bluuebunny/crossref_metadata_2025 huggingface dataset:
Role for Rab11a in mediating TLR dependent cross-presentation responses (106.17)
Authors: Nair Priyanka , Florey Oliver , Baccarini Alessia , Overholtzer Michael , Brown Brian , Blander J.
Published: January 2023

Toll-like receptor (TLR) signaling is critical for accelerated phagosome maturation and enhanced major histocompatibility complex class II (MHC II) presentation of phagocytosed antigens. Since cross-presentation is another consequence of phagosome maturation, we hypothesized that like MHC II presentation, the presentation of exogenous antigens on MHC class I (MHC I) by dendritic cells (DCs) is positively regulated by TLRs. Using antigen derived from bacteria, infected apoptotic cells or antigen-coated beads conjugated to a TLR ligand, our results show that cross-presentation is severely impaired when TLR signaling is abrogated in the cross-presenting DC or when TLR ligands are absent from phagocytic cargo. To delineate the mechanisms behind TLR enhancement of cross-presentation, we studied trafficking of MHC I in the presence or absence of TLR signaling. We observed recruitment of MHC I selectively to phagosomes containing TLR ligands along with the concomitant enrichment of the small guanosine tri-phosphatase (GTPase) Rab11a. In fact, Rab11a was crucial for TLR mediated enhancement of cross-presentation as selective knock-down of Rab11a abrogated the ability of DCs to cross-present antigen. Our results demonstrate a role for Rab11a in trafficking MHC I to phagosomes that engage TLR signaling, thus mediating enhanced cross-presentation of microbial antigens. Results from our studies have implications on anti-viral and anti-bacterial immunity as well as tumor immunotherapy.
The immunoproteasome is expressed by lymphocytes and plays critical roles in lymphocyte development (106.34)
Authors: Zanker Damien , Pang Ken , Chen Weisan
Published: January 2023

The immunoproteasome (IP) differs from the housekeeping proteasome through the substitution of the catalytically active β1, β2 and β5 subunits for LMP2, MECL-1 and LMP7, respectively. The major function of the immunoproteasome is proposed to enhance immune responses by providing peptides optimal for class I binding. Interestingly, LMP2-/- mice have significantly reduced thymocytes and peripheral lymphocytes, a phenotype not easily explained by its proposed role. To explore this, we compared thymocytes and splenocytes from wild type and LMP2-/- mice. In the periphery, LMP2-/- mice have decreased lymphocytes but normal numbers of other BM derived cell types. In the thymus, staging analysis indicated a developmental halt with increased apoptosis, at the CD4-CD8- stage at the transition from DN3 to DN4. Similarly, a halt was found in the B cell lineage at the immature to T1 B cell transition. We confirmed by WB that developing wild type T and B cells express IP subunits. The NFκB pathway plays an important role in lymphocyte survival at these stages. Various NFκB proteins were investigated by WB and confocal microscopy following pathway activation. Subsequently, we detected decreased degradation of IKBα, p105 and p100 from LMP2-/- mice. Together, these results provide a link between the IP and the NFκB pathway at crucial lymphocyte developmental stages. We have therefore demonstrated the IP also plays important housekeeping roles other than simply enhancing antigen processing.
Stimulation of the immune system by small chemicals can be independent of hapten-protein conjugate formation and is regulated by the TCR avidity for the HLA-drug complex (106.51)
Authors: Adam Jacqueline , Eriksson Klara , Schnyder Benno , Fontana Stefano , Pichler Werner , Yerly Daniel
Published: January 2023

Rationale: The antiretroviral drug abacavir (abc) elicits a strong CD8-mediated immune response in HLA-B*5701+ subjects. We used this model to study the interaction of small chemicals with the immune system. Methods: We generated abc-specific T-cell clones (abc-TCC) from HLA-B*5701+ donors and analyzed their specific activation in Ca2+ influx assays and by annexin V staining. Results: Abc-metabolizing enzymes were absent in immune cells and inhibition of the proteasome in antigen presenting cells did not affect TCC reactivity. Five out of 50 abc-TCC reacted instantly (~120 sec) to soluble abc presented on HLA-B*5701, whereas many other TCC reacted after 20 min or later. Increasing the number of HLA-B*5701/abc immunogenic complexes by increasing the abc concentrations and/or using transfected cells with a high HLA-B*5701 expression accelerated the abc-TCC activation kinetic. Analysis of several abc-TCC revealed different TCR Vβ-segments, demonstrating their polyclonality. Conclusions: Since the observed abc-reactivity was metabolism- and processing- independent, an immune activation by small chemicals can occur without covalent drug binding to a carrier protein. Moreover, titration experiments revealed different TCC reactivity patterns. This finding suggests a crucial role of the TCR avidity for the drug in regulating T cell activation. These data have implications in drug regulation of the immune system as well as for understanding drug hypersensitivity.
MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA (108.1)
Authors: Modis Yorgo , Berke Ian
Published: January 2023

MDA5 detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signaling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signaling is not well understood. Here we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16-18-bp footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signaling model in which the CARDs on MDA5-RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry. Ian C. Berke & Yorgo Modis (2012) MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA. EMBO J., in press.
Type I Interferons induce TREX1 in myeloid dendritic cells. (108.10)
Authors: Xu Jun , Gamero Ana , Gallucci Stefania
Published: January 2023

