bluuebunny/crossref_metadata_2025 huggingface dataset:
Asynchronous and modular repression of neutrophil production by IRF8 leads to the development of progenitors with partially overlapping lineage potentials. (153.24)
Authors: Bhattacharya Deepta , Becker Amy , Satpathy Ansuman , Murphy Kenneth
Published: January 2023

Most previous studies have assumed that the maturation of a particular blood cell type downstream of hematopoietic stem cells follows a singular developmental route; therefore the identification of distinct hematopoietic progenitors with partially overlapping lineage potentials has led to considerable controversy. For example, common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) both generate dendritic cells upon adoptive transfer in vivo. To determine the molecular mechanisms by which progenitors with partially overlapping lineage potentials arise, we examined the transcriptional changes that occur as lymphoid-primed multipotent progenitors (LMPPs) differentiate into CLPs, and as CMPs differentiate into common dendritic cell progenitors (CDPs). In both of these independent pathways, we determined that the expression of the transcription factor IRF8 was strongly induced. IRF8-/- mice showed reduced numbers of both CLPs and CDPs. Competitive bone marrow reconstitutions also demonstrated cell-intrinsic defects in the generation of CD8a- dendritic cells, previously reported to be normal in IRF8-/- mice. Microarray comparisons between dendritic cells derived from either wild type CMPs or CLPs demonstrated that these cells are identical. Thus, the asynchronous expression of IRF8 leads to the formation of progenitors with partially overlapping developmental potentials, which can resolve themselves to ultimately generate the same mature cell type.
A role for BAFF during myelopoiesis (153.25)
Authors: Beaudette-Zlatanova Britte , Yeramilli Venkata , Knight Katherine
Published: January 2023

B cell activating factor (BAFF) is expressed in several tissues including the bone marrow (BM). Although BAFF is important for B cell homeostasis and survival, its effects on other leukocytes has not been elucidated. While studying the role of BAFF in rabbit, we discovered that BAFF binds peripheral blood myeloid cells and MHC Class IIneg CD14neg myeloperoxidase (MPO)pos myeloid progenitors (MP) in the BM. Addition of BAFF to co-cultures of unfractionated rabbit BM cells with the murine BM stroma cell line, OP9 stimulated proliferation of rabbit cells resulting in an increase in the total cell number compared to co-cultures lacking BAFF. In addition, BAFF increased the percentage of CD14pos myeloid cells including macrophages in these co-cultures. To determine if BAFF plays a role in human myelopoiesis, we examined Lineageneg CD13pos MP from cord blood (CB) for BAFF binding and expression of BAFF receptors. We found that a small number of these cells express BAFF receptor and bind BAFF. To elucidate the function of BAFF binding to MP, we cultured MP sorted from CB with thrombopoietin, stem cell factor (SCF), and Flt-3L or with SCF and Flt-3L in the presence or absence of BAFF. Total cell number and the number of myeloid cells expressing CD14, CD15, or Class II was not statistically different in cultures containing or lacking BAFF. Experiments to elucidate the function of BAFF binding to MP cells are ongoing. These data suggest a novel role for BAFF in myelopoiesis.
The TREM-2-β-catenin axis is critical to regulate macrophage colony-stimulating factor proliferation and bone homeostasis (153.29)
Authors: Otero Karel , Cella Marina , Shinohara Masahiro , Mizoguchi Toshihide , Takahashi Naoyuki , Takayanagi Hiroshi , Colonna Marco
Published: January 2023

M-CSF regulates proliferation and survival of mononuclear phagocytes through the CSF-1R. DAP12, an ITAM-containing signaling partner for various myeloid receptors, is also critical for mononuclear phagocyte function. In human, deficiency in DAP12 or the immunoreceptor TREM-2 causes Nasu-Hakola disease (NHD), with bone abnormalities including bone cysts and osteoporosis that lead to bone fractures. Paradoxically, DAP12 is necessary for osteoclastogenesis in the mouse and DAP12-deficient mice exhibit an osteopetrotic phenotype. We have previously shown that DAP12 contributes to M-CSF-induced macrophage proliferation through a pathway that involves β-catenin activation. Here we show that the DAP12-associated receptor responsible for the crosstalk of DAP12 with CSF-1R is TREM-2. M-CSF-induced proliferation is impaired in TREM-2-deficient macrophages, and M-CSF-induced DAP12 phosphorylation, but not basal phosphorylation, is abrogated. In contrast to DAP12 deficiency, mice deficient for TREM-2 or β-catenin in osteoclast precursors show accelerated osteoclastogenesis in vitro and osteoporosis in vivo, recapitulating the bone traits of the human NHD. TREM-2 and β-catenin influence osteoclast development by enhancing the proliferative response of osteoclast precursors, thereby inhibiting their differentiation.
Instructional role of ligand in γδ T cell lineage commitment (153.42)
Authors: Coffey Francis , Lee Sang-Yun , Xiong Na , Zuniga-Pflucker Juan-Carlos , Wiest David
Published: January 2023

The processes that influence alphabeta versus gammadelta T cell lineage commitment are not well defined. Evidence suggests that strength and duration of TCR signaling during T cell development can directly influence fate decisions, irrespective of the TCR isotype. Accordingly, T cell progenitors expressing a gammadelta TCR can be directed toward the alphabeta lineage when signaling through the TCR is attenuated. Previously, we showed that KN6 gammadelta TCR transgenic thymocytes can be directed to the alphabeta lineage when the KN6 TCR ligand, T-10/22, is eliminated. Here we investigate the impact of ligand affinity on cell fate as well as whether it regulates fate selection instructively or stochastically. These issues are addressed in vitro using the OP9 stromal cell model where ligand expression can be manipulated. Our data suggest that differences in ligand expression and affinity are important determinants in fate selection. Moreover, ligand appears to be influencing lineage-fate in an instructional manner and is associated with characteristic changes in gene expression. Understanding how modulating TCR signaling affects T cell lineage commitment will provide insight into pathways that control fate decisions of developing alphabeta or gammadelta T cells.
Immune regulation by mycobacterium via delta-like 4 expression on hematopoietic progenitor cells. (153.7)
Authors: Schaller Matthew , Ito Toshiro , Carson IV William , Kunkel Steven
Published: January 2023

