bluuebunny/crossref_metadata_2025 huggingface dataset:
Gp96-Ig/Costimulator Combination Platform Improves T Cell Priming, Enhances Immunity and Memory
Authors: Gonzalez Louis E. , Giffin Louise , Rose Jason , Fromm George , Da Silva Suresh , Schreiber Taylor H. , Jasuja Rahul , Hutchins Jeff
Published: January 2023

The recent excitement in the field of immuno-oncology has been driven largely by the clinical success of checkpoint inhibitors. However, 60–90% of patients are still failing to respond to these therapies. It is widely believed that approaches targeting multiple facets of the immune system, including combinations with checkpoint inhibition and costimulator agonism, will be required to improve patient outcomes. Heat Biologics has developed a next generation T cell activating platform called ComPACT (COMbination Pan-Antigen Cytotoxic Therapy) that incorporates expression of a tumor antigen chaperone (gp96-Ig) and a T cell costimulator (OX40L-Ig) within a single vaccine cell line to effectively prime and boost antigen-specific CD4+ and CD8+ T cells. Furthermore, ComPACT stimulates activation of CD127+KLRG1- memory precursor cells, and lacks the toxic systemic inflammatory cytokine production and proliferation of non-specific CD4+ T cells and Tregs observed with systemic administration of OX40 agonist antibodies. ComPACT also led to high frequencies of IFNγ+, TNFα+, granzyme B+ and IL-2+ antigen-specific CD8+ T cells at both priming and boosting.

Here, we have further assessed ImPACT and ComPACT-OX40L in combination with the anti-PD1 checkpoint inhibitor and with expression of an additional cell-secreted T cell costimulator, TL1A-Ig. We show that these treatments synergize effectively to amplify antigen-specific T cells and program a memory response. The combination of ComPACT-OX40L/TL1A with αPD1 or αPD-L1 may therefore translate into an efficacious approach generate long lasting memory in immuno-oncology settings.
Alum adjuvant is more effective than MF59 in promoting early germinal center formation in response to peptide-protein conjugates and enhancing efficacy of candidate vaccines against opioid abuse in adult and old mice
Authors: Pravetoni Marco , Robinson Christine , Schmiel Shirdi E. , Mueller Daniel L.
Published: January 2023

Heroin and prescription opioid abuse and overdose have been declared a national emergency in the United States. Compared to current medications, vaccines provide a complementary long-lasting, safe and cost-effective therapeutic strategy. Since the efficacy of vaccines against drugs of abuse is contingent upon achieving high antibody levels in immunized subjects, this study tested whether vaccines against oxycodone were more effective when formulated in the well-established aluminum hydroxide or the squalene-based oil-in-water emulsion MF59 adjuvant. Alum was more effective in inducing early expansion of hapten-specific germinal center (GC) B cells, and in inducing oxycodone-specific serum IgG antibodies that were effective in blocking oxycodone-induced motor activity in BALB/c adult mice. Alum was also more effective than MF59 in promoting early, but not late, differentiation of antigen-specific MHCII-restricted GC-Tfh cells in C57Bl/6 adult mice immunized with a model peptide-protein conjugate vaccine. Finally, alum and MF59 adjuvants displayed equivalent stimulation of hapten-specific B cells and peptide-specific T cells in old mice. Alum was significantly more effective, or at least equivalent to MF59 in eliciting protective responses against the highly-abused prescription opioid oxycodone and inducing early germinal center formation in response to hapten and peptide antigens in both adult and aging mice.
Development of a mesothelin-based prophylactic vaccine against ovarian cancer
Authors: Scholler Nathalie , Sharma Khushboo , Yin Catherine , Kamat Kalika , Fernando Romaine Ingrid , Sei Shizuko , Shoemaker Robert H. , Stein Paul L. , Sambucetti Lidia
Published: January 2023

Mesothelin is a tumor-associated antigen overexpressed in ovarian cancer and normally expressed by mesothelial linings. While mesothelin function remains to be fully elucidated, the lack of phenotype of mesothelin-knockout mice makes it plausible that mesothelin can safely serve as a vaccine target. We designed a new vaccination strategy based on mesothelin targeting with activation of type I IFN signaling via cyclic dinucleotides (CDN). Vaccines first combined human mesothelin protein with alum- vs. CDN-based adjuvants. C57Bl/6 mice received a priming immunization followed by 2 boosts; two weeks later, immunized mice were injected orthotopically with luciferase-transduced mouse ovarian cancer cells (Luc-ID8). More than 2/3 of the mesothelin/CDN-immunized mice were protected against invasive cancer and presented increased cytotoxic T cells (CTL) and plasmacytoid dendritic cells in spleens. Effector memory T cells were also increased and MDSC were profoundly decreased in peritoneal lavages after immunization with mesothelin +/− CDN compared with all the other groups. IFNγ ELISPOT assays performed with protected animal splenocytes stimulated with mesothelin peptides identified 2 immunogenic regions. We next tested a peptide-based subunit vaccine derived from mouse mesothelin sequence. Immunization and 2 boosts of 25-mer peptides with CDN were sufficient to mount an immune response against 2 overlapping mouse mesothelin peptides. Immunizations with peptide pools mapping in the immunogenic regions are currently assessed for protection against Luc-ID8 growth and progression. Peptide-based prophylactic vaccination against ovarian cancer may be possible in combination with adjuvants that trigger cellular immunity.
Development of adjuvant scaffolds from small molecule contact allergens
Authors: Kimani Flora , Kim Seong-Min , Esser-Kahn Aaron
Published: January 2023

