bluuebunny/crossref_metadata_2025 huggingface dataset:
NKT cells support B lymphocyte functions independently of expression of the adapter SAP. (106.45)
Authors: Detre Cynthia , Keszei Marton , Kis-Toth Katalin , Castro Wilson , Magelky Erica , Tsokos George , Wang Ninghai , Terhorst Cox
Published: January 2023

Patients with X-linked lymphoproliferative disease (XLP) who lack functional SLAM-associated protein (SAP) have both impaired NKT cells development and progressive agammaglobulinemia. We report that α-galactosylceramide (αGalCer) activated NKT cells positively regulate antibody responses to haptenated protein antigens at multiple checkpoints, including germinal center formation and affinity maturation. NKT cell-dependent B cell responses were absent in SAP-/-.B6 mice that lack NKT cells. However, the small number of SAP-deficient NKT cells in SAP-/-.BALB/c mice adjuvated antibody production, but not the germinal center reaction. To test the hypothesis that SAP-deficient NKT cells can facilitate humoral immunity, SAP was deleted post-development in SAPfl/fl.tgCreERT2.B6 mice. These animals have normal numbers of SAP-deficient NKT cells, which efficiently support early antibody responses in the presence of αGalCer. NKT cell help during late responses does not depend upon the NKT cell intrinsic expression of SAP, provided that the conventional T cells express the adapter, as judged by adoptive transfer experiments. Thus, SAP-dependent signaling is only critical for those NKT-helper functions that act via CD4+ T cells. We conclude that SAP expression in NKT cells is dispensable for their ability to help antibody production, which suggests that residual NKT cells might contribute to the variable dysgammaglobulinemia in XLP patients.
Regulation of cross priming and anti-tumor immunity in vivo (107.2)
Authors: Shen Lianjun , BelleIsle Janice , Rock Kenneth
Published: January 2023

Cross priming plays an important role in immune surveillance against invading pathogens and cancers. The underlying mechanisms and regulation of this process, especially in vivo, are still not fully understood. The cross priming process in vivo is regulated and influenced by a number of factors at several levels. One factor is the state of the antigen donor cells. Another component is the contribution of innate immune receptors (TLR, CLR and NLR). We find that although the innate adaptor MyD88 is dispensable for cross priming of primary CD8 T cell responses, it is essential to generate long lasting anti-tumor immunity. Yet another factor that has been suggested is the participation of CD8α+Clec9a+ dendritic cells in cross presentation. However, we find that the primary CTL response primed by cell-associated antigen is largely unaffected or marginally compromised in mice lacking the CD8α+Clec9a+ DC subset, suggesting that other types of APCs also make a significant contribution. Taken together, many factors are involved in cross priming and its outcome.
Mapping a dynamic innate immunity protein interaction network regulating type I interferon production (108.2)
Authors: Li Shitao , Wang Lingyan , Berman Michael , Dorf Martin
Published: January 2023

We use co-immunoprecipitation and mass spectrometry to identify antiviral signaling networks which regulate innate immune responses. Fifty-eight baits were associated with 260 interacting proteins forming a Human Innate Immunity Interactome of 401 unique interactions; 21% of interactions were remodeled following RNA, DNA, or LPS stimulation. Overexpression and depletion analyses identified 22 novel genes which regulate NF-kB and ISRE reporter activity, viral replication, or virus-induced interferon production. The innate immune interactome provides a dynamic physical and regulatory network which serves as a foundation for mechanistic analysis of antiviral signaling and links innate immunity with other cell processes.
Role of BST-2/Tetherin in Hepatitis C Virus Infection (108.6)
Authors: Amet Tohti , Grantham Ayslinn , Byrd Daniel , Hu Sishun , Yu Qigui
Published: January 2023

Bone marrow stromal cell antigen 2 (BST-2, also known as tetherin/CD317/HM1.24) is an interferon (IFN)-inducible membrane protein with a potential role in innate immunity against virus infection. BST-2 has recently been identified as a host restriction factor for diverse families of enveloped viruses including retroviruses, filoviruses, arenavirus and herpesvirus. In theory, any lipid-enveloped virus could be restricted by BST-2. However, BST-2 antiviral activity against hepatitis C virus (HCV), a lipid-enveloped RNA virus, has not yet been studied. Here we demonstrate that human hepatic cell line Huh7.5.1 expresses a low level of BST-2. However, robust expression or induction of BST-2 in Huh7.5.1 can be elicited upon stimulation with IFN-α, IFN-γ or both. Over-expression of BST-2 by either interferon treatment or BST-2-expressing plasmid DNA transfection suppressed culture-type infectious HCV (JFH1) production in Huh7.5.1 cells. In addition, shRNA-mediated depletion of endogenous BST-2 resulted in enhanced HCV production. Our results have demonstrated a previously unknown anti-HCV activity of BST-2, and have also identified BST-2 as a host restriction factor against HCV infection. These results indicate that the induction of BST-2 in hepatic cells could be one of the mechanisms by which current HCV standard therapy (IFN-α plus ribavirin) achieves a sustained virological response. Our data also point out a novel therapeutic strategy to control HCV infection.
Role of innate signaling pathways in the regulation of antiviral IgG2c responses (108.9)
Authors: Raval Forum , Lien Egil , Pitha Paula , Szomolanyi-Tsuda Eva
Published: January 2023