Cells in the myeloid compartment are the first responders of the immune system, and primarily uninfluenced by previous immune responses. This is due to a rapid turnover of the myeloid compartment, with cells constantly arising from the bone marrow. We observed a functional change in the bone marrow of mice exposed to mycobacterium, characterized by decreased ability of bone marrow derived DCs to cause IL-2 production from OT-II cells. This reduction in IL-2 was dependent on the Notch ligand delta-like 4. Additionally, the granulocyte/monocyte progenitor population (GMP) had a higher percentage of cells expressing delta-like 4 in mice exposed to mycobacterium. Transfer of GMP cells from mice exposed to mycobacterium into naïve mice caused a reduction in IL-2 production from T cells during an unrelated immune response. Bone marrow chimeras revealed that progenitors expressing delta-like 4 gave rise to mature myeloid cells in the spleen that also expressed delta-like 4. Splenic DC from CFA primed mice also caused less IL-2 production from OT-II cells via a delta-like 4 dependent mechanism. These studies conclusively demonstrate that respiratory mycobacterium infection can impact on future immune responses by upregulating the expression of delta-like 4 on myeloid progenitor cells, which maintain increased expression as they mature into fully differentiated cells.
The capsid protein of foot-and-mouth disease virus interacts with host toll-like receptor 2 to induce innate immunity (154.14)
Authors: Chang Yu-Ching , Chen Yen-Po , Liang Chi-Ming , Liang Shu-Mei
Published: January 2023

The mechanism of interaction between foot-and-mouth disease virus (FMDV) with the host immune system is not clear. In this study, we investigated the interaction between FMDV and Toll-like receptor 2 (TLR2), an important pattern recognition receptor that regulates host immune function. We found that binary ethylenimine-inactivated FMDV (I-FMDV) activated NF-κB only upon expression of TLR2 but not other TLRs in HEK293T cells. I-FMDV induced IL-6 production in macrophages from wild-type but not TLR2 knockout mice. Coexpression of TLR6 or CD14 with TLR2 markedly enhanced NF-κB activation induced by I-FMDV. Further study indicated that VP1 and VP3 but not VP42, all of which are the capsid proteins of FMDV, are the ligands of TLR2. Co-immunoprecipitation assay also demonstrated that VP1 and VP3 interacted with TLR2 as well as CD14, but not TLR6. These results suggest that TLR6 and CD14 may serve as coreceptors of TLR2 which interacts with VP1 and VP3 of FMDV to induce pro-inflammatory cytokines production and immune responses.
Binding of complement regulator C4b binding protein to Flavivirus Nonstructural Protein NS1 modulates its function in solution and on the cell surface (154.22)
Authors: Avirutnan Panisadee , Hauhart Richard , Somnuke Pawit , Blom Anna , Diamond Michael , Atkinson John
Published: January 2023

The complement system plays an important role in the innate immune response to many pathogens including Flaviviruses. Flavivirus NS1 is a secreted glycoprotein that is absent from viral particles but accumulates at high levels in the blood of infected patients where it may also bind back to the cell surface. Our previous work defined an immune evasion role of Flavivirus NS1 in limiting classical/lectin pathway activation by forming a complex with C1s and C4 to promote the cleavage of C4 to C4b. Herein, we demonstrate a second mechanism by which NS1 antagonizes complement activation. In this case, dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 directly associate with C4b binding protein (C4BP), a plasma complement regulatory protein that attenuates the classical/lectin pathway. C4BP is a cofactor for the serine protease factor I to cleave C4b and thereby inhibits classical/lectin pathway C3 convertase (C4b2a) activity. Specifically, binding of NS1 to C4BP results in more efficient factor I-mediated degradation of C4b by C4BP in solution. Furthermore, NS1 recruits C4BP to the surface of cells to inactivate C4b on the plasma membrane. These studies define immune evasion mechanisms by which NS1 reduces C4’s activating capacity. They point to the classical/lectin pathway activation being critical in host defense against Flavivirus infections.
Soluble FasL therapy reduces Herpetic Stromal Keratitis (154.23)
Authors: Stuart Patrick , Rogge Megan
Published: January 2023

Fas ligand (FasL) signals apoptosis in Fas+ cells, and plays a vital role in controlling the entrance of inflammatory cells and neovascularization of ocular tissue. We tested the therapeutic value of soluble FasL in both an acute and recurrent model of HSK using BALB/c and BALB-lpr mice. sFasL was administered in a topical ointment and by subconjunctival injection following infection or reactivation. The controls were treatment with ointment alone or with soluble TRAIL (sTRAIL). These mice were evaluated for corneal disease by a masked observer who scored the corneas for opacity and neovascularization. Following acute infection, wild-type BALB/c mice treated with sFasL displayed reduced incidence of opacity and neovascularization when compared to the sTRAIL and ointment groups post infection. When the Fas deficient BALB-lpr mice were used, results showed no difference in HSK between sFasL and sTRAIL treated groups, as expected. When BALB/c mice undergoing recurrent HSK were treated with sFasL, we saw a decrease in both opacity and neovascularization as compared to the sTRAIL treated mice. These data we believe are due to the apoptotic signaling by sFasL of the Fas molecule found on inflammatory cells and vascular endothelium of newly growing vessels which are attempting to invade the cornea following infection or reactivation.
KLF2 expression modulates susceptibility to HIV-1 infection (154.25)
Authors: Riley James , Richardson Max
Published: January 2023