Dendritic cells (DCs) are activated by endogenous and exogenous danger signals to stimulate T and B cell responses. Type I Interferons (IFNα/β) are endogenous danger signals able to activate DCs and induce the expression of IFN-stimulated genes (ISGs) that include genes important in host defense against viruses. Although some ISGs have been demonstrated to have antiviral functions, the antiviral activities of the majority of ISGs have not been confirmed. TREX1 (Three prime Repair EXonuclease1) is an exonuclease acting on ssDNA or mispaired 3’ termini. It has been associated with autoimmune diseases including Systemic Lupus Erythematosus and Aicardi-Goutieres Syndrome, which show over-expression of ISGs. TREX1 also facilitates HIV-1 infection by degrading cytosolic HIV-1 DNA, suggesting that HIV-1 may use TREX1 to escape host detection. The role of TREX1 in DC function is unknown. We have found that IFNα stimulates TREX1 expression in C57BL/6 bone marrow derived Dendritic Cells (myeloid DCs). TREX1 gene expression is induced within 1 h of IFNα treatment and continues to increase up to 6 h after stimulation, indicating that TREX1 is an early responsive ISG with sustained expression possibly due to autocrine IFNβ production. In addition, transcription of TREX1 by IFNα is impaired in myeloid DCs from Stat2-deficient mice, indicating that TREX1 expression is dependent on the canonical IFNAR/STAT2 signaling pathway. We are now studying TREX1 role in the antiviral response of DCs.
molecular mechanisms of innate signaling disruption by human metapneumovirus M2-2 protein (108.15)
Authors: Bao Xiaoyong , Ren Junping , Wang Qingrong , Kolli Deepthi , Prusak Deborah , Wood Thomas
Published: January 2023

hMPV is a leading cause of respiratory infections in pediatric populations globally. Neither an effective cure for hMPV nor a vaccine to prevent hMPV infection is available. Recently, a recombinant hMPV lacking the M2-2 protein (ΔM2-2) demonstrated reduced replication in the respiratory tract of animal models, making ΔM2-2 a promising live vaccine candidate. However, the exact nature of the interaction between the M2-2 protein of hMPV and host cells that regulates viral infection and propagation is largely unknown. In this study, we found that, in addition to its role as a critical regulator of viral RNA synthesis, M2-2 is also an important virulence factor that antagonizes RIG-I/MAVS-mediated cellular signaling, revealing a novel function of M2-2. In reporter gene assays, the M2-2 protein specifically inhibited RIG-I- and MAVS-dependent IFN-β gene transcription, but not the one induced by downstream effectors of MAVS, suggesting that M2-2 targets MAVS in a viral-RNA-independent manner. Coimmunoprecipitation assays revealed that the M2-2 protein interacts with MAVS. In agreement with these findings, ΔM2-2-infected airway epithelial cells produced higher levels of type I interferon and other pro-inflammatory mediators than WT-infected cells. Activation of transcription factors IRF-3 and NF-κB, and induction of their dependent gene transcription were also enhanced by ΔM2-2 infection, confirming the inhibition of hMPV-induced innate signaling by M2-2.
The effects of indoleamine 2,3 dioxygenase on the immune response to Influenza A infection. (108.4)
Authors: Rose David , Tompkins S. , Mellor Andrew , Klonowski Kimberly
Published: January 2023

Indoleamine 2,3 dioxygenase (IDO) is the enzyme responsible for the first step of tryptophan metabolism in the Kynurenine pathway and is a known immunosuppressant. IDO can suppress the T cell response to various antigens via tryptophan starvation, production of toxic byproducts of the kynurenine pathway, or a combination of both. Conversely, 1-methyl-tryptophan, a tryptophan analog that cannot be metabolized by IDO, has been shown to enhance immune responses in both pregnancy and tumor models. While IDO has primarily been studied in the context of adaptive immune responses, little is known about its effect on NK cells. While IDO is expressed at a basal levels by dendritic cells in many tissues, IDO levels are enhanced in the lungs early after influenza A infection where NK cells are not only positioned as the first line of anti-viral defense but also act to bridge the innate and adaptive immune response. Using IDO-1 deficient mice we will report the qualitative and quantitative differences of the NK cell response to influenza A infection in an IDO deficient environment.
B cell development and immunoglobulin production are altered in the absence of IκBNS (109.4)
Authors: Touma Maki , Saito Ibuki , Reinherz Ellis , Clayton Linda
Published: January 2023

NF-κB transcription factors are key mediators of immune function as well as inflammatory responses. The activation of NF-κB is regulated by several IκB proteins, a family comprising classical cytoplasmic as well as nuclear IκBs. In this study, we focused on the role of IκBNS, a nuclear IκB protein, in B lymphocyte development and function. Analysis of lymphoid tissues from IκBNS knockout (KO) mice revealed that a lack of IκBNS affects the development of peritoneal B-1 cells and splenic marginal zone B cells. Development of B-2 cells was essentially normal. We also found that B cell proliferative activity and immunoglobulin production were altered in the absence of IκBNS. These defects were confirmed by the analysis of B cells in bone marrow chimeric mice constructed by the transfer of wild type or IκBNS KO bone marrow cells into B cell-deficient mice. Furthermore, histological analysis of spleens from chimeric mice revealed that re-constitution of B cell areas, marginal zones and germinal centers were markedly reduced in IκBNS KO cell recipient mice. Examination of the functions of IκBNS KO B cell subsets suggested that IκBNS is important for antibody production by both marginal zone and follicular B cells. These results indicate that IκBNS plays a key role in peripheral B cell development and function.
IL-2 regulates tissue-dependent differences in CD8 T cell apoptosis during and after- viral Infection. (110.19)
Authors: Kapoor Varun , Welsh Raymond
Published: January 2023