Long-term antibody responses provide protection from re-infection and recrudescence of persistent viruses. IgG2a/c is a predominant immunoglobulin isotype in most virus infections. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88 KO mice generate low levels of virus-specific IgG after the acute phase of infection, have reduced numbers of long-lived plasma cells and make very low levels of IgG2c, but normal levels of IgG1. Investigating the pathways upstream and downstream of MyD88, here we show that in response to PyV infection (i) Unc93B1 mutant mice, which are triple deficient in TLR3, 7 and 9 have diminished IgG2c, but normal IgG1 responses; (ii) TLR9 KO and TLR7 KO mice do not have greatly altered IgG isotype responses as observed in MyD88 KO mice or the Unc93B1 mutant mice, suggesting that these receptors play a redundant role; and (iii) mice lacking IRF5, a transcriptional regulator downstream of MyD88, have greatly diminished IgG2c and normal IgG1 responses, similar to MyD88 KO mice. The isotype switching defect observed in IRF5 KO mice is B cell intrinsic, as transfer of IRF5 KO B cells and WT T cells into RAG KO mice results in diminished antiviral IgG2c secretion compared to mice reconstituted with B6 B and T cells. Therefore, our studies suggest that TLR7/9-MyD88-IRF5 pathways in B cells play an essential role in the generation of IgG2a/c responses to virus infection.
Prostaglandin E2 augments p53 mRNA transcription and protein expression in activated human B lymphocytes (109.12)
Authors: Haque Shabirul , Nieto Jennifer , Chiorazzi Nicholas , Mongini Patricia
Published: January 2023

p53 suppresses malignancy by controlling the cell cycle and apoptosis. COX-2 and downstream prostaglandin E2 (PGE2) are upregulated in malignancies and activated B cells; in the latter, PGE2 augments or diminishes survival and can elevate activation-induced cytosine deaminase (AID) and isotype-switching. Because p53 and COX-2 are co-expressed in B cells, we tested if co-expression was mechanistically linked. PGE2 was added to cell density-limiting cultures of purified B2 cells from human tonsil/spleen, pre-activated with surrogate C3dg-coated antigen + IL4 + BAFF. p53 protein was monitored by intracellular staining of CFSE-labeled cultures and p53 mRNA by q-RT-PCR. Pulses of PGE2, or the EP2 receptor-specific analog Butaprost, at d2 significantly augmented p53 protein only in d5 cycling blasts; this was preceded by elevations in p53 mRNA. Consistent with greater expression of EP2 in cycling blasts, PGE2-promotion of p53 mRNA synthesis occurred only in the latter. Studies involving Actinomycin D indicate that PGE2 augments p53 mRNA synthesis without notably affecting mRNA stability. Because transcriptionally active genes are more prone to mutation, our data suggest that autocrine or paracrine PGE2 may promote p53 mutation in activated B cells.
Congenital B cell lymphocytosis linked to a novel germline CARD11 mutation (109.13)
Authors: Snow Andrew , Xiao Wenming , Chaigne-Delalande Benjamin , Pittaluga Stefania , Stinson Jeffrey , Matthews Helen , Lu Wei , Schmitz Roland , Jhavar Sameer , Jing Huie , Su Helen , Staudt Louis , Lenardo Michael
Published: January 2023

Nuclear Factor-kappa B (NF-κB) governs the transcription of many genes for normal lymphocyte functions, but constitutive NF-κB activation is most often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we recently investigated a family with a unique, hereditary polyclonal B cell lymphocytosis. Here we report our discovery of a novel, germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a lymphocyte-specific scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. Similar to somatic, gain-of-function CARD11 mutations described in diffuse large B cell lymphoma, E127G CARD11 spontaneously oligomerizes and activates NF-κB when upon ectopic expression in lymphoid cell lines. Primary lymphocytes from these patients also display evidence of CARD11 aggregation and constitutive NF-κB activity. In contrast to B cells, however, we observed that E127G CARD11 contributes to patient T cell hyporesponsiveness upon AgR stimulation, even though the proximal CARD11-associated signaling machinery is highly similar between B and T cells. Our findings suggest that an activating, germline CARD11 mutation can selectively induce B cell expansion and preferentially predispose to B cell malignancy without perturbing T cell homeostasis.
Processing of AID-induced uracils in distinct cell cycle phases (109.8)
Authors: Jolly Chris , Sharbeen George , Yee Christine , Smith Adrian
Published: January 2023

Immunoglobulin (Ig) affinity maturation requires the enzyme AID, which converts cytosines in Ig genes into uracils. This alone produces C:G to A:T transition mutations. Processing of U:G base pairs via UNG2 or MutSα generates further point mutations, predominantly at G:C or A:T base pairs, respectively, but it is unknown why processing is mutagenic. We aimed to test whether the cell cycle phase of uracil processing determines fidelity. Accordingly, we ectopically restricted UNG2 activity in vivo to pre-defined cell cycle phases by fusing a transduced inhibitor peptide to cell cycle regulated degradation motifs. We conclude that excision of AID-induced uracils by UNG2 occurs exclusively in G1-phase, leading to mutation alongside predominantly correct repair. Surprisingly, UNG2 does not seem to process U:G base pairs at all in Ig genes undergoing replication. Preliminary data indicates that processing of AID-induced uracils via MutSα is also cell cycle regulated.
The asymmetric partitioning of mTORC1 activity during cell division facilitates CD8+ effector versus memory T cell generation (110.18)
Authors: Pollizzi Kristen , Delgoffe Greg , Lo Ying-Chun , Patel Chirag , Powell Jonathan
Published: January 2023

