bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding NKT cells through sulfatide analogues
Authors: Hsu Kevin , Olkhanud Purevdorj B , Camara Kaddy , Luvaga Irunga K , Kanyo Emese , Richardson Steward K , Sassu Nicole , Howell Amy , Berzofsky Jay A
Published: August 2023

The goal of this research is to further investigate NKT cell activation using sulfatide analogues. NKT cells have been shown to play a significant role in mediating inflammatory immune responses and because of this, there has been strong interest in pharmacologically regulating this compartment to fight cancer. NKT cells recognize lipids that are loaded on CD1d molecules on the surface of cells, and it has been shown that structural modifications of these lipids directly affect NKT cell activation and polarization. Our work is focused on the impact of modifications of sulfatide, which is a glycolipid that can be loaded onto CD1d which is known to activate sulfatide-reactive (generally type II) NKT cells when presented by CD1d monomers on plastic. We have numerous sulfatide analogues with modifications (based on previous work with alpha-galactosylceramide analogues) on the acyl chain, sphingosine chain, and changes of beta-anomeric to alpha-anomeric. These analogues have been screened with NKT cell hybridomas, DN32 and XV19, to assess their effect on activation of type I and type II NKT cells, respectively. We observed that alterations in the sphingosine or acyl chain in sulfatides do not always mimic the effects on activity of similar changes in alpha-galactosylceramide analogues and that numerous sulfatide analogues can activate DN32 (not sulfatide-reactive) when presented on CD1d monomer or bone marrow-derived dendritic cells. Our results provide further insight into NKT cell activation and have identified sulfatide analogues that may be applicable to cancer therapy by activating type I NKT cells.

NCI, NIH intramural research program (NIA-C-004020), and institutional grants of NIH (R01 GM111849)
Protein-polymer Bioconjugate Nanoparticles To Investigate Innate and Antigen-Specific CD4 T Cell Responses
Authors: Scotland Brianna L , Cottingham Andrea L , Lasola Jackline J , Pearson Ryan M
Published: August 2023

Nanoparticles (NP) comprised of poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) have been shown to be effective delivery systems for peptide and protein antigens (Ags) capable of modulating Ag-specific T cell responses. Encapsulation and surface conjugation are common Ag association methods using NPs. However, these methods are associated with caveats such as imprecise Ag loading, high Ag burst release, and immune recognition. To improve the efficiency of Ag delivery and address these limitations, we prepared ovalbumin (OVA)-PLGA conjugates that were systematically combined with unmodified PLGA to produce NPs (acNP-OVA) with three precise Ag loadings, negligible immune recognition, and a reduced burst release. acNP-OVA was used to elucidate how NP formulation parameters, such as Ag content, influence innate and Ag-specific CD4+ T cell responses. We observed Ag loading-dependent innate immune cell activation (MHCII, CD80/86) and Ag-specific CD4+ T cell proliferation, cytokine secretions, and regulatory T cell induction in vitro. Ag-encapsulated NPs were employed as a control. CD25 expression and T cell expansion were achieved using both Ag delivery methods and observed at low Ag loading. However, high Ag loading was required to induce IFNg and IL-2 cytokine secretions. Notably, nanostring analysis identified inflammation-associated genes, such as S100a9, was significantly downregulated in dendritic cells due to NP treatment. We anticipate this study will aid in designing NP-based immunotherapies for Ag-specific immunomodulation.
Gene Set Enrichment Analysis Reveals That Fucoidan Induces Type I IFN Pathways in BMDC
Authors: Choi Suyoung , Jeon Sol A , Heo Bu Yeon , Kang Ju-Gyeong , Jung Yunju , Thuy Duong Pham Thi , Song Ik-Chan , Kim Jeong-Hwan , Kim Seon-Young , Kwon Jaeyul
Published: August 2023

Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has been proposed to effectively treat and prevent various viral infections. However, the mechanisms behind its antiviral activity are not completely understood. We investigate here the global transcriptional changes in bone marrow-derived dendritic cells (BMDCs) using RNA-Seq technology. Through both analysis of differentially expressed genes (DEG) and gene set enrichment analysis (GSEA), we found that fucoidan-treated BMDCs were enriched in virus-specific response pathways, including that of SARS-CoV-2, as well as pathways associated with nucleic acid-sensing receptors (RLR, TLR, NLR, STING), and type I interferon (IFN) production. We show that these transcriptome changes are driven by well-known regulators of the inflammatory response against viruses, including IRF, NF-κB, and STAT family transcription factors. Furthermore, 435 of the 950 upregulated DEGs are classified as type I IFN-stimulated genes (ISGs). Flow cytometric analysis additionally showed that fucoidan increased MHCII, CD80, and CD40 surface markers in BMDCs, indicative of greater antigen presentation and co-stimulation functionality. Our current study suggests that fucoidan transcriptionally activates PRR signaling, type I IFN production and signaling, ISGs production, and DC maturation, highlighting a potential mechanism of fucoidan-induced antiviral activity.