Virus-specific CD8 T cell populations in lymphoid organs contract by apoptosis or by dissemination into peripheral tissues, and those residing in non-lymphoid organs, including the peritoneal cavity (PEC: peritoneal exudate cells), are more resistant to apoptosis than those in the lymphoid organs, including the spleen. We questioned whether tissue-dependent apoptotic differences in LCMV-specific T cells were due to environment factors or intrinsic stability of the CD8 T cells. The PEC environment conferred an anti-apoptotic effect, as shown by mixing PEC with excess splenic leukocytes or vice versa. This anti-apoptotic effect correlated with Stat5 phosphorylation induced by IL-2 spontaneously secreted by CD4 T cells. Spontaneous IL-2 production by CD4 T cells occurred in vivo during the contraction and the long term memory stage after LCMV infection in PEC and Fat pads, but much less so in spleen and lymph nodes. Virus-specific CD8 T cells in the PEC were also intrinsically more stable, as most had a memory precursor effector cell (MPEC) phenotype and higher expression of CD27, both correlating with decreased apoptosis. However, regardless of their phenotype, PEC CD8 T cells remained less apoptotic than their spleen cell counterparts. We conclude that long after the resolution of viral infections, IL-2-producing CD4 T cells linger in peripheral sites and locally contribute to the stability of memory CD8 T cells, thereby enhancing resistance to re-infection.
Autocrine IFN-gamma promotes limited differentiation of naïve CD8 T cells and synergizes with Type I IFN to stimulate optimal effector functions (110.5)
Authors: Curtsinger Julie , Lins Debra , Mescher Matthew
Published: January 2023

Autocrine IFN-gamma signaling has been shown to be important for differentiation of CD4 T helper 1 effector cells, but it has been unclear whether it contributes to CD8 T cell differentiation. We have found that naive transgenic murine OT-I CD8 T cells produce low levels of IFN-gamma shortly after stimulation with antigen and B7-1, reaching a peak within 8-12 hours. This early IFN-gamma production is distinct from effector IFN-gamma production in both timing and the amount of cytokine produced. Similar results were obtained using CD8 T cells from pmel and P14 transgenic mice. We found that early IFN-gamma contributes signals for upregulation of expression of Tbet, granzyme B, and weak cytolytic effector function. Type I IFN acts synergistically with IFN-gamma to support development of strong effector functions, while IL-12 induces optimal effector functions in the absence of IFN-gamma signaling. An early IFN-gamma signal is not sufficient to program CD8 T cells to survive in vivo and develop a memory population, nor is it necessary for IL-12- or Type I IFN-dependent programming for survival. Thus, IFN-gamma is not only an important CD8 T cell effector cytokine, it is an autocrine/paracrine factor whose contributions to differentiation vary depending on whether the response is supported by IL-12 or Type I IFN.
Role of Pax5 and YY1 in regulation of V(D)J recombination (111.1)
Authors: Malshetty Vidyasagar , Chen Jian , Hanna Mary , Cortes Patricia
Published: January 2023

The infinite variety of antigen-binding receptors originates from the rearrangement of B and T-cell receptor loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. Deregulation of this process, leads to a spectrum of diseases including immunodeficiency and leukemia. These leukemias are believed to arise from RAG1/2 mediated oncogenic chromosomal translocations. The transcription factor Pax5 is critical for B cell development and is known that, it interacts with RAG1/2 and mediates V to DJ recombination. Pax5 is also been implicated in human B cell malignancies. Another multifunctional transcription factor, YY1, is known to play a role in V to DJ recombination similar to Pax5. We were able to see an interaction of YY1 with RAG1/2 and Pax5, when these proteins were over-expressed in 293T cells. When we performed a plasmid based recombination assay in presence of Pax5, YY1 or both a consistent decrease in recombination levels was observed. To elucidate the mechanism of inhibition, we analyzed the recombination intermediates and found that the initial cleavage step is inhibited by these two factors. In addition, purified Pax5 and YY1 inhibit the in vitro cleavage activity of RAG1/2 recombinase. The expression of a mutant Pax5 increased the RAG recombinase activity, suggesting the importance of these two factors in regulation of RAGs mediated recombination.
Hes1-mediated constraint of C/EBPα is essential for T-cell development (111.22)
Authors: De Obaldia Maria , Bell J. Jeremiah , Zlotoff Daniel , Sultana Dil Afroz , Bhandoola Avinash
Published: January 2023

Notch signaling in the thymus induces expression of T-cell genes and discourages progenitors from adopting alternative fates. Recent work has suggested that T-cell progenitors arrive at the thymus with T and non-T (B, NK) lymphoid potential as well as dendritic cell potential and a degree of myeloid potential; however, the importance of actively repressing alternative gene expression programs in T-cell development has not been adequately addressed. To examine the role of Notch-mediated gene repression in T-cell development, we used mice deficient for the Notch target and transcriptional repressor Hes1. We found that Hes1-deficient progenitors failed to make normal numbers of T-cells upon culture with Notch ligands, but instead failed to downregulate the myeloid transcription factor C/EBPα and gave rise to increased numbers of Mac1+ myeloid cells. To test whether the primary role of Hes1 in T-cell development is to repress expression of the myeloid transcription factor C/EBPα, we deleted floxed alleles of C/EBPα in Hes1-deficient progenitors using a retroviral Cre recombinase and co-cultured transduced progenitors with Notch-ligand expressing stromal cells. We found that deletion of C/EBPα restored T-cell development in Hes1-deficient progenitors. These results establish that an essential function of Hes1 is to constrain progenitors from activating myeloid lineage programs early in T-cell development.
IL-33-induced hematopoietic stem and progenitor cell mobilization and anti-fungal activity in mice (111.24)
Authors: Kim Wonyoung , Kim Juyang , Le Hongnga , Kim Hyunju , Cho Hong , Kwon Byungsuk
Published: January 2023