CD8+ T cells are an essential component of the adaptive immune response, generating both effector cells necessary for clearance of acute infection and long-lived memory cells primed to provide a rapid response upon secondary infection. The mechanism by which CD8+ T cell effector versus memory differentiation arises remains unclear. Here we show that upon antigen stimulation, mTORC1 activity is asymmetrically partitioned during cell division. Cells fated to become effector cells show high mTORC1 activity, while memory precursors have low mTORC1 signaling. These results correlate with experiments generated from T cell specific knockouts of Rheb, an mTORC1 activator, and TSC2, an mTORC1 negative regulator. CD8+ T cells deficient in mTORC1 activity fail to differentiate into effector cells, but retain memory characteristics such as surface marker expression and longevity in vivo. In contrast, CD8+ T cells with hyper-active mTORC1 signaling have potent effector capabilities both in vitro and in vivo but retain the phenotype of terminally differentiated effector cells incapable of transitioning into memory, being short lived and unable to proliferate upon secondary infection. Our data highlight a role of mTOR as a critical regulator in determining CD8+ T cell fate. Further, we suggest that the generation of CD8+ T cell effector vs. memory lineages is dependent upon the asymmetric distribution of mTORC1 signaling during cell division.
IL-1 signals regulate effector CD8 T cell expansion and memory differentiation (110.23)
Authors: Sarkar Surojit , Penny Laura , Khan Arif , Kalia Vandana
Published: January 2023

Inflammation is a critical bridge between innate and adaptive immunity. However, how CD8 T cells sense and get programmed by inflammation remains to be clearly understood. The role of the prototypic pro-inflammatory cytokine, Interleukin-1 (IL-1), in regulating CD8 T cell responses during acute infections is not known. Our studies demonstrated a necessary requirement of IL-1 signals in programming the initial expansion of CD8 T-cells following antigenic encounter. The absence of IL-1 signals resulted at least 10-fold lower Ag-specific effector and memory cells. Moreover, effector functions such as cytotoxicity and polyfunctionality (co-production of IFN-γ, TNF-α and IL-2) were also compromised in the absence of IL-1 signaling. Together, this defect in CD8 T-cell expansion and effector differentiation led to delayed pathogen clearance. In line with this, hallmark memory properties of recall proliferation and homeostatic maintenance were also significantly compromised in the absence of IL-1 signaling. While, IL-1 signals were essential for programming CD8 T-cell expansion, differentiation of memory cells intrinsically from effector cells was not affected. Collectively, these data demonstrate a critical and novel role of IL-1 signaling in promoting CD8 T cell expansion and effector differentiation, and in programming functional memory responses. This knowledge will have a major impact on our strategy of adjuventing vaccines that will effectively program protective immunity.
Enhanced memory development in mice by aptamer targeted mTORC1 inhibition in CD8+ T cells (110.3)
Authors: Berezhnoy Alexey , Castro Iris , Malek Thomas , Gilboa Eli
Published: January 2023

Recent studies have highlighted the importance of immunological memory in mediating protective immunity in chronic infectious diseases and cancer. Inhibition of intracellular mediators of T cell differentiation with pharmacological agents was shown to promote memory differentiation in mice. However, given their often pleiotropic effects, non-targeted administration of pharmacological agents could compromise their immune promoting properties. We have shown that a CD8+ T cell targeted 4-1BB aptamer -raptor siRNA conjugate downregulated mTORC1, while preserving mTORC2, activity in 60% of adoptively transferred OVA-primed OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced proliferative recall response. However, the rapamycin-generated memory cells were defective in their in vivo cytotoxic effector functions which correlated with reduced perforin expression. Likewise, rapamycin or aptamer-siRNA conjugate treatment enhanced the in vivo differentiation of gp100-primed Pmel-1 cells into memory cells. Consistent with a cytotoxic defect in the rapamycin-generated memory cells, the rapamycin-treated mice were not able to inhibit a challenge with gp100-expressing B16 melanoma tumor cells. These observations support the premise that in vivo cell-targeted inhibition of mTOR, and conceivably other mediators of T cell differentiation, will circumvent undesirable effects of non-targeted inhibition by pharmacological agents.
T Cell Factor-1 and beta-catenin regulate the development of memory-like CD8 thymocytes (110.6)
Authors: Sharma Archna , Chen Qinghua , Nguyen Trang , Yu Qing , Sen Jyoti
Published: January 2023

In addition to conventional alpha-beta T cells, recent studies have demonstrated the presence of a subset of CD8 single positive (SP) mature thymocytes, referred to as memory-like or innate-like thymocytes that acquire effector functions in the thymus as a result of the maturation process. These cells have been described in mice with mutations in KLF2, Itk, CBP genes and SLAM family members and in non-mutant Balb/c mice in response to IL-4 produced by thymocytes. Here, we demonstrate that enhanced function of transcription factors T Cell Factor (TCF)-1 and beta-catenin regulate the frequency of IL-4 producing thymocytes that promote the generation of Eomes-expressing memory-like CD8 thymocytes in trans. By contrast, TCF1-deficient mice show a paucity of these cells demonstrating an essential requirement of TCF1 for the generation of Eomes-expressing memory-like CD8 thymocytes. Generation of TCF1 and beta-catenin-dependent memory-like CD8 thymocytes requires the expression of IL-4 and IL-4 receptor. CD8 memory-like T cells migrate to the periphery and are functional. Thus, TCF1 and beta-catenin regulate the generation of memory-like CD8 T cells in the thymus in a non-cell intrinsic manner.
Mef2c is required for the development of B cells at multiple points during B cell maturation and for the expression of B cell-specific surface proteins (111.12)
Authors: Roundy Kirstin , Debnath Irina , Pioli Peter , Weis Janis , Weis John
Published: January 2023