Here, we show that the transcription factor KLF2 is a host factor with a role in modulating susceptibility to HIV-1 infection. We observed a strong correlation between expression of KFL2 and CCR5. Transformed CD4 T cell lines lack CCR5 as well as KLF2 expression. Forced expression of KFL2 in transformed cell lines induced CCR5 expression and rendered them susceptible to R5 HIV-1 infection, providing insight into why most transformed lines fail to support CCR5 dependent HIV-1 replication. In primary human CD4 T cells, KFL2 expression is rapidly downregulated after T cell activation and is undetectable after anti-CD3/28 costimulation, whereas trace amounts remain after PHA + IL-2 stimulation. This residual amount of KLF2 present after T cell activation correlates well with CCR5 expression and susceptibility to R5 infection. Primary human CD4 T engineered to constitutively express KLF2 retained CCR5 expression and susceptibility to R5 infection after CD3/28 costimulation. These studies implicate KLF2 expression levels present after T cell activation as the determinant of whether a T cell is susceptible to R5 HIV-1 infection. Thus, KLF2 is a host factor that could affect an individual’s susceptibility to HIV-1 infection and the rate by which one progresses to AIDS.
Filamentous phage as a model immunogen for tracking B-cell responses to infection and vaccination (154.30)
Authors: Murira Armstrong , Henry Kevin , Holland Jessica , Scott Jamie
Published: January 2023

Humoral immunity to infection can be categorized into four response stages: (i) innate-like, (ii) early-induced, (iii) adaptive, and perhaps (iv) the response to chronic infection (ChI). The first three stages are mediated by B-cell lineages distinguishable by phenotypic, genetic and functional characteristics. Our analysis of antibodies (Abs) produced during ChI indicates that “ChI Abs” have unique genetic features including high somatic mutation (SM) and usage of distal germline VH genes; ChI is also associated with aberrations in B-cell subsets. Here, we use filamentous phage as a model antigen to determine the genetic features and cellular origins of “ChI Abs” in the mouse. The phage’s surface consists of ~3,000 copies of the major coat protein, pVIII, and a majority of the anti-phage Ab response cross-reacts with a 12-aa peptide comprising the N-terminus of pVIII, allowing easy isolation of phage-specific B cells. After a single immunization, two “waves” of Ab-secreting cells (ASCs) appeared: first, IgM and IgG ASCs expressing proximal unmutated genes, followed by IgG ASCs expressing more distal genes bearing SMs. Phage-specific ASCs and serum IgG increased with repeated immunization, peaking after the fifth immunization and declining thereafter. Thus, we have validated the approach of combining cellular, flow cytometric, serological and immunogenetic analyses to characterize the dynamics of the B-cell response after initial and repeated encounter with antigen.
Vγ4+ T cells regulate host immune response to West Nile virus infection (154.33)
Authors: Wang Tian , Welte Thomas , Aronson Judith , Gong Bin , Rachamallu Aparna , Tesh Robert , Born Willi , O'Brien Rebecca
Published: January 2023

Vγ4+ cells, a subpopulation of peripheral γδ T cells are involved in West Nile virus (WNV) pathogenesis but the underlying mechanism is unclear. Here, we further investigated the role of Vγ4+ cells in WNV encephalitis and found that Vγ4+ cell-depleted mice had a lower viremia and reduced inflammation in the brain following WNV infection. Although Vγ4+ cells contributed to interleukin (IL-17) production during infection, blocking IL-17 signaling did not affect host susceptibility to WNV encephalitis. Furthermore, there was an enhanced magnitude of protective Vγ1+ cell expansion in the spleen and more Vγ1+ cell infiltration into the brains of Vγ4+ cell-depleted mice than controls during WNV infection. Vγ4+ cells of WNV-infected mice had a higher potential for producing transforming growth factor (TGF)-β. We noted that the difference in anti-CD3-stimulated γδ T cell proliferation between those of controls and Vγ4+ cell-depleted mice was diminished in the presence of TGF-β inhibitor. Moreover, Vγ4+ cells contributed to the production of IL-10, which is known to play a negative role in immunity against WNV infection, either directly or by increasing IL-10-producing αβ T cells. These results suggest that Vγ4+ cells may suppress Vγ1+ cell expansion via TGF-β and promote IL-10 production, which leads to higher viremia and enhanced brain inflammation during WNV infection.
The herpes simplex virus 1 encoded latency associated transcript promotes dysfunctional virus specific CD8+ T cells in latently infected trigeminal ganglia (154.39)
Authors: Alami chentoufi Aziz , Kritzer Elizabeth , Tran Michael , Dasgupta Gargi , Afifi Rasha , Jiang Xianzhi , Osorio Nelson , Carpenter Dale , Nesburn Anthony , Wechsler Steven , BenMohamed Lbachir
Published: January 2023