IL-33 has been implicated in the pathogenesis of asthma, atopic allergy, anaphylaxis and other inflammatory diseases by promoting the production of pro-inflammatory cytokines and chemokines or Th2 immune responses. In this study, we investigated the role of IL-33 in hematopoietic stem and progenitor cell (HSPC) mobilization and host anti-fungal response in mice that received autologous HSPC transplantation. IL-33 induced HSPC mobilization through increasing vascular cell CXCL1 and CXCL2 productions. IL-33-mobilized HSPCs were capable of homing to and of long-term reconstitution in the BM of irradiated recipients. Immune cells derived from these recipients had normal anti-fungal activity. When injected after IL-33-mobilized HSPC transplantation, IL-33 had an additional protective effect against lethal Candida albicans infection. The anti-fungal effect of IL-33 was due to the rapid recruitment of neutrophils to the infection site via release of CXCR2 chemokines in peritoneal cavity and blood and prevention of CXCR2 down-regulation induced by Toll-like receptor (TLR) signaling in neutrophils and their enhanced phagocytic activity. The ability of IL-33 to promote HSPC mobilization and host anti-fungal activity represents a previously unrecognized role of IL-33 in innate immunity. These properties of IL-33 have clinical implications in hematopoietic stem cell transplantation.
Endogenous mouse mammary tumor viruses influence the development of thymic αβ NKT cells and innate CD8+ T cells (111.26)
Authors: Holt Michael , Punkosdy George , Blain Melissa , Glass Deborah , Lozano Mary , Dudley Jaquelin , Shevach Ethan
Published: January 2023

Mounting evidence has demonstrated the presence of a population of thymic CD8+ T cells that share phenotypic and functional similarity to “innate-like” or “memory-like” T cells. Innate CD8+ T cells have increased expression of CD44, CD122, and Eomesodermin (Eomes), as well as the capacity to rapidly produce interferon (IFN)-γ upon stimulation. In addition to several gene-deficient models, including KLF2-, Itk-, and Id3-deficient mice, innate CD8+ T cells have also been documented in naïve BALB/c mice. The development of such cells is dependent on the production of interleukin (IL)-4 from an expanded population of αβ or γδ NKT cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF). Interestingly, this innate phenotype among CD8+ T cells is lost in congenic BALB/c mice lacking all endogenous mouse mammary tumor proviruses (BALB/Mtv-null). In the absence of endogenous Mtv proviruses, the expression of CD44, CD122, CXCR3, and Eomes, as well as the production of IFN-γ, was greatly reduced among thymic CD8+ T cells. The loss of this innate CD8+ T cell population in BALB/Mtv-null mice correlated with a reduction in PLZF+, IL-4 secreting αβ NKT cells within the thymus compared to wild type BALB/c. Our findings suggest a potential Mtv-mediated role in modulation of thymic αβ NKT cell development that subsequently influences the generation of innate CD8+ T cells.
Does androgen signaling cause thymic involution? (111.34)
Authors: Cooper John , Bryson Jerrod , Li Jie , Narayan Prema , Manley Nancy
Published: January 2023

The thymus is the main source of naïve T cell output. As the body ages, the thymus undergoes involution, causing a drop in naïve T cell availability in the peripheral T cell pool. The molecular mechanisms underpinning thymic involution are not well characterized. However, androgen signaling has been associated with involution based on increases in thymus size and thymocyte number in both sex steroid ablated (SSA) mice and those lacking functional androgen receptor (Ar). However, to date there is no clear cellular target or mechanism by which androgen signaling affects involution. The available data consistently point to a thymic stromal target as the major site of androgen action in the thymus. Among these stromal cell types, thymic epithelial cells (TECs) express Ar and have been shown to increase proliferation and production of specific effectors such as CCL25 in response to castration. We have used a Cre-LoxP strategy to selectively delete Ar in TECs. Evaluations of these mice present compelling evidence that TECs are directly involved in androgen signaling within the thymus. While TEC-selective Ar deletion does cause increased thymus size and thymocyte numbers, these mice differ in several ways from previous reports on global Ar deletion or SSA, leading to the possibility that current models of the role of androgens in postnatal thymus may need to be re-evaluated.
STAT3 and STAT5 guide key transcriptional and epigenetic responses during dendritic cell development (111.37)
Authors: Li Haiyan , Yang Cliff , Nallaparaju Kalyan , Zhang Huiyuan , Liu Yong-Jun , Goldrath Ananda , Watowich Stephanie
Published: January 2023

STAT proteins regulate homeostatic development of dendritic cells (DCs), yet information about their role in demand situations and their mechanisms of action remains limited. We found that STATs control cytokine-driven production of plasmacytoid (pDCs) and conventional DC (cDC) subsets in vivo. In DC progenitor cultures, STATs induce expression of the transcriptional regulators E2-2/Tcf4 and Id2, which have essential and distinct activities in pDCs, CD8α+ and CD103+ cDCs. Tcf4 is stimulated by Flt3 ligand- (Flt3L) activated STAT3. Id2 is induced by Flt3L or GM-CSF; both cytokines utilize C/EBPβ while GM-CSF also employs STAT5. These results are consistent with previous reports that Flt3L regulates pDCs, CD8α+ and CD103+ DCs while GM-CSF is required for the development of CD103+ DCs in skin and peripheral lymph nodes. The status of promoter chromatin modifications suggests pDC-restricted access for Tcf4 and an open configuration for Id2. These epigenetic marks are influenced by STATs independent of Tcf4/Id2 mRNA expression. Thus, our results reveal mechanisms by which STAT3 and STAT5 control the transcriptional networks that define pDC and cDC lineages.
Analysis of Hematopoietic Stem Cell populations in control and autoimmune mouse strains. (111.48)
Authors: Elia Jeanne , Chronopoulou Efthalia , Carson Christian
Published: January 2023