The Mef2 family transcriptional regulator Mef2c is highly expressed in maturing bone marrow and peripheral mature B cells. To evaluate the role of this transcription factor in developing B cells, we generated a B cell specific conditional deletion of Mef2c using the Mb-1-Cre transgene that is expressed during the early stages of immunoglobulin rearrangement. Young mice possessing this defect demonstrated a significant impairment in B cell numbers in the bone marrow and spleen. This phenotype was evident in several B cell subsets; however, this deficit diminished with B cell maturation and migration to peripheral B cell compartments. The absence of Mef2c also produced a unique CD23- B cell subset that persisted with maturation and could not be induced to express CD23 in cultured, differentiated B cells. Genome wide expression analysis of immature and mature B cells lacking Mef2c indicated altered expression of several key regulatory factors for B cell function including Ciita, Fcer2α, Cr1/Cr2, and Tnfsf4. The altered expression of these genes was confirmed by transcript analysis and flow cytometry while the specificity of Mef2c involvement was confirmed with ChIP analysis. In summary, the data presented here point to a role for Mef2c as a key transcriptional regulator during multiple stages of B cell development and demonstrate its ability to control the expression of a number of proteins required for appropriate mature B cell functions.
Mi-2/NuRD chromatin remodeling complexes maintain transcriptional attenuation during B cell development (111.17)
Authors: Hagman James , Ramirez Julita , Dege Carissa , Eshleman Emily , Musselman Catherine , Kutateladze Tatiana
Published: January 2023

Our laboratory has previously demonstrated the importance of epigenetic mechanisms for activating mb-1 (Ig-α/Cd79a) promoters in B cell progenitors. In response to the transcription factors EBF1 and Pax5, mb-1 promoters undergo stepwise epigenetic remodeling prior to their transcription. This regulation includes at least two components: enhancement of chromatin accessibility and the loss of CpG methylation. These mechanisms require SWI/SNF chromatin remodeling complexes. In opposition to SWI/SNF, Mi-2/NuRD complexes provide a barrier to activation in the absence of transcriptional activators. To better understand how Mi-2/NuRD controls transcription, we developed an in vitro cell system that allows for depletion of endogenous Mi-2β(CHD4), the catalytic component of Mi-2/NuRD, concurrent with exogenous expression of wild type or mutated Mi-2β and transcriptional activators in B cells. Our data demonstrate that multiple domains in Mi-2β are required for its association with chromatin and transcriptional attenuation. Furthermore, the data suggest that Mi-2/NuRD complexes bind and stabilize DNA methylation. Knockdowns of methyl-binding proteins in Mi-2/NuRD complexes confirmed their importance for functions of Mi-2/NuRD. These data suggest a hierarchy of function in which recruitment or exclusion of chromatin remodeling complexes by transcription factors including EBF1 regulates gene expression during B cell ontogeny.
Pyruvate kinase M2 plays distinct roles in hematopoietic stem cells and leukemia (111.19)
Authors: Wang Ying-Hua , Scadden David
Published: January 2023

It has been realized that different cell states employ different metabolic modes. Whether metabolism is a determinant of cell state is unclear, but studies in cancer suggest that altering metabolic activity can modify cellular function. Cancer cells metabolize glucose through glycolysis even in the presence of oxygen, a phenomenon called Warburg effect. Normal somatic cells thought to also preferentially use glycolytic metabolism are tissue stem cells, particularly the hematopoietic stem cells (HSC) resident in the hypoxic microenvironment of the bone marrow. Recently the molecular basis for Warburg effect in cancer cells has been identified as due to a specific isoform of pyruvate kinase M2 (PKM2). Interestingly, HSC also express PKM2. Using a mouse strain that allows conditional deletion of PKM2, we observed that PKM2 depletion enhances normal HSC function as revealed by a competitive transplantation assay. The altered cellular function is accompanied with decreased glycolysis and increased oxidative phosphorylation specifically in the primitive stem/progenitor cell population. Notably however, PKM2 deletion markedly disadvantages disease development in mouse models of both acute and chronic myeloid leukemia. Such a distinction in response between normal and malignant cells is unprecedented to our knowledge and may offer a highly unique opportunity to advantage normal while disadvantaging malignant cells of the hematopoietic lineage by targeting a single metabolic enzyme.
CD27 expression patterns on human developing B cells in fetal and adult tissues (111.27)
Authors: Su Kuei-Ying , McWilliams Laurie , Liang Xiaoe , Floyd Serina , Amos Joshua , Moody M. , Kuraoka Masayuki , Kelsoe Garnett
Published: January 2023

CD27 is an accepted marker for memory B cells in humans. However, CD27 expression was recently demonstrated on substantial populations of human B cells in cord blood and fetal liver (FL), tissues that contain few if any memory B lymphocytes. In addition, CD27 expression was recently proposed to identify natural B cells, the human counterpart of murine B-1 B cells. These observations led us to explore the characteristics of human, CD27+ B cells, and particularly developing B cells. Comparing B cells from FL, cord blood, and adult bone marrow (BM), we found CD27 expression as early as the pro-B cell stage. Moreover, the frequency of CD27+ pro-B cells was higher in FL than in BM. Expression of TdT, RAG1 and VpreB in CD27+ pro-B cells was similar to conventional, CD27- pro-B cells, confirming their identity as B-lineage progenitors. Developmentally immature CD27- and CD27+ B cells showed no significant differences in VH gene usage, VH mutation frequencies, or CDRH3 length, excluding the possibility of contamination by memory B cells. Interestingly, when placed in an in vitro culture system optimized to support the maturation of human fetal pro-B cells and murine B-1 B cells, CD27+ pro-B cells differentiated into IgM+ immature and transitional B cells significantly more efficiently than did autochthonous CD27- pro-B cells. In conclusion, we have demonstrated functional CD27+ pro-B cells in FL and BM that may identify a predominantly fetal, B-cell lineage in humans.
Characterization of glucocorticoid receptor in B lymphocytes (111.3)
Authors: Gruver-Yates Amanda , Cidlowski John
Published: January 2023