Small molecule contact allergens are an understudied class of immunostimulatory compounds that have the potential of being developed into adjuvants. Vaccines remain the most effective tool in preventing disease and adjuvants serve to make vaccines more efficacious by activating the innate immunity. To develop contact allergens molecules as adjuvants, we began by first elucidating the mechanism of allergic contact hypersensitivity. Using contact allergens and their analogues we wanted to define the structural elements necessary for the activation of immune cells. Our initial experiments sought to correlate sensitization potential to chemical reactivity where the latter influenced the initial phase of sensitization, hapten formation. We showed that there is a non-linear relationship between the rate of reactivity of the compounds and the ability to induce IL-8 expression on THP-1 cells. This result suggested that contact allergens modify specific target proteins and hence sterics, as well as electronic nature of the contact allergen, play an important role. Using the insights from this initial study we are developing probes that will allow the identification of the hapten-protein complex formed during the sensitization process in order to understand the recognition mechanism of this complex to immune cell receptors. We envision this study as the first step towards the development of potent adjuvants from contact allergen scaffolds. Additionally, the elucidation of the protein targets of small molecule contact allergens will reveal therapy avenues for the treatment of allergic contact hypersensitivity.
Targeted delivery of antigens to CD11b+ cells via nanobodies induces strong antigen-specific T cell and anti-tumor responses
Authors: Woodham Andrew W. , Cheloha Ross W. , Ling Jingjing , Rashidian Mohammad , Kolifrath Stephen C. , Mesyngier Maia , Duarte Joao N. , Bader Justin M. , Skeate Joseph G. , Da Silva Diane M. , Kast W. Martin , Ploegh Hidde L.
Published: January 2023

The variable regions of camelid heavy chain-only antibodies (nanobodies or VHHs) are the smallest antibody fragments that retain full antigen binding. Owing to their small size, ease of expression, and high tissue penetration, VHHs that recognize cell surface proteins on antigen-presenting cells (APCs) can serve as targeted delivery vehicles for antigens conjugated to them. We site-specifically attached antigens to the C-terminus of an anti-CD11b VHH (VHHCD11b), and then investigated the ability of the VHH-antigen conjugates to elicit antigen-specific T cell and anti-tumor responses in a mouse model of human papillomavirus (HPV)-induced cancer. This VHH had high affinity for CD11b+ dendritic cells (apparent KD = 204 ± 90 pM), and enhanced cross-presentation of a conjugated antigen (OVA257–264) in vitro. Mice immunized with the VHH conjugated to an immunodominant HPV16 E7 epitope (E749–57) had more E7-specific cytotoxic T cells compared to mice immunized with E7 peptide alone, conferring protection against HPV16 E7+ tumor cell challenge. Therapeutically, VHHCD11b-E7 vaccination resulted in greater numbers of CD8+ tumor infiltrating lymphocytes compared to mice receiving E7 peptide alone in HPV16+ tumor-bearing mice, as measured by in vivo non-invasive VHH-based positron emission tomography (PET), which correlated with tumor regression and increased survival. Thus, targeting CD11b for the delivery of antigens via VHHs may be a promising cancer immunotherapeutic strategy.
Recovery of HIV-mediated Immune Aging of T Cells in Virologically Controlled HIV-Infected Patients on Antiretroviral Therapy
Authors: Bradley Nicole Elizabeth , Wertheimer Anne , Klotz Stephen , Nikolich-Zugich Janko , Ahmad Nafees
Published: January 2023

Several studies have shown that HIV infection induces an accelerated aging of T cells, immunosenescence, and alterations in T cell homeostasis resulting in dysregulation of T cell function and rapid HIV disease progression in infected individuals. We have investigated whether long term antiretroviral therapy (ART), which successfully controls viremia and restores CD4 T cell counts in HIV-infected individuals, is correlated with reversal of immunosenescence and/or improved homeostasis of T cells in 100 virologically controlled HIV-infected individuals. We show that the median frequencies of immunosenescence of T cells (CD4+/CD8+ CD28−, CD27−, CD57+ and CD28−CD57+ T cells), were higher in the HIV-infected cohort than the uninfected cohort, however, senescent T cells significantly decreased with increasing CD4 T cell counts in HIV-infected individuals. Although overall levels of naïve CD4+ and CD8+ T cells were lower in HIV-infected than uninfected individuals, there was a significant increase in both CD4+ and CD8+ naïve T cells with increasing CD4 T cell counts in HIV-infected individuals. In addition, the increase in naïve CD4+ and CD8+ T cells in HIV-infected individuals was due to recent thymic immigrants expressing CD31+ marker; however, with increasing CD4 T cells counts accumulation of CD4+CD31− T cells were seen. Moreover, the CD4+ T cells of HIV-infected individuals produced cytokines, including IL-2, IL-10 and IFN-g comparable to uninfected individuals. In conclusion, HIV-infected individuals with controlled viremia and restored CD4 T cell counts due to prolonged ART showed significant reduction in T cell immunoscenesce and improvement of T cell homeostasis and function.
Co-inhibitory receptor TIGIT inhibits CD8+T mediated hepatocellular carcinoma
Authors: Zong Lu , Li Fenglei , Wei Haiming , Sun Rui , Tian Zhigang
Published: January 2023