Hematopoietic stem cells (HSC) are cells capable of self renewal and multilineage differentiation. Flow cytometry is often used to isolate very pure populations of HSC. To successfully isolate live HSC using FACS, cells are typically identified using a combination of cell surface markers, a dye or both. Several methods have successfully been used for the isolation of these cells in mice, including KLS (c-kit/Sca-1/Lineage negative), side population (Hoechst Red vs. Hoechst Blue), and more recently the SLAM panel (CD150+/CD48-/CD41-). It has been demonstrated that transplantation of all these cell populations results in bone marrow reconstitution in lethally irradiated mice. Using combinations of these signatures, we examined various mouse strains to determine whether there were strain-specific differences in the HSC populations. Specifically, we isolated HSCs by FACS from two control (C57BL/6 and Balb/C) and two autoimmune (SJL and NZB) strains. The data suggest that certain signatures are more suitable for the isolation of prospective HSC populations from certain strains of mice and that the HSC compartment is variable is size from strain to strain. In addition we will discuss interesting findings with respect to expression of these markers in the spleens of the different strains.
Regulation of B lymphopoiesis by Zinc Finger Protein 521 (ZFP521) (111.9)
Authors: Lei Hong , Lukin Kara , Rodriguez Valerie , Straign Desiree , Feeney Ann , Warming Søren , Hagman James
Published: January 2023

The ZFP521 (Evi3/ZNF521/EHZF) transcription factor has been identified as an inhibitor of Early B cell factor 1 (EBF1), which is essential for B cell lineage specification and commitment. ZFP521 contains 30-zinc finger (ZF) domains, including C-terminal ZF’s that bind to EBF1. However, whether ZFP521 is required for appropriate B cell development and function has not been addressed. We demonstrated that Zfp521 transcripts correlate inversely with Ebf1 transcripts. Zfp521 transcripts are expressed at the highest levels in pre-pro-B and pro-B cells and decrease progressively with differentiation. In contrast, Ebf1 transcripts are detected only weakly in pre-pro-B cells and are increased at later stages in the bone marrow. In 3-wk old Zfp521 knockout (KO) mice, pre-pro-B and pro-B cells are increased substantially, while late pro-B, pre-B and immature B cells are reduced significantly. Re-circulating mature B cells accumulate prematurely in the bone marrow of Zfp521 KO mice relative to WT mice. We are addressing two possible models in these mice. ZFP521 may modulate EBF1 function through direct protein:protein interactions. Alternatively, because ZFP521 is itself a DNA-binding protein, ZFP521 may direct transcription of cognate target genes independently of EBF1. We obtained evidence that ZFP521 co-occupies functional promoters with EBF1 in pro-B cells. This indicates that ZFP521 regulates B cell development through interactions with EBF1 on target genes.
Characterization of live, attenuated rubella viral vectors expressing SIV Gag or HIV MPER determinants (113.16)
Authors: Berkower Ira , Ni Yisheng , Virnik Konstantin
Published: January 2023

Infection with HIV or SIV can elicit potent and broadly reactive neutralizing antibodies against HIV, as well as disease modifying CTL against SIV. In order to elicit similar responses by vaccination, we have developed live attenuated rubella viral vectors to express HIV and SIV antigens. Safety and immunogenicity of rubella vaccine is well known; it elicits mucosal immunity, and one dose protects for life (against rubella). We have identified two insertion sites, in the nonstructural (ns) and structural regions, where foreign genes can be expressed without affecting viral growth. We have made 15 vectors expressing HIV MPER and SIV Gag antigens containing multiple CTL epitopes. Inserts at the ns site were expressed as rubella early genes. Inserts at the structural site were over-expressed as late genes and processed with the structural polyprotein. Some of the structural inserts were incorporated into virions and displayed on their surface. The vector should be safe, since loss of the insert would revert to the vaccine strain. We are using rubella because it grows well in rhesus macaques, which provide a good model of immunogenicity and protection against SIV or SHIV challenge. In preliminary studies, one of the vectors is growing in vivo, and it has elicited antibodies to the vector. These vectors will be useful in determining the role of a live vector in eliciting protective immunity and for optimizing its use in a prime and boost strategy.
Engineered microneedle arrays for transcutaneous HIV vaccine delivery (113.17)
Authors: DeMuth Peter , Huang Bonnie , Min Younjin , Hammond Paula , Irvine Darrell
Published: January 2023