Glucocorticoids play an important role in the resolution of inflammation. They are routinely used to treat allergies, asthma, sepsis, and autoimmune diseases. In addition to immune suppression, glucocorticoids can negatively impact the development and function of the immune system by inducing apoptosis, an effect well-studied in mature and developing T cells. Little is known however about glucocorticoid function specifically in B lymphocytes. We have shown that glucocorticoid receptor (GR) is present in enriched B cell populations isolated from mouse spleen. GR protein was present in all B cell (B220+) developmental subsets (Mature IgM+IgD+, Immature IgM+IgD-, and Pro/Pre IgM-IgD-) isolated from spleen. Intracellular staining for GR analyzed with flow cytometry also confirmed the presence of GR in all B cell subsets from cells isolated both from mouse spleen and bone marrow. Mean fluorescence intensity data indicated that Pro/Pre B lymphocytes had more intense staining of GR than either mature or immature populations. Ex vivo cell culture of murine splenocytes indicated that dexamethasone, a synthetic and potent glucocorticoid, reduced cell viability in culture in all B cell developmental subsets. In vivo administration of dexamethasone reduced total cell numbers of all B cell subsets present in the mouse spleen within 24 hours. These data suggest that B cells glucocorticoid signaling through GR on B cells have an important role in B cell function.
BTB-POZ transcription factors recruit the E3 ubiquitin ligase Cullin 3 to initiate lymphoid effector programs (111.38)
Authors: Mathew Rebecca , Sieler Michael , Scanlon Seth , Constantinides Michael , Bertozzi-Villa Clara , Singer Jeffrey , Bendelac Albert
Published: January 2023

The differentiation of several T and B cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodeling. We report that PLZF, the BTB-POZ transcription factor directing the innate-like effector program of NKT thymocytes was prominently associated with cullin 3 (Cul3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF induced the transport of Cul3 from the cytosol to the nucleus where the two proteins were associated within a chromatin associated/modifier protein complex. Furthermore, PLZF expression in thymocytes resulted in selective changes of ubiquitination of multiple components of this complex. Cul3 was also found associated with another BTB-POZ transcription factor, Bcl6, which directs the B cell germinal center program. Conditional deletion in mice demonstrated an absolute, cell-intrinsic requirement of Cul3 for the development of NKT cells and germinal center B cell responses. We conclude that distinct lineage-specific BTB-POZ transcription factors recruit Cul3 to alter the ubiquitination pattern of their associated chromatin associated/modifier complex. We propose that this novel function is essential to direct the differentiation of several T and B lymphocyte effector programs, and may also be involved in the oncogenic role of PLZF and Bcl6 in leukemias and lymphomas.
Cellular and molecular profiling of CD8 T cell differentiation with respect to positive and negative selection in vitro reveals a role for Notch signaling (111.45)
Authors: Dervovic Dzana , Liang Haydn , Cannons Jennifer , Elford Alisha , Ohashi Pamela , Schwartzberg Pamela , Zuniga Pflucker Juan Carlos
Published: January 2023

Differentiation of functionally mature CD8 single positive (SP) T cells is predicated by the ability of lymphocyte progenitors to integrate multiple signaling cues provided by the thymic microenvironment. In thymus, signaling through Notch receptors directs T cell progenitors to commit to the T cell lineage, undergo early T cell differentiation, and progress to the CD4+CD8+ (DP) stage of T cell development. However, it is still unclear whether Notch signals are prerequisite for differentiation or commitment of DP cells to CD8 SP stage of T cell development. Here, we demonstrate that ectopic expression of the Notch-ligand by OP9 stromal cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- bone marrow (BM)-derived hematopoietic stem cells (HSCs). We also prove that OP9-DL1 stromal cells restrict differentiation of innate CD8 T cells but allow for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. We further reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 stromal cells. Finally, using a T cell receptor (TCR) transgenic model we disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.
The role of IRF4 in regulating effector CD8+ T cell development via repression of EOMES expression. (111.51)
Authors: Nayar Ribhu , Enos Megan , Prince Amanda , Shin Hyun Mu , Hemmers Saskia , Klein Ulk , Rudensky Alexander , Berg Leslie
Published: January 2023

CD8+ T cell development in the thymus generates a predominant population of conventional naïve cells, along with minor populations of ‘innate’ T cells that resemble memory cells. Recent studies analyzing a variety of knockout or knock-in mice have indicated that impairments in the TCR signaling pathway produce increased numbers of innate CD8+ T cells, characterized by their high expression of CD44, CD122, CXCR3, and the transcription factor Eomesodermin. One component of this altered development is a non-CD8+ T cell-intrinsic role for IL-4. To determine whether reduced TCR signaling within the CD8+ T cells might also contribute to this pathway, we investigated the role of the transcription factor IRF4. IRF4 is upregulated following TCR stimulation in wild-type T cells; however, this upregulation is impaired in Itk-/- T cells, which have reduced responses to TCR signaling. Further, analysis of IRF4-deficient CD8+ thymocytes showed normal development of thymic CD8+ T cells. Interestingly, IRF4-deficient peripheral CD8+ T cells acquired a memory phenotype and expressed the transcription factor Eomesodermin. We also show that activation of naïve IRF4-deficient CD8+ T cells leads to rapid and robust expression of Eomesodermin. Together, these data indicate that IRF4 upregulation following CD8+ T cell activation normally suppresses Eomesodermin expression, thereby regulating the differentiation pathway of CD8+ effector T cells.
PAD4 regulates proliferation of hematopoietic multipotent progenitors by directly controlling c-Myc expression. (111.7)
Authors: Nakashima Katsuhiko , Arai Satoko , Miyazaki Toru
Published: January 2023