Chronic hepatitis B virus (HBV) pathogenesis is widely accepted as numerous immune response inducing prolonged inflammation, cirrhosis and lastly hepatocellular carcinoma(HCC). However, the potential theory of HBV tolerance and the immune-mediated HCC development has not been clearly defined currently. We found here that hepatic CD8+T cells of HBsAg-transgenic mice (HBs-tg) express remarkably high levels of co-inhibitory receptor T cell Ig and ITIM domain (TIGIT) with age. Co-inhibitory pathway blockade in HBs-tg mice initiated regional chronic inflammation sequentially inside the liver which reflected by gradually rising ALT level and hepatic fibrosis. TIGIT blockade gave rise to the emerging of HBsAg-specific CTLs and rescued CD8+T cells function by increased proliferation and interferon-γ(IFN-γ) and CD107a production in liver. Besides, HBsAg vaccination to TIGIT-blockade mice triggered the development of HCC. Breeding HBs-tg TIGIT−/−mice showed spontaneous chronic inflammation in life and got HCC toward HBsAg vaccination. CD8+T depletion during TIGIT blockade alleviates liver inflammation in HBs-tg mice. While depleting CD8+T cells start from the vaccination stage till harvest in TIGIT-blockade mice abolishes HCC formation.

Conclusion

High co-inhibitory receptor expression on CD8+T cell may be a contributing factor for HBV tolerance. Our studies reveal a new molecular pathway which causes chronic hepatic diseases.
Increased reactivation of murine gammaherpesvirus-68 during Heligmosomoides polygyrus infection
Authors: Zarek Christina M. , Reese Tiffany A.
Published: January 2023

A vast majority of people are infected with a herpesvirus. These viruses are double stranded DNA viruses that avoid eradication by the immune system by becoming latent. Certain signals, such as immune repression, can lead to reactivation of the virus. Reactivation of gamma herpesviruses is associated with pathologies, such as lymphomas and tumors. Past studies have shown that infection with helminths, extracellular parasitic worms, during gamma herpesvirus latency can cause reactivation. Helminths are known to secrete immunomodulatory factors that suppress host inflammation. The aim of the current study is to determine how prior helminth infection leads to increased reactivation of murine gammaherpesvirus-68 (MHV68). In this study, mice were or were not infected with Heligmosomoides polygyrus (HP), and then infected with MHV68. We found that infection with HP increased the reactivation of virus and the number of infected cells. However, there was no difference in acute replication of the virus between HP infected mice and HP uninfected mice. This suggests that the helminth infection specifically alters viral reactivation. The increased reactivation was only seen in a conventional mouse facility, but not in a barrier mouse facility, suggesting dependence on a microbiota component. Further work is required to determine how HP infection alters immune signals, such as IL-4 and STAT6 signaling, to cause increased MHV68 reactivation. Whether HP alters the permissiveness of cells to MHV68 infection should also be explored.
Development of an in vitro cell culture model to investigate HCMV priming of CD8+ T cells
Authors: Abad Maria , Lenarcic Erik , Xu Yinyan , Wong Jason , Clutton Genevieve , Warren Joanna A. , Savoldo Barbara , Moorman Nathaniel J. , Goonetilleke Nilu
Published: January 2023

Human cytomegalovirus (HCMV)-specific CD8+ T cells are characterized by an unique, non-exhausting, effector memory phenotype. How HCMV induces this phenotype is poorly characterized. We hypothesized that HCMV may skew T cell priming producing functionally distinct CD8+ T cells.

To investigate this question, we have established an autologous 3-cell culture system using human umbilical tissue in which naïve CD8+ T cells are isolated from cord blood, myeloid DCs (mDCs) are generated from cord blood CD34+ precursor cells and fibroblasts, which are a primary target of HCMV infection, are generated from matching umbilical cord (UC-F).

Previous studies have reported IFN-γ treatment of fibroblasts induces MHC-II expression, leading to suggestions that fibroblasts can acquire APC-like activity. To investigate this, we used flow cytometry to examine the effects of HCMV infection +/− IFN-γ on MHC-II, CD40, CD80 and CD86 surface expression on fibroblasts. HCMV alone did not induce MHC-II or costimulatory molecules. HCMV infection +/− IFN-γ induced MHC-II but costimulatory molecules remained undetectable. This suggests HCMV-infected fibroblasts do not acquire APC-like phenotype and that fibroblasts mediate T cell priming through cross-presentation.

Fibroblasts release extracellular vesicles (EVs), which are important mediators of cell signaling, including antigen cross-presentation. We used electron microscopy to confirm that fibroblasts release EVs. Following AD169-GFP infection, 5.5–8% of EV collected were GFP-positive by ImageStream. Future studies will investigate whether UC-F modulate mDC priming of CD8+ T cells through direct cell-cell contact and/or indirectly, including through the release of EVs.
Defining the mechanisms responsible for the induction of cytotoxic CD4 T cell responses during viral infection
Authors: Knudson Cory , Kappes Dietmar , Sigal Luis J.
Published: January 2023