Following ocular herpes simplex virus type 1 infection, HSV-specific CD8+ T cells are induced, selectively retained in latently infected trigeminal ganglia and appear to decrease HSV-1 reactivation. The LAT gene increases HSV-1 reactivation. We hypothesized that increased numbers and/or function of HSV-specific functional CD8+ T-cells in TG might be involved in the decreased reactivation seen with LAT(-)mutants. Mice were ocularly infected with either LAT(+) or LAT(-) viruses and the number and function of CD8+ T-cells in TG were examined during latency. Although fewer total CD8+ T-cells were found with LAT(-) viruses, TG had more functional HSV-gB-specific CD8+ T cells compared to LAT(+) TG which had many exhausted HSV-gB-specific CD8+ T cells, as judged by high levels of PD-1, impaired cytotoxicity, and decreased IFN-γ and TNF-α production. In addition, mouse Neuro2A cells expressing LAT, had elevated PD-L1 and MHC-I compared to LAT(-) Neuro2A cells and were resistant to lysis by allogeneic CD8+ T cells. Collectively, our findings suggest that TG from mice latently infected with LAT(-) viruses had more HSV-specific functional CD8+ T-cells than did TG from mice infected with LAT(+) viruses. In addition LAT appeared specifically able to upregulate both PD-L1 and MHC-I. These findings may constitute a novel immune evasion mechanism whereby LAT promotes dysfunctional HSV-specific CD8+ T cells in latently infected TG, resulting in more virus reactivation
Differential regulation of post-transplant viral host defense by donor versus host regulatory T cells (154.40)
Authors: Lindell Dennis , White Maria , Lukacs Nicholas , Berlin Aaron , Torgerson Troy
Published: January 2023

Respiratory syncytial virus (RSV) infection is a significant cause of morbidity and mortality following hematopoietic cell transplant (HCT). We used a murine model of autologous HCT to investigate the roles played by regulatory T cells (Treg) in host defense against RSV infection post-transplant. Regulatory T cells were highly enriched in the CD4+ T cell compartment up to five weeks post-transplant (>30% of lung CD4+ T cells at week 2 post-transplant, versus 5-10% in controls). The increased frequency of Tregs resulted from 1) survival recipient-derived cells, 2) increased proliferation of Tregs compared to Teff, and 3) differentiation of induced Treg (iTreg). Using Foxp3-DTR mice, DT- mediated ablation of recipient-derived Tregs led to increased lung leukocytes (CD45+, CD4+, CD8+, and CD11b+CD11c+ cells). Total numbers of IFNγ+ CD4+, IL-17+CD4+, IFNγ+CD8+ T cells were all increased in recipient Treg ablated mice. Remarkably, ablation of donor Tregs led to decreases in the same pulmonary leukocyte subsets, and decreased effector T cell (CD4+IFNγ+ and CD8+IFNγ+) responses. Ablation of donor (but not recipient) Tregs impaired viral clearance. In other studies, mature Foxp3+ CD4+ T cells co-transferred at transplant lost Foxp3 expression (>30% by week 4), suggesting instability of Tregs in the post-transplant environment. Together, these studies indicate differential (and possibly, competing) functions for Tregs in post-transplant host defense.
Oligomeric procyanidins stimulate innate antiviral immunity (154.9)
Authors: Hedges Jodi , Kimmel Emily , Jerome Maria , Holderness Jeff , Snyder Deann , Kemoli Sharon , Jutila Mark
Published: January 2023

Oligomeric procyanidins (OPCs) have been shown to have antiviral and immunostimulatory effects. OPCs isolated from non-ripe apple peel, found in the dietary supplement Applepoly®, were tested for capacity to reduce dengue virus (DENV) titers. Similar to published accounts, OPCs exhibited direct antiviral activity. OPCs also enhanced expression of critical innate antiviral products during DENV infection of human PBMCs. Specifically, expression of MXI and IFNB transcripts in DENV infected cells, and phosphorylation of STAT2 in response to recombinant type I IFN (IFN I), were increased by OPC treatment. During low MOI infection, addition of OPCs also increased expression of STAT1 transcripts, MHC I and TNFα protein production. Thus, OPCs exhibited innate immune stimulation of DENV-infected human cells, bystander cells in infected cultures, and uninfected cells treated with IFN I. To test the effects of OPC ingestion in vivo, mice were fed a rich source of OPCs and injected with polyIC to mimic innate responses to viral infection. Ingestion of OPCs resulted in enhanced innate antiviral responses in vivo. While OPCs from a number of sources are known to exhibit antiviral effects, their mechanisms are not precisely defined. The capacity of OPCs to enhance innate antiviral immunity could be broadly applicable to many viral infections and two separate antiviral mechanisms, direct and immunomodulatory, suggest that OPCs may represent a novel, robust antiviral therapy.
VRC01 provides sterilizing protection to non human primates from mucosal SHIV challenges (155.11)
Authors: Pegu Amarendra , Yang Zhi-yong , Chen Xuejen , Todd John-Paul , McKee Krisha , Rao Srinivas , Mascola John , Nabel Gary
Published: January 2023

VRC01 is an exceptionally broad neutralizing monoclonal antibody targeting the CD4 binding site of HIV gp120. It was isolated from an HIV-1 infected donor and is able neutralize over 90% of circulating HIV-1 isolates at high potency in vitro. Using a nonhuman primate lentiviral immunodeficiency virus infection model, we assessed the ability of VRC01 to protect animals from mucosal challenge with a pathogenic chimeric simian-human immunodeficiency virus (SHIV). The animals were first administered either VRC01 or a control IgG i.v. at a dose of 20 mg/kg and 2 days later were mucosally challenged with SHIV. We used two different mucosal routes - intrarectal (i.r.) and intravaginal (ivag) for mucosal challenge to mimic natural HIV exposures in humans. The plasma viral loads and sera VRC01 levels in animals were measured weekly by real time RT-PCR and ELISA, respectively. We observed that 3 out of the 4 animals in both the control groups were infected, with peak viremia occurring at week 2 for the i.r. challenge and at week 3 for the ivag challenge. In contrast, none of the 4 animals that received VRC01 got infected in both mucosal challenge groups (p=0.04). These results demonstrate that a broadly neutralizing CD4 binding site mAb, VRC01, protects against mucosal exposure to pathogenic viruses in vivo. This finding provides support for the CD4 binding site as a target of AIDS vaccine development and the possible use of VRC01 to prevent mucosal transmission of HIV-1 in humans.
The anti-infective Nitazoxanide shows strong immumodulating effects (155.21)
Authors: Clerici Mario , Trabattoni Daria , Pacei Michela , Biasin Mara , Rossignol Jean-Francois
Published: January 2023