Peptidylarginine deiminase 4 (PAD4) functions as a transcriptional co-reporessor by catalyzing the conversion of arginine to citrulline residues of histone H3. Although the high expression of PAD4 in the bone marrow may suggest its involvement in the hematopoiesis, its precise contribution remains unclear. Here we found that PAD4 is highly expressed in the hematopoietic stem and multipotent cells (Lineage-Sca1+c-kit+; termed LSK cells). Microarray and RT-qPCR analysis showed that the mRNA level of c-myc in PAD4-deficient (PADI4-/-) LSK cells was increased compared to PADI4+/+ LSK cells. Chromatin immunoprecipitation assay showed that in LSK cells, PAD4 targeted to the upstream of c-myc gene, suggesting that PAD4 might catalyze the citrullination and reduce the arginine methylation in histone H3. Furthermore, PAD4 was associated with the transcription factor LEF1 as well as histone deacetylase 1 (HDAC1). These results indicated that PAD4 suppressed c-Myc expression by histone citrullination acting as a suppressor complex with LEF1 and HDAC1 in LSK cells. Supporting these findings, PADI4-/- LSK cells showed an advanced proliferation in a cell-autonomous fashion, resulting in increase in number compared to PADI4+/+ LSK cells. Together, our results strongly suggest that PAD4 regulates the proliferation of LSK cells via controlling c-Myc expression, implying that PAD4 may be involved in pathogenesis of some myeloproliferative diseases.
MAP Kinase Phosphatase 5 Protects Against Sepsis-induced Acute Lung Injury (112.10)
Authors: Qian Feng , Deng Jing , Gantner Benjamin , Dong Chen , Christman John , Ye Richard
Published: January 2023

Mitogen-activated protein kinases (MAPKs) play a critical role in inflammation. While activation of MAPK in inflammatory cells has been studied extensively, much less is known about the inactivation of these kinases. MAPK phosphatase 5 (MKP5) is a member of the dual specificity phosphatase family that dephosphorylates activated MAPKs. Here we report that MKP5 protects sepsis-induced acute lung injury. Mice lacking MKP5 displayed severe lung tissue damage following LPS challenge, characterized with increased neutrophil infiltration and edema compared to wild type controls. In response to LPS, MKP5-deficient macrophages produced significantly more inflammatory factors including inflammatory cytokines, nitric oxide (NO), and superoxide. Phosphorylation of p38 MAPK, JNK and ERK were enhanced in MKP5 deficient macrophages upon LPS stimulation. Adoptive transfer of MKP5-deficient macrophages led to more severe lung inflammation than transfer of WT macrophages, suggesting that MKP5-deficient macrophages directly contribute to acute lung injury. In addition, mice lacking MKP5 showed higher mortality rate than WT mice following cecal ligation and puncture (CLP), although MKP5 deficiency enhanced bacterial clearance by macrophages. Taken together, these results suggest that MKP5 is crucial to homeostatic regulation of MAPK activation as well as the balance of antimicrobial and inflammatory responses.
Coordinated influence of β-defensins on innate immunity against bacterial infection of the airway (112.12)
Authors: Ryan Lisa , Wu Jichuan , Yim Sunghan , Shah Helly , McMahon Laura , Minhas Abeer , Diamond Gill
Published: January 2023

β-defensins act as antimicrobial agents and as immunomodulators. Human β-defensin (BD) peptides 1, 2 and 3 are differentially modulated in primary tracheal epithelial cells (TEC) by viruses, bacteria, and proinflammatory cytokines. We hypothesized that hBD-1 and virulence factors would influence induction of hBD-2 and hBD-3 during lung bacterial infections. Since BD genes exist in close proximity on chromosome 8, making it impossible to delete more than one BD gene, a model was devised to study their coordination. Since mouse orthologs exist, (hBD-1=mBD-1; hBD-2=mBD-3; hBD-3=mBD14), we used mBD-1(-/-) (KO) mice and the inhibitory capability of the type III secretion system of Bordetella bronchiseptica on NF-κB to study effects of mBD-1 deletion on mBD-3 gene expression using wild-type RB50 and mutant WD3 strains lacking this system. In C57BL/6 wild-type (WT) mice, mBD-3, mBD-14 and kerotinocyte-derived chemokine (KC) mRNA levels were suppressed by RB50 compared with WD3 induction in lung 24 hr after infection, whereas mBD-1 levels were unaffected. Deletion of mBD-1 significantly blunted WD3 induction of mBD-3 and mBD-14, but not KC. Bacteria numbers increased, along with decreased neutrophils in the trachea, when KO mice were infected with RB50 compared with WT mice. In TEC, WD3 induced mBD-3 in WT but not in KO mice, yet with cultured KO TEC, mBD-14 and KC were unaffected. The results illustrate the coordination of BD in TEC and in lung to influence bacterial infection.
Specific modulation of host intestinal epithelial NF-kB signaling and inflammatory response by E. coli O157:H7 effector proteins (112.20)
Authors: Wan Fengyi
Published: January 2023