Induction of cytolytic lymphocytes is a core aspect of the anti-viral immune response. While NK and CD8 T cells are most often associated with the job of directly eliminating virus-infected cells, cytotoxic CD4 T cells can also be a major contributor. Cytotoxic CD4 T cell responses have been observed during a multitude of virus infections in both humans and mice including HIV, cytomegalovirus (CMV), herpes simplex virus, influenza virus, ectromelia virus (ECTV), and lymphocytic choriomeningitis virus. It has been previously demonstrated that the absence of a cytotoxic CD4 T cell response following ECTV infection of mice results in significantly greater viral burden. Runx3, the main transcription factor responsible for induction of cytolytic genes in NK and CD8 T cells, was also upregulated in cytotoxic CD4 T cells as compared to other activated or naive CD4 T cells following ECTV infection. Isolation of CD4 T cells expressing Runx3 revealed upregulated expression of cytolytic genes granzyme B and perforin. This suggests that Runx3 may play a critical role in the induction of cytotoxic effector functions in CD4 T cells. In addition, mice lacking expression of MHC class II on either CD11c+ or monocytic cells had significantly impaired cytotoxic CD4 T cell responses following ECTV infection. Challenge of mice with a mutant strain of ECTV that cannot spread from the footpad does not induce a cytotoxic CD4 T cell response, indicating that viremia or local replication is necessary. Our results highlight cell intrinsic and extrinsic mechanisms that are critical for the induction of the cytotoxic CD4 T cell response during viral infection.
Delayed and highly specific antibody response to nonstructural protein 1 (NS1) revealed during natural ZIKV infection by NS1-based capture ELISA
Authors: Yu Lei , Gao Xiujie , Wen Yingfen , Wang Jian , Hong Wenxin , Li Chunlin , Zhao Lingzhai , Yin Chibiao , Jin Xia , Zhang Fuchun
Published: January 2023

The high degree of cross-reactivity of Envelope (E) protein specific antibody responses between ZIKV and DENV made it difficult to perform differential diagnosis between the two infections using standard ELISA method for E protein. The analyses of the sequential serum samples from ZIKV infected individuals showed NS1 specific Abs appeared two weeks later than E specific Abs. Notably, there is none cross-reactivity to NS1 proteins from all four DENV serotypes in human sera from ZIKV infection. Furthermore, four out of five NS1-specific monoclonal antibodies (mAbs) isolated from ZIKV infected individuals did not bind to DENV NS1 proteins. Only limited cross-binding to ZIKV NS1 was displayed in 108 DENV1 immune sera at 1:100 dilution. The high degree of NS1-specific Abs in both ZIKV and DENV infection revealed here suggest that NS1-based diagnostics would significantly improve the differential diagnosis between DENV and ZIKV.
Determinants of CD8+ T Cell Immunodominance in Acute HIV-1 Infection
Authors: Warren Joanna A. , Xu Yinyan , Constanzo Margaret , Whale Victoria , Liu Michael , Thomas Rasmi , Tovanabutra Sodsai , Kijak Gustavo H. , Eller Leigh A. , Rolland Morgane , Michael Nelson L. , Robb Merlin L. , Eller Michael A. , Goonetilleke Nilu
Published: January 2023

CD8+T cells detect and clear virus-infected cells. During human immunodeficiency virus-1 (HIV-1) infection, different HIV-specific CD8+ T cell populations are expanded, each targeting a discreet epitope. These different HIV-specific CD8+ T cell populations do not arise equally, and instead establish a hierarchy (immunodominance) based on strength of T cell response to a given viral epitope. Our previous work has shown that immunodominance is a major determinant of the rate at which HIV can mutate to escape T cell immune pressure. The precise mechanism of how the first CD8+ T cell response to HIV is generated, and which factors determine T cell dominance is unclear. To investigate determinants of T cell immunodominance in acute HIV-1 infection (~45 days after infection), we determined all primary HIV-1 specific CD8+ T cell responses in 10 individuals from the RV217 early acute HIV-1 cohort. Participants are high-risk volunteers from Kenya and Thailand, and acute HIV-1 infection was determined from twice-weekly blood draws using a nucleic acid test. T cell responses were mapped against the unique infecting HIV-1 virus/es in each individual using IFN-γ ELISpot. Across participants, the immunodominant T cell response mostly targeted the Env, Gag and Pol proteins of the HIV-1 genome. We detected a wide (4–14 epitopes) breadth of T cell response across our cohort, with all HIV-1 proteins targeted and different rates of virus escape observed. The summed magnitude of response ranged from 1,388–16,044 SFU/106 cells. Ongoing studies are focusing on better understanding how factors, such as T cell receptor repertoire, avidity, antigen presentation and function contribute to T cell immunodominance, which in turn will inform HIV-1 vaccine design.
Biochemical and structural characterization of a host protein that binds Ebola VP30
Authors: Liu Dandan , Small Gabe , Hultquist Judd , Krogan Nevan , Batra Joyti , Basler Christopher , Leung Daisy , Amarasinghe Gaya
Published: January 2023

Ebola and Marburg viruses are negative sense RNA viruses that can cause high case fatality rates during outbreaks. Approximately 19 kb genome encodes for 7 open reading frames. During virus replication, 7–10 multifunctional proteins are expressed, but viral replication and pathogenesis also require numerous cellular proteins. While the need for host factors in virus replication and pathogenesis has been long appreciated, our understanding of key host-virus interactions during filoviral infection and replication remain incomplete. In order to address this limitation, our collaborators recently performed a proteomic analysis, which identified RBBP6, an E3 ubiquitin ligase, as a cellular interactor of Ebola VP30 (eVP30). eVP30 is a viral protein critical for transcription initiation. In this study, we generated a series of recombinant RBBP6 and eVP30 truncation constructs. We performed in vitro pull-down assays to validate the initial proteomic identification of the eVP30/RBBP6 protein-protein interaction and defined key regions within RBBP6 that are critical for eVP30 binding. Furthermore, using purified eVP30 protein, we generated crystals with a RBBP6 binding peptide and refined X-ray diffraction data to define a series of key residues on the interface of eVP30/RBBP6. Hence, this study provides insights on host-viral interface that modulates viral pathogenesis and defines a novel target for potential development of antiviral therapeutics that target filoviruses.
Lack of T-cell mediated IL2 and TNFa production is linked with the decreased CD58 expression in intestinal tissue during acute simian immunodeficiency virus infection
Authors: Pahar Bapi , Pan Diganta , Srivastav Sudesh K. , Lackner Andrew A. , Das Arpita
Published: January 2023