이 연구는 충남대학교 연구비와 교육부 조성 한국연구재단(NRF-2019R1I1A3A01060331 to J.K.) 또한, 과학기술정보통신부가 지원하는 한국연구재단(NRF)을 통한 브레인풀 프로그램(NRF-2020H1D3A2A02048437, J.G.K.)과 과학기술부(NRF-2021M3H9A1030267, S.Y.K.)의 지원으로 진행됐다.
Extracellular vesicles deliver feto-placental paternal antigens to maternal secondary lymphoid tissues for T cell recognition
Authors: Powell Juliana Schneider , Shufesky William , Larregina Adriana T , Sadovsky Yoel , Morelli Adrian E
Published: August 2023

Pregnancy constitutes an immunological paradox, where despite the competent maternal immune system, the semi-allogeneic fetus evades rejection. The mechanisms at the maternal-fetal interface and mother’s secondary lymphoid tissues (SLTs) that dodge the immunologic attack to the fetus, remain largely unknown. Passage of extracellular vesicles (EVs) is a mechanism of cell-to-cell communication that transfer antigens (Ags), regulatory mediators, and RNAs. Thus, we tested whether, similar to transplantation, feto-placental EVs could transport fetoplacental Ags via blood to maternal SLT immune cells, in this case in a tolerogenic fashion

Results

In female BALB/c mice impregnated with mOVA C57Bl/6 (B6) males, the surrogate paternal Ag OVA accumulated on follicular dendritic cells (FDCs) in maternal SLTs (E17.5). In a mouse model in which EVs released by the fetoplacental unit are labelled with mNeonGreen linked to EV-CD81, mNeonGreen-CD81 was detected by microscopy in punctate areas on FDCs, conventional DCs, B cells, and macrophages in the mother’s spleen (E17.5). IP followed by western blot of EVs from cultures of trophoblast cells from BALB/c females impregnated with OVA B6 males, revealed OVA on the EVs. Administration of these EVs to non-pregnant females induced defective activation and proliferation of OVA-specific T cells in SLTs. In a human translational mouse model, iv injection of CM-DiI EVs from supernatant of primary human trophoblast cells were captured by B cells, macrophages, and cDCs of human origin in the spleen

Conclusion

In the models tested, systemically released feto-placental EVs carry paternal/trophoblast Ags that reach the maternal SLTs, where the Ags are recognized by maternal T cells. NIH R01 AI148690.
The unexpected effect of the protease CPAF from Chlamydia trachomatis is to increase the global synthesis of the host proteins
Authors: Alvarez Carlos , Mora Valentina
Published: August 2023

MHC-I molecules are involved in the predisposition to several immune diseases. Reactive arthritis (ReA) is strongly linked with Chlamydia trachomatis infection and HLA-B*27. However, how the MHC-I can contribute to the pathology is not well understood.

Chlamydia is an intracellular pathogen and one of its main pathogenic factors is CPAF. This enzyme is secreted to the cytosol of the host during infection and there are no data regarding its global effect on the protein degradation and how could affect the ligands presented by MHC-I molecules.

T-REx-293 cells stably transfected with CPAF were induced with doxycycline and its effects on several known substrates were assayed by Western blot. Moreover, its unknown effects on the global proteome were evaluated by label-free quantitation (LFQ) using a Q-Exactive plus mass spectrometer. Additionally, MHC-I complexes were purified by immunoprecipitation and the eluted peptides were analyzed by MS.

CPAF is highly active degrading known substrates such as Vimentin and RFX5, but also showed an unexpected and profound effect on the cellular proteome increasing the expression of many proteins with a wide range of functions, mostly related to transcription and translation pathways.

More than 3000 MHC-I-restricted peptides were identified showing that the peptidome is altered when CPAF is induced, increasing the diversity and altering the physicochemical properties of the peptides.