Intestinal epithelial NF-kB signal pathways are crucial for host response to pathogen infections by orchestrating the innate and adaptive immune responses. Many successful pathogens have acquired sophisticated mechanisms to regulate host NF-kB signaling in order to favor the continued pathogen survival. A repertoire of novel virulence proteins, namely Nle effectors, was identified in the foodborne pathogen Escherichia coli O157:H7, and most of them appeared to subvert host NF-kB activation. However, the mechanism(s) through which the effectors proteins manipulate NF-kB signaling and inflammatory response in host is still largely unknown. We discovered that the NleH1 and NleH2 effectors from E. coli O157:H7 subvert host intestinal epithelial NF-kB signaling through their interactions with ribosomal protein S3 (RPS3), a non-Rel subunit of NF-kB that confers certain transcriptional specificity. NleH1 selectively attenuates, whereas NleH2 facilitates, RPS3-dependent proinflammatory cytokine genes, although both effectors do not impair p65 nuclear accumulation. We further demonstrated that NleH1 and NleH2 execute distinct function in regulating the Inhibitor of kB kinase 2 (IKK2)-mediated RPS3 phosphorylation and subsequent nuclear translocation. Thus, our results reveal a novel mechanism on the specific modulation of host NF-kB activation and inflammatory response by NleH effectors, and provide new molecular insights into the complex pathogenic mechanisms of E. coli O157:H7.
mTOR modulates host immune responses to Francisella tularensis LVS infection (112.7)
Authors: Edwards Michael , Zhang Ping , Michalek Suzanne , Katz Jannet
Published: January 2023

Francisella tularensis (Ft) is the cause of tularemia, a disease with a high morbidity and mortality rate. The Ft live vaccine strain (LVS) has been used to study disease pathogenesis since infection of mice with LVS resembles tularemia in humans. It has been reported that Ft LVS is a TLR2 agonist and involves PI3K/Akt signaling. However, the involvement of mTOR, a downstream molecule of the PI3K/Akt pathway, has not been determined in Ft LVS infection, nor has the interplay between TLR2 and the mTOR cascade. In the present study, we provide evidence that mTOR exerts a differential regulation on both cytokine mRNA and secreted protein in murine peritoneal macrophages stimulated with Ft LVS. IL-6, IL-10 and IFN-β mRNA production increased with inhibition of the mTOR pathway, while levels of IFN-γ mRNA were independent of mTOR signaling. Secreted IL-6 and IFN-γ but not IFN-β and TNFα was dependent of mTOR regulation. Furthermore, mTOR signaling was critical for the DNA binding activity of CREB, but not of NF-κB p65. Upregulation of the interferon regulatory factors (IRF)-1, IRF-3 and IRF-5 in nuclear fractions of cell lysates was observed upon inhibition of mTOR, and correlated with increased levels of the pro-apoptotic proteins Bax, caspase 3 and caspase 8, indicating mTOR modulation of apoptosis via IRF downreguation. Overall, these studies identify mTOR as a critical regulator of the host immune response to Ft LVS infection.
Anti-vector immunity following vaccination with Venezuelan equine encephalitis replicon filovirus vaccine (113.1)
Authors: Zak Samantha , Kuehne Ana , Barth James , Herbert Andrew , Muhammad Majidat , Ortiz Ramon , Prugar Laura , Hannah Brian , Bakken Russell , Pratt William , Dye John
Published: January 2023

The Venezuelan equine encephalitis replicon (VRP) is the lead candidate for development as the DoD filovirus vaccine platform. One concern regarding this vaccine candidate focuses on the need to vaccinate individuals that have been pre-exposed to the VRP through previous vaccinations. To address this concern, cohorts of nonhuman primates (NHPs) were vaccinated with VRP expressing alphavirus proteins and VRP expressing Ebola virus Zaire (EBOV) glycoprotein either concurrently or sequentially. These NHPs were challenged with either 1000pfu of EBOV or 10^8 pfu of Venezuelan equine encephalitis virus (VEEV). Animals that were vaccinated with both vaccinations independent of sequence or co-administration were protected from each challenge. This data indicates that anti-vector immunity is not a concern with the VRP vaccine candidate as the product transitions into phase 1 clinical trials.
Alum adjuvant rescues HPAI vaccine immunogenicity and survival in BALB/c mice following H5N1 infection (113.30)
Authors: Vaughan Sarah , Stout-Delgado Heather , Pollock Zemmie , Plourde Jennifer , Pyles John , Harrod Kevin
Published: January 2023

Highly pathogenic avian influenza (HPAI) is an emerging infectious virus with an approximate 60% fatality rate in humans. In the United States, a vaccine for H5N1 has been developed and stockpiled in case of a pandemic. This vaccine demonstrated low immunogenicity in human clinical trials with only 22% of recipients developing protective antibody titers. Previous studies using a lethal challenge ferret model have shown that supplementing the H5N1 vaccine with an alum adjuvant can improve immunogenicity and survival. We developed a lethal challenge mouse model of H5N1 vaccination to examine differences in innate immune responses to the vaccine alone or in the presence of an alum adjuvant. Mice treated with the adjuvanted vaccine showed significantly lower weight loss and higher survival than mice treated with the nonadjuvanted vaccine. Further, mice treated with adjuvanted vaccine had higher neutralizing antibody titers when compared to mice that received the nonadjuvanted vaccine. In vitro, dendritic cells (DCs) cultured with the adjuvanted vaccine had increased expression of the activation markers CD40 and CD86 compared to DCs treated with the nonadjuvanted vaccine. Interestingly, neither treatment induced activation of Toll-like receptors or the production of cytokines IL-6, IL-12, or TNF-α. Taken together, our results suggest that the alum adjuvant rescues immunogenicity and improves survival in the host.
Protective efficacy of CD4 memory T cells depends on route of administration of inactivated influenza vaccine (113.31)
Authors: Jelley-Gibbs Dawn , Winfrey Carolyn , Eaton Sheri , Haynes Laura
Published: January 2023