Human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection causes a progressive impairment of the immune system characterized by massive CD4+ T-cell loss, CD8+ T-cell expansion and sustained immune activation and inflammation. Our data also suggest that pathogenic SIVMAC251 infection in rhesus macaques (RMs) leads to decreased production of several T-helper 1 (TH1) and TH2 cytokines and increased production of IL-17, IFNg, CCL4 and GM-CSF by intestinal CD8+ T-cells 21 days after infection. For an effective T-cell activation and response, costimulation is required in addition to the antigen-specific signal from their antigen receptors. The CD2/CD58 interaction is considered as one of the important T-cell costimulatory pathway for T-cell activation and proliferation. Intestinal lamina propria T cells were reported to be highly responsive to a stimulus delivered by CD2 pathway compared to the conventional CD3/TCR pathway. The role of CD2/CD58 in regulating intestinal T-cell function of acute and chronic SIV infected RMs is poorly documented. Here, we demonstrated a significant reduction of CD58 expression in both T- and B-cell population during acute SIV infection along with the loss of intestinal CD4+ T-cells and high plasma viral load compared to SIV-uninfected normal RMs. The reduction of CD58 expression in T-cells was positively correlated with the reduced expression of T-cell mediated IL2 and TNFa production. Together, these results indicate that the reduction in CD2/CD58 interaction pathway in mucosal lymphocytes might play a crucial role in mucosal T-cell dysfunction during acute SIV/HIV infection.
Autoimmune Risk Allele of PTPN22 influences early events post viral infection
Authors: Willenbring Robin , Pratumchai Isaraphorn , Marquardt Kristi , Mowen Kerri A. , Teijaro John R. , Sherman Linda A.
Published: January 2023

The PTPN22 alternative allele, PTPN22 1858T, is present in 5–10% of the North American population and is considered the highest autoimmune, non-HLA, risk allele. Extensive work has been done to better understand this allele in the context of autoimmune disorders. However, the role of PTPN22 1858T in the context of viral infection remains incompletely defined. Our lab has previously defined a role for Ptpn22 in generating T cell exhaustion during chronic viral infection. Yet, the mechanism by which it is acting and how the alternative allele effects T cell exhaustion remains unknown. To directly determine this we used CRISPR/Cas9 technology to independently generate mice expressing the human-relevant Ptpn22 alternative allele. Using the well-established LCMV-cl13 infection model in our mice, we tested the hypothesis that pleiotropic effects of Ptpn22 contributes to T cell exhaustion and chronic viral infection. We address this through examination of the role of Ptpn22 in numerous immune cells contributing to viral clearance in vivo. Since early events post-infection drastically influence persistent viral infection we began our studies characterizing splenic organization, cytokines, immune cell infiltration of target organs, and viral load at 1 and 3 days post infection in our mice. Next, we further define how the Ptpn22 alternative allele effects APC functions required for proper anti-viral immune response. Lastly, we determine the state of viral clearance and T cell exhaustion at late time points post-infection in our system. Results of these studies give valuable insight into the role of Ptpn22 in multiple immune cell types that contribute to chronic viral infection and T cell exhaustion.
Dendritic cell intrinsic IRF4 expression regulates production of regulatory cytokines during influenza virus infection
Authors: Hatipoglu Ibrahim , Ainsua-Enrich Erola , Kadel Sapana , Turner Sean , Singh Simar , Kovats Susan
Published: January 2023

Lung resident dendritic cells (DCs) are a heterogeneous population with diverse roles in respiratory infection. The development and function of DC subsets are controlled by distinct transcription factors including IRF4, which regulates CD11b+ cDC2 development, migration, and function. CD11c-cre Irf4fl/fl mice (KO) with a deletion of Irf4 in DCs lack a subset (P1) of lung CD11b+ DCs while other DCs are present but lack IRF4. To test the hypothesis that DC intrinsic IRF4 regulates anti-viral responses, wild type (WT) and KO mice were infected with a sublethal dose of influenza A/PuertoRico/8/34. On days 3–8 post-infection, a novel flow cytometry scheme was used to assess numbers and functional responses of lung and mediastinal lymph node (mLN) DCs, including tissue DC subsets CD103+ and CD11b+ (P1, P2), and inflammatory monocyte-derived Ly6Chi DCs. In addition to lacking CD11b+P1 DCs, KO mice had increased numbers of lung CD103+DCs, a defect in migration of CD11b+P2 DCs to the mLN and reduced numbers of Ly6Chi DCs in the mLN. In WT mice, CD11b+P1 DCs mice produced IL-10, and CD103+DCs preferentially displayed a latent form of TGFβ (LAP) and expressed Itgb8 needed to retain surface LAP for TGFβ activation. KO CD103+DCs showed reduced expression of TGFβ-LAP and Itgb8. Thus, DC production of both IL-10 and TGFβ is deficient in infected KO mice. The reduction of regulatory cytokines is consistent with our earlier finding that infected KO mice harbor decreased numbers of Foxp3+ T regulatory and CD8+ T memory cells, yet greater numbers of effector IFNg+ influenza-specific CD8+ T cells. In sum, IRF4 differentially regulates lung DC subsets to promote regulatory cytokines, and this diversity balances the anti-viral immune response to influenza infection.
IFN-lambda regulates dendritic cell function to mediate protective immunity against influenza A virus infection
Authors: Hemann Emily A. , Green Richard , Langlois Ryan A. , Savan Ram , Gale Michael
Published: January 2023