Microneedle skin patches represent a promising approach for safe, convenient, and rapid delivery of vaccines and therapeutics to the skin through pain-free disruption of the stratum corneum to access epidermal and dermal immune-competent cell populations. We have developed a ‘nano-layer’-coated microneedle (MN) delivery platform in which vaccines/therapeutics encapsulated in surface coatings on biodegradable polymer MNs are rapidly delivered upon application to the skin of mice. Layer-by-layer deposition of plasmid DNA (pDNA) and nucleic acid TLR agonists together with biodegradable polymers on the surface of microneedle arrays gave thin coatings with tunable dosages of vaccine cargo and vaccine release rates. Following application of coated MN arrays to the skin of mice for ~5 min, intact film coatings were rapidly implanted in the skin for sustained release of vaccine components over time. Microneedle delivery of DNA vaccines encoding SIV-gag proteins induced 5-fold increased antigen-specific T-cell expansion and generation of serum IgG titers 5-fold in excess of that observed for traditional i.m. or i.d. immunizations; further, responses obtained with MN delivery were quantitatively similar to those observed for i.m. delivery with in vivo electroporation. MN delivery also induced enhanced generation of long-lived memory T-cells. Together these results demonstrate the potential for MN delivery to improve vaccine performance in the context of HIV immunization.
HIV-Env protein subunits containing native gp41 and gp120 in immunoreactive form are enriched by DC-SIGN lipoparticles. (113.22)
Authors: Vyas Girish , Stoddart Cheryl Stoddart , Killian Michael , Brennan Todd , Goldberg Tiffany , Ziman Alyssa , Bryson Yvonne
Published: January 2023

We prepared infection-free HIV-1 envelope proteins (HIVenv) from genetically homogeneous R5-HIV-1 occurring in plasma (PHIV) of donors before anti-HIV response. Such an immunogen could elicit broadly neutralizing antibodies (bnAb) against viruses transmitted in humans. PHIV was co-cultured with PBMCs and virions from culture supernatants were isolated by ultracentrifugation, and purified to remove contaminating microvesicles with anti-CD45. Membrane cholesterol was extracted with beta-cyclodextrin (BCD) to permeabilize virions and expel p24, RT and virion RNA, removable by 100kD Centricon. The residual host/viral DNA/RNA were hydrolyzed with protease-free Benzonase (BZ) and filtered through 30kD Centricon to obtain mHIVenv. This was tested for infectivity by PBMC cocultures, by inoculating SCID-hu Thy/Liv mice, and by EM for virions. Immunometric analyses of mHIVenv bound to DC-SIGN lipoparticles was done with anti-gp41 (1240) and anti-gp120 (b12) MoAb. A prototype HIV-1280 isolate after inactivation by BCD-BZ yielded mHIVenv with residual 0.28% p24 and retention of 91.9% gp120, and HIV RNA reduced by 7.4 logs. The mHIVenv proved noninfectious in vitro and in vivo and showed no virion particles by EM. The mHIVenv bound to DC-SIGN lipoparticles through gp120 revealed noncovalently bound gp41, with relative luminescence paralleling that of gp120. Infection-free mHIVenv protein subunits concentrated on DC-SIGN lipoparticles fulfil the need for an HIV vaccine candidate.
Adjuvant-dependent cytokine profiles in the context of a DNA prime-protein boost HIV vaccine (113.23)
Authors: Buglione-Corbett Rachel , Pouliot Kim , Marty-Roix Robyn , West Kim , Wang Shixia , Lien Egil , Lu Shan
Published: January 2023

Heterologous prime-boost vaccination approaches have emerged as a promising strategy to generate protective immunity against a variety of pathogens in animal and human studies. Our previous studies demonstrated that HIV-1 gp120 DNA vaccine prime-protein boost elicits improved neutralizing antibody responses. To understand the mechanisms that regulate the immune responses in prime-boost vaccination strategy, current studies were designed to define the cytokine profiles associated with different adjuvants used in such regimens. A polyvalent vaccine DP6-001, including gp120 antigens from 5 different primary viral isolates, was used as the model vaccine. Any influence of endotoxin associated with DNA plasmid preparation was first excluded, as regular DNA preparations did not elicit different immune response when tested in vivo against endotoxin-free DNA preparations. Next we studied the effects of several adjuvants, QS-21, alum, and MPL, on the immunogenicity of DP6-001 in a mouse model. gp120-specific antibody and T cell responses, and innate cytokine production, were monitored at time points after immunizations and at termination. Our results indicate that different adjuvants may generate unique cytokine profiles and improve the immunogenicity of DP6-001. This study may provide valuable information in selection of an adjuvant for inclusion in future prime-boost vaccination strategies, with the goal of enhancing immunogenicity while minimizing reactogenicity.
Influenza virus-like particle vaccination boosts CD8 T cell immunity (113.28)
Authors: Hemann Emily , Song Jae-Min , Kang Sang-Moo , Legge Kevin
Published: January 2023

Influenza virus-like particles (VLPs) have been demonstrated to provide protection against lethal influenza A virus (IAV) infections in mice following a single, intranasal (i.n.) vaccination. Previous work has demonstrated that VLP administration leads to an increase in virus-specific IgG production. However, the effect of VLP vaccination on the influenza-specific CD8 T cell response remains unknown. Given the important role of CD8 T cells in viral clearance, we determined the role of vaccine-induced CD8 T cell immunity in influenza VLP-mediated protection. Our results demonstrate that the HA533-specific CD8 T cell response is significantly boosted in the lungs, spleens and lung-draining lymph nodes of VLP-vaccinated mice following IAV challenge. Further, adoptive transfer of CL4 (HA533-specific) T cells and serum from VLP-vaccinated mice improved mortality following lethal infection compared to transfer of T cells or serum alone. Together, our results suggest that memory CD8 T cell responses contribute to the protection mediated by influenza VLP vaccination and support the potential use of VLPs as an effective influenza vaccine candidate.
Differential immune responses to seasonal IM flu-vaccines (113.36)
Authors: Kasten-Jolly Jane , Lawrence David
Published: January 2023