Studies aimed at determining the impact route of influenza vaccination has on vaccine efficacy revealed reduced protection against influenza virus challenge afforded by intranasal delivery compared to subcutaneous delivery of inactivated H1N1 influenza A virus. Both vaccine routes induced similar anti-viral antibody titers, similar numbers of anti-viral CD4 T cells, and undetectable anti-viral CD8 T cell responses. Further analysis of vaccine-induced CD4 memory T cell responses revealed that CD4 memory T cells primed in the lung draining lymph nodes were defective in recruiting anti-viral CD8 effectors to the lungs during challenge infections compared to the recruitment of CD8 effectors to the lungs by peripherally primed CD4 memory T cells. Since the influx of anti-viral CD8 effector T cells to the airways during influenza virus infection is a key event for virus control and elimination, we assayed for the expression of type-1 chemoattractants in the respiratory tract of vaccinated mice following challenge infection and discovered reduced lung expression of chemoattractants in the presence of intranasally primed CD4 memory T cells. We also determined that the presence of intranasally primed CD4 memory T cells reduced the ability of pulmonary antigen presenting cells to stimulate CD8 effectors directly ex vivo. We are currently assessing whether route of influenza vaccination also impacts susceptibility to secondary bacterial pneumonia following influenza challenge.
Harnessing Sendai virus to induce mucosal immunity (113.32)
Authors: Le Thuc-vy , Garcin Dominique , Compans Richard
Published: January 2023

The human mucosal surface covers greater than 400 m2 and is a target for infection and replication of pathogens such as influenza virus and HIV. Induction of specific immunity at the site of pathogen entry is therefore critical for the prevention of infection. We generated an attenuated recombinant Sendai virus (GP42-H1) in which the hemmagglutinin (HA) gene of A/Puerto Rico/8/1934 (PR/8) influenza virus was introduced into the genomic RNA. Intranasal (i.n.) instillation of mice with 1,000 pfu of the vector did not lead to weight loss. Expression of recombinant protein was observed in vivo, within 4 days post immunization. Heterologous expression of the recombinant gene contributed to high titer of HA-specific IgG and IgA antibodies in blood, saliva, fecal pellet extracts and bronchoalveolar lavage fluid. These antibodies inhibited hemagglutination of chicken erythrocytes and neutralized influenza virus infection of Madin-Darby Canine Kidney cells in vitro. Following challenge with 5LD50 of mouse adapted PR/8 influenza virus, mice that were immunized with 1,000 pfu of the GP42-H1 vector were completely protected from the influenza infection as opposed to mice that were immunized with a vector control that expresses green fluorescent protein. These data demonstrate that i.n. immunization with the GP42-H1 vector is competent to induce immunity at mucosal tissues and immunization with the vector promotes specific protection from lethal challenge in mice.
Role of inflammasome in HSP-mediated innate immunity (114.5)
Authors: Pawaria Sudesh
Published: January 2023

The immunogenic heat shock proteins gp96, hsp70 and calreticulin stimulate antigen presenting cells to provide co-stimulation in the form of cytokines and co-stimulatory molecules. The unique pattern of co-stimulation is dependent on the type of antigen presenting cell and the HSP that is used as the stimulant. The pattern of co-stimulation also determines the T helper response that is primed. A cytokine that is routinely released by HSP-stimulated APCs is IL-1beta. Here we investigate the role of (components of) the inflammasome pathway in HSP-mediated IL-1beta release. Our results show that in response to HSP stimulation and in a CD91-dependent manner, first, antigen presenting cells secrete robust amounts of IL-1β comparable to LPS stimulation, second, the HSP receptor CD91 is phosphorylated, and third, downstream p38 and NF-kB are activated. The source of the second signal which allows for the conversion of the pro-IL1beta to the mature form in this system is being investigated.
IP4 regulates FasL-mediated T lymphocyte death via inhibition of Orai1 (115.1)
Authors: Miller Andrew , Dahlberg Carol , Parker Albert , Schmedt Christian , Beisner Daniel , Gosling Martin
Published: January 2023

Antigen receptor-mediated production of Ins(1,4,5)P3 induces the release of Ca2+ from intracellular stores, resulting in the opening of store-operated Ca2+ (SOC) channels. We previously reported a mouse line deficient in Inositol(1,4,5)P3-3 kinase B (ITPKb), which converts Ins(1,4,5)P3 (IP3) to Ins(1,3,4,5)P4 (IP4). Mice lacking ITPKb exhibit a complete block in T cell positive selection and possess impaired B cell development and activation. Interestingly, ITPKb-/- cells exhibit enhanced Ag receptor-induced SOC signaling, which can be rescued by adding cell-permeable IP4 to ITPKb-/- cells, suggesting that IP4 serves to inhibit SOC channels. Due to developmental blocks, the role of ITPKb in mature lymphocyte function is unknown. Using the ER-Cre-loxP system, tamoxifen-induced deletion of ITPKb (ITPKbfl/fl) demonstrates a crucial role for ITPKb in mature T cell function. While ITPKbfl/fl mice have normal T-independent Ab responses, T-dependent Ab responses are completely abolished. In vitro, mature ITPKbfl/fl T cells exhibit enhanced SOC entry and reduced proliferative responses to TCR signals. Furthermore, we found that ITPKbfl/fl T cells die rapidly after activation, due in part to enhanced FasL upregulation. Using a HEK293 cell line stably expressing Stim1 and Orai1, we demonstrate that IP4 is an inhibitor of open-state Orai1 channels. These data identify ITPKb and IP4 as essential mediators of lymphocyte development and T cell activation by regulating SOC entry via Orai1.