Influenza A virus (IAV) is a public health threat with constant potential for pandemic outbreak in addition to causing annual, seasonal epidemics. While an IAV vaccine is available, it is rapidly subverted through viral genetic drift, necessitating strategies to limit IAV infection and drive robust, protective immune responses. Type III interferon (IFN), IFN-lambda (IFNλ), is the most highly produced IFN in the lung during IAV infection and restricts IAV in vitro and in vivo. Further, IFNλ influences CD4 T cell skewing following IAV vaccination, but the contribution of IFNλ to the critically protective CD8 T cell response against IAV has not been defined. Here we evaluated the role of IFNλ signaling in the development of CD8 T cell responses against IAV infection in a murine model. Our studies reveal that IFNλ signaling is essential to support cytokine-producing IAV-specific effector CD8 T cells as the CD8 T cell response against IAV is significantly reduced in IFNλ receptor knock out (IFNλR KO) mice. Importantly, IFNλR KO mice exhibited reduced immune protection upon heterologous IAV re-challenge that is dependent upon T cell responses. This immune defect was not T cell intrinsic but instead mapped to altered function and migration of dendritic cells (DCs) in IFNλR KOs, thus linking IFNλ signaling in DCs with modulation of the T cell response. Transcriptomic analysis of DCs revealed the dysregulation of a set of genes implicated in antigen uptake, processing and/or presentation, downstream of IFNλ, but not type I IFN. The function of these genes in the IFNλ response for programming CD8 T cells against IAV will be presented. Our findings reveal a unique role for IFNλ in the governance of DC function and subsequent downstream T cell function.
Anti-insulin B cells are poised for antigen-presentation in pro-inflammatory subsets in type 1 diabetes
Authors: Felton Jamie Lynn , Bonami Rachel , Hulbert Chrys , Thomas James W.
Published: January 2023

Type 1 diabetes (T1D) results from T-cell mediated autoimmune destruction of insulin-producing beta cells. Early breaches in B cell tolerance are central to T1D progression in mouse and man. Conventional transgenic mouse models (VH125.Tg NOD) suggested that B cell specificity is a powerful driver of disease. However, the precise staging of B cell development is limited in the conventional IgM-only model by the inability to class-switch. To investigate the developmental fate and functional status of anti-insulin B cells in NOD mice, we generated a new model (VH125SD.NOD) in which anti-insulin VDJH125 is targeted to the IgH chain locus and recombines with endogenous Vk chains to produce fully functional BCRs. This single copy VH transgene generates a small (1–2%) population of class-switch-competent anti-insulin B cells. Tracking their developmental fate reveals that anti-insulin B cells are preferentially skewed into marginal and late transitional zones which are known to have increased sensitivity to innate signals and enhanced capacity for antigen-driven expansion. Functional studies demonstrate that VH125SD.NOD anti-insulin B cells are anergic to antibody production; however, proliferation in response to B cell mitogens remains intact, diabetes is accelerated, and autoaggressive T cell responses to islet epitopes are increased. These findings validate the power of BCR specificity to drive T1D in a new mouse model and highlight the pathogenic potential of coupling a critical beta cell autoepitope to pro-inflammatory B cell subsets. We believe these findings may have implications for the discovery of new therapeutic targets in sources of innate signals that enhance autoreactivity in these B cell subsets, such as gut microbiota.
NZB;NZW chimeras develop strong autoimmunity earlier than NZB/NZW F1
Authors: Hankins Julia , Liu Aijie , Choi Jin , Li Xiaohong , Turer Emre , Beutler Bruce
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by an immune response to many self-antigens, including DNA, histones, and RNA-binding proteins. One of the most physiologically relevant mouse models is the NZB/NZW F1 hybrid mouse, which develops SLE at high penetrance, in contrast to either parental strain. However, the critical genetic contributions of each parent, which must somehow interact to cause SLE, have remained unresolved. We have determined that NZB;NZW embryonic chimeras develop an accelerated form of autoimmunity characterized by the presence of auto-antibodies 6 months earlier, and at titers 10-fold greater, than the NZB/NZW F1 hybrids. The chimeras also developed glomerulonephritis, severe lymphadenopathy, and splenomegaly. Within each chimera, expansion of activated T cells of NZB/BINJ origin and a predominance of B cells of NZW/LacJ origin was observed with respect to the relative proportions of myeloid cells. Furthermore, the expansion of NZB/BINJ T cells correlates with autoantibody production. This SLE phenotype was specific for the NZB/BINJ;NZW/LacJ combination, and occurs despite the fact that neither parental strain develops SLE-like autoimmunity in a non-chimeric environment. Understanding the pathogenesis of SLE in the NZB;NZW embryonic chimeras may elucidate causes of autoimmunity by identifying the intercellular transactions between these two strains that lead to disease.
Comparison of immune cells from germinal centers specific to protein or lipid antigens
Authors: Galicia Viridiana Galicia , Manzano Oscar Arturo Angeles , Castro Ma Natividad Flores , Rostro Elizabeth Martínez , Wong Carlos , Wong-Baeza Carlos , Baeza Isabel , Reséndiz-Mora Albany
Published: January 2023