T (total, CD4 and CD8), B, and NK lymphocytes were enumerated by flow cytometry (4-color MultiTest) before and after intramuscular injection of the 2005/2006, 2006/2007, 2007/2008, 2009/2010, and 2010/2011 vaccines as an initial step in the characterization of the immune response to vaccine preparations. Study subjects (yrs 2005-2007) ranged in age from 25-68 yrs, while the 2009/2010 flu-shot subjects were 59-80yrs with one 30yr young control. The first 3 seasons included 11 subjects common to all seasons and 8 others common to the 2006/2007 and 2007/2008 vaccination season. Blood lymphocyte numbers were assessed 5-7 days before, and 3-6 days and 12-14 days post-vaccination. The 2010/2011 study included 2 subjects from previous years and 1 (35 yr) new to the study. Most of the vaccines, except 2010/2011, which induced an increase of B cells and CD4 and CD8 T cells, induced no detectable increase in these lymphoid subsets; the 2006/2007 and 2007/2008 vaccines actually caused a decline in T cells at 2 wks post vaccination. However, the 2006/2007 vaccine did increase NK cell numbers at 5 days post vaccination, which returned to pre- numbers after 2 wks. An increase in B and T-cell numbers after the 2010/2011 vaccine was associated with the highest influenza virus-specific antibody titer. Since most of the individuals in these studies remained constant, the results indicate that different vaccine preparations affect the immune system in different ways.
Evaluation of HCMV vaccines in rhesus macaques by a novel micro-neutralization assay (113.4)
Authors: Tang Aimin , Li Fengsheng , Freed Daniel C. , He Xi , Finnefrock Adam , Casimiro Danilo , Wang Dai , Fu Tong-Ming
Published: January 2023

Human cytogemalovirus (HCMV) causes wide-spread infection in adult human populations. Although no symptoms observed in healthy persons, HCMV infection can cause severe and even life-threatening diseases in immune-compromised individuals. There are lines of evidence that humoral immunity can provide protection against HCMV infection. We have tested several versions of HCMV vaccines based on laboratory strain AD169 with its epithelial tropism restored in rhesus macaques. Vaccines were administered intramuscularly three times. In comparison, recombinant HCMV gB protein vaccine, which showed marginal protections in previous clinical trial, was given with adjuvant MF59. To facilitate the evaluation of these vaccines, we have developed a novel micro-neutralization assay based on the detection of a dominant HCMV antigen expressed in ARPE19 cells, using near infrared dye-labeled immune reagents, to measure HCMV neutralizing activities in serum samples of vaccinated monkeys. Our results showed that AD169 revertant vaccines induced much stronger neutralizing activities than recombinant gB vaccine in rhesus monkeys. The addition of ISCOMATRIX adjuvant (CSL, Ltd) in the vaccine formulation further enhanced its immunogenicity. There were significant boost effects after the second injection, while the third injection did not increase the neutralizing titers above those obtained post the second vaccination.
HSV-2 antigens formulated with Matrix M-2 induce balanced neutralizing antibody and durable polyfunctional T cell responses in mice (113.5)
Authors: Lee Alexander , Larson Shane , Zielinski Veronica , Long Deborah , Flechtner Jessica , Skoberne Mojca
Published: January 2023

An effective HSV-2 vaccine is still needed despite the prevalence of antiviral drugs. Recent HSV-2 vaccine clinical trials highlighted the need to elicit potent T cell responses since neutralizing antibodies alone were incapable of providing complete protection. Through proteomic screens using T cells of HSV-2-exposed donors several antigens were identified as promising vaccine targets. Two of these antigens, GB208 and GB217 (GEN-003), were formulated with a saponin-derived adjuvant Matrix M-2, which in recent avian influenza human clinical trials induced cellular and humoral immune responses. In preclinical studies, C57BL/6 mice were used to evaluate the ability of GEN-003 combined with Matrix M-2 to induce antigen-specific T cell and antibody responses. T cell effector functions were evaluated by IFNγ ELISPOT, intracellular cytokine staining, and in vivo cytolysis of fluorescently labeled peptide-pulsed targets. Neutralizing antibody responses as well as total and subclass IgG titers were also assessed. Cellular and humoral immune responses were primed in mice after administering a wide range of protein and adjuvant doses. GB208- and GB217- specific T cell responses were polyfunctional, cytolytic, and persisted for more than 3 months. Antibody responses were neutralizing, and represented balanced Th1/Th2 phenotypes. In conclusion, GEN-003 combined with Matrix M-2 form a promising vaccine candidate that activates both arms of the immune system necessary for viral clearance.
Impaired NLRP3 inflammasome activity in elderly hosts contributes to the prevalence of primary and secondary Streptococcus pneumoniae infection. (114.4)
Authors: Stout-Delgado Heather , Shirali Anushree , Jaramillo Richard , Harrod Kevin
Published: January 2023