Nitazoxanide (Alinia®, NTZ) and its active circulating metabolite tizoxanide (TIZ) are anti-infectives thiazolide with activity against parasites, anaerobic bacteria, and viruses. NTZ is licensed in the US for the treatment of diarrhea caused by Cryptosporidium parvum and Giardia lamblia. NTZ and TIZ also inhibit replication of RNA and DNA viruses including influenza A and hepatitis C viruses. In clinical trials, NTZ was shown to be effective in treating rotavirus gastroenteritis, norovirus gastroenteritis and chronic hepatitis C, and is in late-stage clinical development for treatment of influenza. We evaluated the immunomodulatory effect of TIZ. PBMC from 10 healthy donors were cultured in the presence/absence of 3 different doses of TIZ (0.5, 1.0 and 10 mg/ml) in both unstimulated and flu-stimulated conditions and analysed for T helper and CTL activity as well as for TLR7 and TLR8 expression and type I IFN responses. TIZ showed potent immunomodulatory effects inducing an increase in: 1) IFNg- and IL2-secreting CD4+ T cells, 2) CTL degranulation, 3) Fas-expressing CD8+ T cells, 4) TLR8-expressing monocytes, 5) IFNa- and IFNb- mRNA expression, 6) mRNA specific for type I IFN inducible genes (MXA, PRKCZ, ADAR, CXCL10, IRF1, PRKRA), and 7) mRNA specific for gene involved in MHC class I presentation (HLA-A, HLA-B, TAP1).The antiviral effects of TIZ result from immumodulatory activity stimulating a strong antiviral immune response mediated by both native and acquired mechanisms
Dual antigen target based immunotherapy for prostate cancer eliminates the growth of established tumors in mice (155.3)
Authors: Karan Dev , Dubey Seema , Thrasher Brantley
Published: January 2023

Generation of antigen-specific CD8 T cells is considered optimal for an effective immunotherapy against cancer. Knowing that heterogeneity of prostate cancer limits the therapeutic benefit, we developed a recombinant adenovirus type 5 (rAd5) co-expressing a fusion of PSA (prostate-specific antigen) and PSCA (prostate stem cell antigen) genes. The selection of these genes is based on their restricted distribution within the prostate and their association with the development and progression of prostate cancer. Immunization of mice with rAd5 vector co-expressing PSA and PSCA antigens (Ad5-PSPA) simultaneously induces the expansion of anti-PSA, and anti-PSCA T cells as measured by intracellular cytokine staining for IFN-y. To analyze the impact on therapeutic efficacy of Ad5-PSPA vaccine against the tumor cells co-expressing cognate antigens (RM11-PSA/PSCA cells), injection of mice with Ad5-PSPA vaccine inhibited the growth of established tumors. Initially, the mice developed slow growing tumors and eventually upto 80% of the mice remain tumor free following immunization with Ad5-PSPA vaccine. These data provide useful information that antigen-specific effector T cells can be generated simultaneously and their additive anti-tumor effect has the ability to eliminate the growth of established tumors. Therefore, the immunotherapy approach of using the simultaneous targeting of dual antigens associated with prostate cancer may have important implications for human clinical trials.
Signaling via neurokinin-1 receptor is required for cellular immunity induced by melanoma vaccines (156.12)
Authors: Larregina Adriana , Mathers Alicia , Tkacheva Olga , Morelli Adrian
Published: January 2023

Tumor vaccines are promising alternatives to current therapies for melanomas. Yet, in spite of initial enthusiasm, melanoma vaccines not always provide the positive results expected. Hence, there is a need for in-depth study of the mechanisms underlying effective vaccines for melanocyte Ags. The neuroimmune function of the skin, is ideal for efficient cancer vaccines and agonistic signaling of skin cells via neurokinin-1 receptor (NK1R), by the inflammatory substance-P (SP) released by cutaneous nerves and immune cells, favor CD4+Th1 and CD8+Tc1 immunity to foreign Ags. Here, we demonstrate that elicitation of cellular immunity by skin gene gun (GG) immunizations needs agonistic signaling via NK1R.Indeed, GG-vaccines encoding the tyrosine-related-protein-2 (TRP-2) (GG-TRP-2), delivered to NK1RKO mice failed to induce TRP-2-specific immunity. Conversely, NK1R agonist (Sar9Met O2)11-SP (SarSP) used as adjuvant at the vaccination site in BL/6 WT mice significantly enhanced the ability of GG-TRP-2 to elicit TRP-2-specific effector and memory Th1 and Tc1 immunity. Importantly, this resulted in prevention of melanoma growth and increased survival of mice challenged with B16F0 melanoma cells and overcame a potential tumor promoting effect of SarSP. Our results shed light on the mechanisms involved in efficient elicitation of cellular immunity through cutaneous delivery of weakly immunogenic tumor self Ags are relevant for development of effective tumor vaccines.
Decitabine and Interferon-γ enhance neuroblastoma cells susceptibility to CTL-mediated killing (156.26)
Authors: Bao Lei , Dunham Kimberly , Lucas Kenneth
Published: January 2023

MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma, but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen specific T cells. If expression of cancer antigens as well as MHC molecules could be enhanced pharmacologically, the efficacy of CT antigen specific immunotherapy could be improved. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to the demethylating agent 5-aza-2’-deoxycytidine (decitabine, DAC) in 10/10 neuroblastoma cell lines. Culture of neuroblastoma cell lines with IFN-γ was associated with an increased expression of either MHC Class I or II by cytofluorometry, with MHC Class I upregulated in 10/10 of these cell lines, although to varying degrees. MAGE-A1, MAGE-A3, and NY-ESO-1 specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full length proteins, and these CTL lysed HLA partially matched, DAC treated neuroblastoma and glioblastoma cell lines. These studies confirm that DAC and IFN-γ can be used to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL mediated killing.
High avidity T cells have increased potential to convert to FoxP3+ T cells in vitro and in the tumor microenvironment (156.29)
Authors: Sklavos Martha , Zhu Ziqiang , Singh Vinod , Hurwitz Arthur
Published: January 2023

We have previously reported that CD8+ T cells that enter the tumor microenvironment and become tolerized can also acquire suppressive function. More recently, we reported that low avidity (TCRlo) T cells were unable to control B16 melanoma tumor growth, despite retention of effector function. In contrast, high avidity (TCRhi) T cells initially delayed tumor growth until they became tolerized, when tumor growth resumed. We hypothesized that TCRhi T cells may be more likely to differentiate into FOXP3+ regulatory T cells, which could contribute to restored tumor growth. To examine our hypothesis, B16 tumor-infiltrating TCRlo and TCRhi T cells were tested for FOXP3 expression. An increased frequency of intratumoral FOXP3+ TCRhi T cells (p=0.012 vs. FOXP3+ TCRlo cells) were detected approximately 2.5 weeks after T cell transfer, suggesting that conversion to a FOXP3+ phenotype contributed to restored tumor growth. We next tested the potential of TCRlo and TCRhi T cells to differentiate into FOXP3+ T cells. After a 10 day in vitro culture with TGF-β, an increased frequency of TCRhi T cells expressed FoxP3 compared to TCRlo T cells (p=0.019). On-going studies are characterizing the function of these unique tumor-infiltrating CD8+ T cells. Circumventing the conversion of high avidity T cells to FoxP3-expressing T cells in vivo may extend the beneficial effects of adoptive T cell therapy and produce more effective cancer therapies.
Autophagy of Listeria monocytogenes within macrophages is dependent on Nod/Rip2 pathway (157.2)
Authors: Anand Paras , Kanneganti Thirumala-Devi
Published: January 2023

Listeria monocytogenes is an intracellular pathogen that escapes from the phagosome and replicates in the cytosolic compartment. Intracellular bacteria are often targeted for autophagy, an essential component of the innate immune response. Autophagy involves engulfment of the pathogen within double-membrane structures called autophagosomes wherein the contents are degraded by acidification upon fusion with the lysosomes. The role of innate immune receptors, such as the Nod-like receptors, in autophagy of Listeria monocytogenes is not well known. We observed that upon Listeria infection autophagy is induced within macrophages as observed by LC3-II immunoblotting, GFP-LC3 puncta formation and electron microscopy. However, in macrophages deficient in Nod1 or Nod2, autophagy induction upon Listeria infection was found to be inefficient. Further, autophagy induction was dependent on the Nod1/Nod2 downstream adaptor Rip2 kinase. Defective autophagy in Nod2-/- macrophages resulted in fewer bacteria co-localizing with the GFP-LC3 autophagosomes. Furthermore, Nod2-/- macrophages were found to be more susceptible to Listeria replication. These findings demonstrate a mechanistic link between the innate immune receptors and antibacterial autophagy.
Sequential phosphorylation and ubiquitination regulate NOD2:RIP2 signaling (157.7)
Authors: Tigno-Aranjuez Justine , Abbott Derek
Published: January 2023

NOD2, the Crohn’s Disease and sarcoidosis susceptibility protein, is an intracellular sensor of muramyl dipeptide (MDP), a breakdown product of bacterial peptidoglycan. Upon exposure to MDP, NOD2 binds to the kinase RIP2 to modulate cytokine responses. We have recently described that NOD2 activation results in the tyrosine autophosphorylation of RIP2 and have demonstrated that pharmacologic inhibition of RIP2’s tyrosine kinase activity blunts activation of downstream NOD2 signal transduction pathways. In complementarly work, the E3 ubiquitin ligase, ITCH, downregulates NOD2 signaling, and in ITCH-/- macrophages or ITCH knockdown cells, both MDP-induced cytokine release and RIP2 tyrosine phosphorylation are aberrantly high. Our current work extends these findings by demonstrating that ITCH-/- mice are hypersensitive to sarcoidosis models. We molecularly map the direct site of ITCH-induced downregulation of the NOD2:RIP2 pathway and find that ITCH mediates regulation of NOD2 signaling and RIP2 tyrosine phosphorylation through ubiquitination and degradation of cIAP1. Lastly, we show that by mimicking the action of ITCH through the use of pharmacological inhibitors of cIAP1 (MEBS), we can decrease levels of cIAP1, decrease RIP2 tyrosine phosphorylation, and negatively regulate downstream NOD2 signaling. Agents such as MEBS may be useful in hyperactive NOD2 states such as in individuals with Crohn’s Disease harboring WT NOD2, Early Onset Sarcoidosis, and ITCH-deficiency.
Analysis of phosphorodiamidate morpholino oligomer chemisrty CpG effects in murine and human cells (158.12)
Authors: Hauck Laura , Mourich Dan , Iversen Patrick
Published: January 2023