Anti-lipid IgG antibodies can participate in the physiopathology of some autoimmune diseases. In our research group, we developed a mouse model of Lupus by the administration of liposomes bearing non-bilayer phospholipid arrangements NPA induced by some drugs like chloropromazine CPZ. The mice of this model produce anti-NPA IgG antibodies; these antibodies are mainly formed via germinal center GC. However, the cells that participate in a GC reaction against lipidic antigens are unknown; therefore, we study these cells in the mouse model of Lupus induced by NPA and compared them with a GC reaction against ovalbumin OVA, because the cells that participate in a GC reaction against protein antigens are well known. We studied germinal center B cells, plasma cells, dendritic cells, follicular dendritic cells FDC, T follicular helper cells TFH, T follicular regulatory cells TFR and macrophages from the groups administered with OVA or NPA and compared them with mice without administration. Then we extracted the spleen and the mesenteric lymph nodes and perform flow cytometry and H&E staining at days 5, 10, 15 and 30 postadministration. Histologically, germinal centers were found in both groups at day 5. The mice administered with NPA, present an increase of germinal center B cells 15 days post-administration and a bigger size of the secondary lymphoid organs, the mice administrated with OVA present an increase of these cells and bigger lymph organs at day 10. The higher percentage of plasma cells was found 30 days after the administration of OVA; while dendritic cells and TFH cells presented similar percentages in both groups at all times. For both antigens, the FDC increase at day 5 and 15, the macrophages from day 10 to 15 and the TFR cells increase at day 15.
The macrophages in the pancreatic islets are activated and sense pathogen associated products in blood
Authors: Zakharov Pavel , Ferris Stephen T. , Wan Xiaoxiao , Unanue Emil R. , Zinselmeyer Bernd , Carrero Javier A.
Published: January 2023

Using RNAseq, we examined the transcriptome of endocrine macrophage in the islets of Langerhans in NOD (non-obese diabetic) and in two non-diabetic controls: NOD.Rag1−/− and B6.g7. The NOD mice spontaneously develop diabetes at 20–24 weeks of age. By gene expression analysis we found that at 3 weeks of age the macrophages from all 3 strains of mice were already activated. They expressed high levels of MHC-II, costimulatory molecules, and the pro-inflammatory cytokines IL-1b, TNF, both at protein and transcript levels. Leveraging the ImmGen database, we found that this activation signature is common for resident macrophages from barrier surfaces of the body, in particular lung and gastrointestinal tract. In addition to the basal level activation found in all islet macrophages at 3 weeks of age, we found an elevated activation signature in NOD when comparing to non-diabetic control. This finding indicates that the diabetic inflammatory process was already underway at as early as 3 weeks of age. These results were confirmed by single cell qRT-PCR that showed a distinct activated population of Cxcl9, Ccl5, Ly6a high (~28%) macrophages in NOD, but not in NOD.Rag1−/−. Additionally, supporting the idea of a barrier phenotype in islet macrophages, we found that they responded to intravenous injection of lipopolysaccharide by rapid upregulation of inflammatory gene expression. In imaging experiments we showed that macrophages were constantly extending filopodias into the blood lumen and capturing microparticles. In conclusion, the islet macrophages are in a highly activated steady state, sense pathogen associated products in blood, and may have a protective barrier function in the endocrine pancreas.
Interleukin-7 receptor pathway controls human T cell homing to the gut and predicts response to anti-TNFα therapy in IBD
Authors: BELARIF LYSSIA , Danger Richard , Kermarrec Laetitia , Daguin Véronique , Boureille Arnaud , Naveilhan Philippe , Vanhove Bernard , Poirier Nicolas
Published: January 2023

Background and aims

Signaling networks perpetuating chronic gastrointestinal inflammation in Crohn’s disease (CD) and ulcerative colitis (UC), the two main forms of inflammatory bowel diseases (IBD), remain unclear in man.

Methods and results

According to an analysis of nearly 500 patients with IBD and 100 controls, we report here that key transcripts of the IL-7 receptor (IL-7R) pathway accumulate in inflamed colon tissues of severe IBD patients not responding to either immunosuppressive/corticosteroids or anti-TNFα therapies. High expression of both IL-7R and IL-7R signaling signature transcripts in the colon before treatment are strongly associated with nonresponsiveness to anti-TNFα therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controls α4/β7 integrin expression and imprints gut-homing specificity on T cells. IL-7 receptor blockade in vivo reduced human T cell homing to the gut and colonic inflammation in humanized mouse models, and decreased production of IFNγ by colon explants from UC patients grown ex vivo.

Conclusion

Our findings suggest that the failure of current treatments for IBD, including anti-TNF, may occur at least in part by dysregulated IL-7R signaling and points to the IL-7R as a relevant novel therapeutic target and biomarker to fill an unmet need in clinical IBD detection and treatment.
Lactoferrin inhibits immune cell adhesion via suppression of cell adhesion molecules expression in hypoxia/reoxygenation animal model
Authors: Lee Tae Hoon
Published: January 2023

Lactoferrin (Lf), an 80 kDa iron binding protein, belongs to transferrin family found in secretions such as milk and in plasma secreted from neutrophils granules upon inflammatory stimulation. Lf is a multifunctional protein mainly involved in both the innate and adaptive immune defenses. In this study, we investigated the anti-adherent activity of Lf in hypoxia/reoxygenation (H/R)-stimulated human endothelial cells (HUVECs). Lf dramatically reduced the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by hypoxia-situation HUVECs. Our results also demonstrated that Lf was reduced the expression of cell adhesion molecules on H/R-induced vascular inflammation mouse model. Lf significantly reduced the gene expression of pro-inflammatory cytokine such as IL-6, IL-1β and TNF-α in hypoxia-induced HUVECs. These results demonstrate that Lf inhibits the expression of cell adhesion molecules including ICAM-1, VCAM-1 and pro-inflammatory cytokines. It also suggests that Lf may play an important role in the development of drug for anti-vascular inflammation by inhibiting the expression of cell adhesion molecules.
Evaluating the Effects of Systemic, Exercise-Induced Skeletal Muscle Injury using Whole Transcriptome Sequencing
Authors: Tanner Elizabeth A. , Kim Tracy A. , Gary Melody A. , Stelly Steven , Davis Asheal , Bowman Erin M. , McFarlin Brian Keith
Published: January 2023