Lethal pneumonia has become a serious problem in elderly populations. Although influenza infection alone may lead to pneumonia, secondary bacterial infections, such as Streptococcus pneumoniae, during and shortly after recovery from influenza infections are more common. Previous work has demonstrated that the NLRP3 inflammasome is activated in the lung during influenza viral infection by the M2 ion channel as well as during S. pneumoniae bacterial infection by pneumolysin; however, the impact of aging on inflammasome function during primary or secondary S. pneumoniae infection has not been examined. Here, we show that elderly mice infected with S. pneumoniae alone or shortly after influenza infection produced lower levels of IL-1β and IL-18 during infection when compared to young mice. Further, elderly mice had increased morbidity, decreased cellular infiltration to the lung, and higher bacterial titers in lung during both primary and secondary bacterial infections. Examination of whole lung tissue collected from elderly mice exhibited decreased expression of ASC, NLRP3, TXNIP, and capase-1, but similar expression of pro-IL-1β and pro-IL-18, when compared to young, similarly infected mice during both primary and secondary bacterial infections. Taken together, our study demonstrates that during primary and secondary S. pneumoniae infections, elderly mice have impaired NLRP3 inflammasome activity which may contribute to increased susceptibility for developing pneumonia.
IL-4 has a critical, mouse strain-limited role in innate memory CD8+ T cell development. (115.19)
Authors: Morris Suzanne , Heidorn Stephanie , Perkins Charles , Hildeman David , Finkelman Fred
Published: January 2023

Mice have large numbers of innate memory cells: CD8+ T cells that develop functional and phenotypic memory cell characteristics in the absence of purposeful immunization. The role of IL-4 production by iNKT cells in innate memory cell development is shown by an ~80% decrease in splenic memory phenotype CD8+ T cell number in IL-4-, IL-4Rα-, Stat6- and CD1d KO BALB/c mice and an ~1/3 normal basal IL-4 level in CD1d KOs. The stimulatory effect of IL-4 on innate memory cell development is first observed in CD8hiCD4lo thymocytes and persists through adulthood. Anti-IL-4 mAb decreases innate memory cell number in thymus and spleen in immature mice, but only in thymus in adults. In contrast to BALB/c mice, IL-4Rα deficiency has little effect on CD8+ memory phenotype T cell number in C57BL/6 mice. This BALB/c - C57BL/6 difference is not associated with appreciable differences in basal total body or thymic IL-4 expression, CD8+ T cell IL-4Rα expression or IL-4 responsiveness, but is associated with greater IL-15Rα and IL-2/15Rβ expression and IL-15 responsiveness by C57BL/6 than BALB/c CD8+ splenocytes. In 5 additional mouse strains studied, 3 resemble BALB/c and 2 resemble C57BL/6 mice with regard to the IL-4 stimulation of innate memory cell development; an IL-4 effect correlates positively with thymic memory phenotype CD8+ T cell number. Thus, IL-4 produced by thymic NKT cells has a major role in memory phenotype CD8+ T cell development that varies among mouse strains.
Enhanced cell cycle activation in response to stimulation in RGC-32-/- T lymphocytes (115.3)
Authors: Tegla Cosmin , Cudrici Cornelia , Nguyen Vinh , Khan Ali , Vlaicu Sonia , Rus Violeta , Badea Tudor , Rus Horea
Published: January 2023

We have previously identified Response Gene to Complement (RGC)-32 as a factor induced by complement activation, growth factors and cytokines. To study the in vivo function of RGC-32, we have generated mice carrying a null allele of the gene. Mice lacking RGC-32 were generated by replacing most of the second exon of the RGC-32 gene with sequences encoding neomycin resistance. These mice develop normally, have a similar size as wild type (WT) mice and a normal life span. Although RGC-32 was found to be involved in cell cycle regulation in vitro and is considered a tumor suppressor gene, RGC-32-/- mice do not spontaneously develop overt tumors. Little is known about the roles played by RGC-32 in T-cell proliferation. The effect of the lack of RGC-32 on cell cycle activation was investigated in CD4 cells isolated from spleen. Proliferation of anti-CD3/anti-CD28 stimulated CD4 cells from RGC-32-/- mice measured by [3H]-thymidine incorporation was significantly increased when compared with WT mice. In addition, by flow cytometry, both CD4 and CD8 T-cells from RGC-32-/- mice displayed a significant increase in the percentage of proliferating Ki-67 cells upon TCR stimulation. Furthermore, Akt phosphorylation was induced significantly in CD4 cells of RGC-32-/- when compared with WT mice. Our data indicate that in T-cells, RGC-32 plays an important role in inhibiting cell cycle activation by suppressing Akt activation.
The acute effects of sub-lethal gamma irradiation on the thymus and T cells were directly affected by age, sex and radiation exposure dose (115.8)
Authors: Xiao Shiyun , Zhang Wen , Manley Nancy
Published: January 2023

To study the effects of A-bomb radiation on immunosenescence, we developed a mouse model to evaluate the effects of sub-lethal gamma irradiation, varying exposure dose and analysis time points in males and females. The current data report the effects on 2 and 6-month old mice exposed at 0, 0.5, 1, 2, or 4 Gray (Gy), analyzed 1.5 months after irradiation. We saw a dose dependent reduction of DN1 thymocytes, especially of DN1a,b subsets. Although DN cells were reduced, the DP subset was increased, with a dose dependent restoration of total thymocyte numbers at all but the highest exposures. Significantly, there was a dose dependent increase of CD4 and decrease of CD8 SP cells, resulting in an increased CD4/CD8 ratio. Thymic NK cell numbers were increased at low doses, but decreased at the 4Gy dose. In males, total peripheral T cell numbers showed a dose-dependent decline while thymocyte numbers showed the opposite effect. In contrast, peripheral T cells showed the same increased CD4/CD8 ratio as thymocytes. Newly produced thymocytes contributed more to restoration of peripheral T cells at low doses, but peripheral homeostasis appeared to have more effect at 4Gy. Thymic epithelial cells (TECs) were also affected, with an increase of total TEC number at low doses followed by a dose dependent reduction at higher doses. These results suggest that age, sex and exposure dose all affect the restoration of the immune system during early recovery period after sub-lethal irradiation.