Many RNA-based therapeutic oligomer backbone chemistries will elicit undesirable immunological response through a number of pathways. Additionally, it has been shown that certain sequences can also elicit cellular pro-inflammatory responses in the form cytokine and chemokines production. For example, non-methylated CpG DNA motifs are considered pathogen-associated molecular patterns (PAMPS) due to their abundance in microbial genomes and their recognition by TLR-9 receptors. Some synthetic oligonucleotides, containing modified backbone or other synthetic nucleotide motifs, will still retain potent immunostimulatory activity which may be inappropriate within the desired therapeutic effect. The purpose of this study was to look in vitro for immune activation resulting from exposure to optimized CpG sequences incorporated into the phosphorodiamidate morpholino oligomer (PMO) chemistry. Human and murine dendritic cells (DCs) were treated with varying concentration of PMO and culture media was assayed by multiplex cytokine and chemokine detection platform for simultaneous detection of multiple analytes. There was no evidence for a sequence- or chemistry-specific activation of the DCs indicating that PMO do not provide an innate stimulus to the immune system.
Regulation of host innate immune receptors by influenza (158.23)
Authors: Vigerust David , Shepherd Virginia
Published: January 2023

Influenza virus A (IVA) is a significant cause of morbidity and mortality each year and is one of the most important infectious diseases in the United States Very little is known about the interaction between influenza and the macrophage. Interactions between influenza and macrophages may affect acute symptomology, inmmune evasion and disease outcome by preventing the proper function of the macrophage in the clearance of harmful pathogens. Our hypothesis is that host innate defense molecules are utilized as a receptor of entry and target of down-regulation by influenza A viruses leading to dysregulation of macrophage function. The HIV-1 Nef protein was shown to interact with MR tail to down-regulate the expression of the MR on macrophages. Sequence comparison of the interacting domains of the HIV-1 Nef molecule and the sequence of influenza A NS1 and PB1 reveals similarity in several motifs that indicate a potential for MR regulation by influenza. Our initial data demonstrates that infection of MR expressing cells results in a significant decrease in surface expression of MR by 48 hours post-infection. Further, in vitro transient transfection of influenza proteins leads to removal of MR from the surface at levels approaching 30% for NS1 and PB1. Removal of MHC-I from the cell surface was also seen as early as 5 hours post-transfection. Our data suggests that influenza regulates critical host innate defense molecules in a fashion that is similar to that seen with HIV-1 Nef.
Ligation of CD4 on human blood monocytes triggers macrophage differentiation (158.8)
Authors: Kitchen Scott , Krutzik Stephan , Levin Bernard , Rezek Valerie , Kitchen Christina , Zack Jerome
Published: January 2023

Human monocytes/macrophages (M/M) have an important role in innate immune responses and in controlling adaptive immune responses. One unique aspect of human M/M is that they express the CD4 molecule. The role of the CD4 molecule in human monocyte/macrophage function and development is not known. To investigate the role of CD4 expressed on human M/M, human blood monocytes were treated with soluble HLA Class II (sHLA II), a natural CD4 ligand. We determined that sHLA II:CD4 ligation triggered the differentiation of human monocytes into functional mature macrophages. These newly developed macrophages had an increased ability to induce T cell activation, suggesting enhanced immune function following CD4 ligation of these cells. We observed a calcium flux and the modulation of gene and cytokine protein expression following CD4 ligation on peripheral monocytes. This indicates that ligation of CD4 transmits a functional signal in this cell type. We found that intracellular signaling of CD4 in primary monocytes occurs through src family kinases as well as via the MAPK pathway to trigger macrophage differentiation. We show that HLA II on IFN-γ-induced HUVECs can ligate CD4 on monocytes and trigger differentiation of these cells into functional macrophages. In all, our results have identified a new function for the CD4 molecule on human peripheral monocytes in triggering the differentiation of these cells into functional macrophages following their interaction with HLA II.
Increased caspase activity primes human lyme arthritis synovial γδ T cells for proliferation and death (160.11)
Authors: Thai Phan , Collins Cheryl , Fortner Karen , Koenig Andreas , Hayes Sandra , Budd Ralph
Published: January 2023

γδ T cells function between the innate and adaptive immune responses, promoting antigen-presenting cell function, presenting antigen, and manifesting cytolytic activity. Their numbers increase in infections, such as HIV, and at sites of chronic inflammation. However, the turnover dynamics of human γδ T cells are poorly understood. Here we find that despite more rapid proliferation, human Lyme arthritis synovial γδ T cells of the Vδ1 subset have reduced cell number expansion in vitro compared to αβ T cells due to increased cell death by the γδ T cells. Because caspases are involved in cell proliferation and death, and signaling is more efficient through the γδ-TCR than the αβ-TCR, we examined active caspases during cell cycling and following TCR restimulation. We observed higher overall caspase activity in Borrelia-reactive γδ T cells than comparable αβ T cells. This was paralleled by greater spontaneous and TCR restimulation-induced cell death of the γδ T cells. Furthermore, the transition from cell growth to death was associated with a transition of active caspases from the cell membrane to the cytoplasm. Our current findings thus are consistent with a model where human γδ T cells evolved to function quickly and transiently, in an innate fashion.
Atypical T cell receptor usage defines a population of Natural Killer T cells with distinct glycolipid antigen recognition properties (160.13)
Authors: Godfrey Dale , Uldrich Adam , Patel Onisha , Cameron Garth , Pellicci Daniel , Day E. , Sullivan Lucy , Kyparissoudis Konstantinos , Kjer-Nielsen Lars , Vivian Julian , Brooks Andrew , Besra Gurdyal , Turner Stephen , Porcelli Steven , McCluskey James , Smyth Mark , Rossjohn Jamie
Published: January 2023

