection will provide a better understanding of the etiology of childhood herpes simplex encephalitis.
Mechanism of induction and genotype-phenotype correlation of IL28B and ISG expression in HCV-infected primary human hepatocytes and liver (P1383)
Authors: Thomas Emmanuel , Noureddin Mazen , Rotman Yaron , Liang T. Jake
Published: January 2023

Background: IL28B gene polymorphisms associated with hepatitis C virus (HCV) clearance suggests a role for type III interferons (IFNs. However, the intrinsic innate immune response following HCV infection remains poorly understood. Methods: Immunofluorescence staining of innate immune molecules (IRF3, ISG15) were performed on HCV infected PHHs. PHHs were also studied for correlation of IL28 genotype with type III IFN and ISG expression. In addition, RNA was extracted from 60 pre-treatment archived liver biopsies and hepatic expression of IFNs was measured by qPCR. Rs12979860 genotype was determined in PHHs and patients. Results: Primary human hepatocytes infected with HCV, but not contaminating non-parenchymal cells, demonstrate activated nuclear IRF3, supporting a direct anti-viral response to HCV pathogen associated molecular pattern (PAMP). In the patient cohort, there was a significant correlation between the hepatic expression of type III IFNs (IL28B & IL29, r=0.60, P <0.0001). ISGs expression was positively correlated with Type III IFNs (e.g. for ISG115 r=0.55, p<0.001). Conclusions: Our study demonstrates that HCV infection results in a rapid and robust intrinsic innate response in the liver. Although non-parenchymal cells may subsequently participate in this response, it is the infected hepatocyte that signals the initial danger signal. Finally, the hepatic expression of ISGs in HCV patients is mainly associated with type III IFNs.
Salt-dependent chemotaxis of macrophages (P5122)
Authors: Mueller Silke , Quast Thomas , Hucke Stephanie , Klotz Luisa , Schroeder Agnes , Jantsch Jonathan , Titze Jens , Gerzer Rupert , Hemmersbach Ruth , Kolanus Waldemar
Published: January 2023

Recent studies support the hypothesis that macrophages are not only essential for efficient immune responses, but are also regulators of local tissue electrolyte composition and hence arterial hypertension. Blockade of this macrophage-dependent regulatory axis resulted in skin electrolyte accumulation and blood pressure increase. Based on the observation that macrophages accumulate in skin tissue of rats fed under high salt diet conditions, the question arose whether macrophages actively migrate to sites of high sodium storage. We therefore investigated the migratory behavior of RAW264.7 macrophages, murine bone marrow-derived macrophages and murine peritoneal macrophages toward different NaCl gradients, showing that NaCl may act as a chemotactic stimulus per se. We also assessed the reorganization of the actin cytoskeleton, a potential NaCl-induced chemokine CCL2 production and the role of the osmoprotective transcription factor TonEBP in salt-dependent chemotaxis. Although the underlying mechanism remains unidentified, we propose that salt-dependent chemotaxis might be an important functional feature of macrophages within the context of excess salt clearance in vivo.