Recent advances in next generation sequencing have dramatically reduced the cost of whole transcriptome sequencing to measure differential gene expression. Extreme physical performances represent a unique model for investigating the combined effects of oxidative stress and eccentric muscle contraction on systemic skeletal muscle injury outcomes. Studying changes in whole transcriptome RNA expression may allow identification of specific potential treatment targets for a variety of disease states associated with chronic inflammation and oxidative stress. The purpose of this study was to investigate changes in whole transcriptome RNA expression in response to prolonged endurance running. The protocols were approved by the University IRB committee (in accordance with the latest Declaration of Helsinki) and subjects gave written informed consent to participate. Blood samples were collected in PAXgene RNA tubes at baseline, 4-h, and 24-h after performing a half-marathon race. After collection, tubes were frozen and total RNA was extracted and verified using accepted methods. RNAseq analysis was conducted using an Illumina NextSeq 500 sequencing platform. RNAseq analysis revealed distinct changes in injury, inflammation, anti-oxidant defense, and stress-associated RNA expression with responses appearing more pronounced at 24-h compared to 4-h post-race. These results confirm the systemic changes that occur following whole-body exercise-induced muscle injury in a human model. The next step in this research is to test potential therapeutic strategies to determine their effectiveness on altering the changes in the transcriptome.
TREML4 affects lesion composition but not calcification in the Apolipoprotein E deficient model of experimental Atherosclerosis
Authors: Cotto Marieli Gonzalez , Guo Liang , Karwan Megan , Palmieri Erika M. , Boelte Kimberly , Quigley Laura , Sen Shurjo K. , Biesecker Leslie G. , McVicar Daniel W.
Published: January 2023

The Triggering Receptor Expressed on Myeloid cells-like 4 (Treml4) is a member of the TREM receptor family which are recognized fine-tuners of the inflammatory response. We have previously found that Treml4 expression correlates to increased risk of Human Coronary Arterial Calcification and Acute Coronary Syndrome. Moreover, we show that carriage of either one of the eQTL SNPs previously identified to permit Treml4 expression, confers the highest treml4 expression in human inflammatory macrophages when compared to alternative activated macrophages. However, the exact role of Treml4 in the pathogenesis of coronary diseases remains incompletely defined. To elucidate the role of Treml4 in atherosclerosis, we investigated whether treml4 deficiency affected pathogenesis in the Apolipoprotein E knockout mouse (apoe−/−). We found that aortas from apoe−/− mice had increased treml4 expression when compared to wild type and that treatment with either LDL or oxLDL upregulated macrophage treml4 expression in vitro. After a western diet, overall plaque burden and calcification in the aortic sinus of apoe−/−/treml4−/− mice remained unchanged compared to controls. However, we found that treml4 deficiency resulted in less complex lesions as indicated by decreased foam cell content, plaque necrosis and collagen deposition as well as dysregulation of matrix metalloproteinases expression within the plaques. Moreover, less collagenous metaplasia was observed in the brachiocephalic artery of apoe−/−/treml4−/− mice. Taken together, our results suggest that Treml4 contributes to the inflammatory mechanisms and extracellular matrix regulation associated with lesion complexity without affecting lesion burden or calcification.
PLAG ameliorates LPS-induced ALI by attenuation of neutrophil infiltration into alveolar via a prompt resolution of TLR4 signaling.
Authors: Shin Su-Hyun , Lee Ha-Reum , Jeong Jinseon , Kim Joo Heon , Lee Do Young , Moon Byoung-Gon , Sohn Ki-Young , Yoon Sun Young , Kim Jae Wha
Published: January 2023

Acute lung injury (ALI) is an acute respiratory failure that is associated with excessive neutrophil recruitment into the bronchoalveolar space and results in severe mortality. To evaluate 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) as a therapeutic agent for ALI, PLAG was administered orally to mice intranasally challenged with lipopolysaccharide (LPS). The excessive neutrophil infiltration in the bronchoalveolar lavage fluid (BALF) was detected in LPS-treated mice. Intranasally introduced LPS especially stimulates tissue-resident macrophages in the pulmonary tissue and induces neutrophil-attracting chemokine production such as MIP-2. Co-administrated PLAG dramatically ameliorated neutrophil infiltration into the bronchoalveolar region via modification of TLR4 signaling. We found that PLAG effectively accelerated endocytosis of LPS-TLR4 complex and promoted the NADPH oxidase activity through the formation of Rac, p47phox assembly into membrane for ROS production which results in elimination of exotoxin in Raw264.7 cells. PLAG also triggered a prompt TLR4 signals occurred in endosome mediated by TRIF and terminated signaling including MIP-2 expression when endocytosed exotoxin is cleared. Moreover, immunofluorescence microscopes of RAW264.7 cells showed that PLAG accelerated the endocytosis of the LPS-TLR4 complex and the clearance of the internalized LPS. Our results suggest that PLAG promotes the clearance of invaded exotoxin and eventually triggers an earlier termination of MIP-2 secretion in the exotoxin-cleared macrophages, and might be used as a potential therapeutic agent to prevent ALI through speedy resolution of exotoxin.