Lipid Ag reactive T cells represent an important component of the T cell repertoire. The most well studied example is the classical (or Type-1) NKT cells that respond to the glycolipid Ag α-galactosylceramide (α-GalCer) presented by CD1d. While often referred to as 'invariant' due to the expression of an invariant TCR-α chain (Vα14-Jα18), in fact, Type-1 NKT cells carry a diverse TCR-β repertoire that influences Ag recognition. Furthermore, we have identified a completely novel population of NKT cells (Type-1A cells), that expresses a previously unidentified, canonical, Vα10-Jα50 TCR α-chain. These cells exhibit a different pattern of glycolipid Ag reactivity, including a preference for α-glucosylceramide (α-GlcCer) over α-GalCer and a bias towards Th2 cytokine production. Two populations of Type-1A NKT cells (TCRhi and TCRlo) are present in the thymus and they also have diverse TCR-β chains. We also provide the first structural analysis of a non-classical NKT TCR-CD1d-glycolipid complex, showing that the Type-1A TCR-CD1d-α-GlcCer complex displayed a similar docking mode to that of Type-1 NKT TCRs, although differences at the Ag-binding interfaces accounted for the altered specificity. The discovery of a second invariant TCR α-chain expressed by CD1d-restricted NKT cells provides new insights into the repertoire diversity and its influence on glycolipid recognition by NKT cells, and has significant implications for the immunologic role of glycolipid-specific T cell responses.
Regulation of memory CD4 T cell repertoire diversity upon peptide and protein vaccination (160.18)
Authors: Baumgartner Christina , Malherbe Laurent
Published: January 2023

Diverse antigen-specific memory TCR repertoires are essential for protection against pathogens. Subunit vaccines that combine peptide or protein antigen with TLR agonists are very potent in inducing T cell immune responses but their capacity to elicit stable and diverse memory CD4 T cell repertoire has not been evaluated. Here, we tracked the development of antigen-specific memory CD4 T cells after peptide or protein vaccination. Both vaccination regimens induced equally diverse effector CD4 TCR repertoires but a skewing toward high affinity clonotypes was observed in the memory TCR repertoire elicited by peptide vaccines. This skewing did not occur after protein vaccination leaving the antigen-specific TCR repertoire essentially intact in the transition from effector to memory. Clear understanding of the factors regulating the TCR repertoire diversity after peptide and protein vaccination contributes to our knowledge of T-cell mediated immune responses and has practical relevance to the design of vaccines against infections requiring robust T cell immunity.
Cross-reactivity and repertoire connectivity of CD8+ T cells in adults (160.19)
Authors: Petrova Galina , Naumova Elena , Gorski Jack
Published: January 2023

Measuring the extent to which a T cell repertoire raised against one epitope can recognize slightly different epitopes is important for questions of response to escape variants or novel pathogens. Analyzing the MHC-peptide structures that can be recognized can provide insight into the nature of the cross-reactive TCR in the initial repertoire. We measured the cross-reactivity of the highly polyclonal BV19 T cell repertoire that responds to influenza M158-66 in HLA-A2 individuals by using an incremental epitope modulation approach. In three HLA-A2 adult subjects we identified the BV19 clonotypes in the recall response to the influenza epitope M158-66 and then in the response to twelve M1 peptides substituted at TCR contact positions 63 or 65. Six of the twelve peptides were recognized by some fraction of the M158-66 repertoire. The fraction of cross-reactive clonotype as defined by recognizing at least one additional peptide varies from 45 to 58%. The maximal extent of cross-reactivity defined as an additional number of peptides recognized is six. The BV19 cross-reactive clonotypes form a completely connected network identifying a well defined set of related MHC-peptide structures that can be recognized if they should be part of a novel pathogen or escape variant.
Antibiotic treatment reduces intestinal non-defensin protein and facilitates Klebsiella pneumoniae translocation (161.11)
Authors: Chen Lee-Wei , Wu Ying-Ying , Hsu Ching-Mei
Published: January 2023

Effects of microbiota disruption on intestinal permeability and immunity were examined in C57BL/6 mice receiving combined antibiotic treatment. Antibiotic decreased the total number of bacteria including specific aerobic group Enterobacteriaceae and Enterococcus, and specific anaerobic group Lactococcus/Bifidobacterium and Bacteroides in intestinal mucosa and lumen. Although only a slight increase in the intestinal permeability, the bacterial translocation (BT) to mesenteric lymph nodes (MLN) increased significantly. Antibiotic treatment decreased the mucosal killing activity and increased the translocation of pathogen K. pneumonia, suggesting that the increase of BT to MLN after antibiotic treatment is likely due to a reduction in gut immunity. This is further supported by that antibiotic treatment decreased the expression of non-defensin family including RegIIIβ, RegIIIγ, CRP-ductin and RELMβ but not the defensin family. Both Myd88-/- mice and germ-free mice demonstrated decreased non-defensin protein expression and increased K. pneumonia translocation. Furthermore, killed E.coli supplementation reversed antibiotic effect on intestinal immunity and K. pneumonia translocation. Collectively, antibiotic treatment facilitates K. pneumonia translocation through the reduction of intestinal non-defensin protein. Oral dead E.coli supplementation restores antibiotic-induced innate immune deficit and decreases K. pneumonia invasion by increasing intestinal non-defensin protein.