As conclusion, CPAF deeply alters the host proteome contributing to alter the peptide supply of the antigen processing pathway which affect the composition of the MHC-I-associated immunopeptidome. These data suggest that the generation of “new ligands” could be a new pathogenic mechanism of C. trachomatis in ReA.

Supported by grants Fondecyt 11170969 and Fondequip EQM190142 from ANID, Chile.
Genetic immunization with self-assembling SARS-CoV-2 Spike-HBsAg nanoparticles
Authors: Merriam Jonah S , Liu Catherine , Wang Lingshu , Shi Wei , Yang Eun-Sung , Zhang Yi , Kong Wing-Pui , Tsybovski Yaroslav , Stephens Tyler , Verardi Raffaello , Leung Kwanyee , Stein Cody , Chen Man , Olia Adam , Harris Darcy , Pegu Amarendra , Graham Barney , Kwong Peter , Mascola John , Koup Richard
Published: August 2023

As the COVID-19 pandemic continues to pose a great challenge to global health, more effective and durable vaccines are needed owing to the waning protection offered by the current available vaccines. Here we report highly immunogenic self-assembling hepatitis B surface antigen (HBsAg) nanoparticles displaying the ectodomain of SARS-CoV-2 WA1 S6P, the prefusion spike stabilized by six proline substitutions. Three self-assembling nanoparticle constructs with different linkers named CL1, CL2 and CL3 expressed well-formed nanoparticles in mammalian cells. These purified nanoparticles can bind diverse monoclonal antibodies (mAbs) specific to different domains of SARS-CoV-2 spike. Immunization by DNA electroporation of CL2 and CL3 in mice elicited significantly more potent and durable neutralizing antibody responses against diverse SARS-CoV-2 strains than non-nanoparticle forms of stabilized spikes, including SARS-CoV-2 S2P and S6P ectodomain and full-length S2P with its coding sequence matching mRNA-1273. Our data showed that genetic delivery of HBsAg based nanoparticle vaccines displaying SARS-CoV-2 spike can elicit potent and durable neutralizing antibody responses and highlight their potential as next generation genetic vaccine candidates against SARS-CoV-2.

NIH Intramural Funding
Controlled release of inactivated influenza virus and α-galactosylceramide from PLGA microparticles results in long-term protection against influenza infection
Authors: Wen Yuhan , Sparks Zachary , Chauhan Anuj , Nelson Corwin , Driver John
Published: August 2023

The objective of this study was to develop biodegradable microparticles with continuous release that can enhance and extend the long-term protection of single-dose vaccines. Several formulations of Poly (lactic-co-glycolic acid) (PLGA) microparticles were synthesized using a double emulsion solvent evaporation technique to encapsulate inactivated A/PR/8/34 (PR8) H1N1 influenza virus in combination with the invariant natural killer T (NKT) cell agonist α-galactosylceramide (α-GalCer). The most efficient controlled release particle formulations were injected into C57BL/6J mice. Additional control mice were injected with PBS alone, inactivated PR8 dissolved in PBS, or inactivated PR8 and α-GalCer dissolved in PBS. Thirty weeks after vaccination, mice were challenged with a lethal dose of live homologous virus. Mice immunized with the microparticle vaccine survived the infection and had reduced clinical disease and decreased weight loss compared to unvaccinated mice. After vaccination, mice injected microparticles presented a gradual increase production of PR8-specific IgGs that was dominated by IgG1 isotype that is usually produced by T helper (Th) 2 responses. In contrast, mice that received soluble vaccine presented a rapid antibody response that was dominated by antibodies of the Th1-associated IgG2a isotype. Collectively, these results demonstrate the potential of PLGA-based single-dose microparticle vaccines for providing long-term protection against influenza infection and potentially other pathogens.

Supported by grants from USAMRAA (W81XWH-19-1-0005)
The slow-release platform, VacSIM, shapes host immune response against influenza infection after vaccination
Authors: McCormick Anna L , Mousa Jarrod J , Harn Donald A
Published: August 2023

Influenza virus is a leading cause of respiratory infections known to cause global morbidity and mortality, despite the widespread use of multiple influenza vaccines. Current influenza vaccines target the hemagglutinin (HA) envelope protein responsible for the attachment and entry of influenza into host cells. However, high mutation rates during influenza replication lead to mutations that cause the HA protein to be unrecognizable by host antibodies, increasing levels of infection within both vaccinated and unvaccinated individuals. A promising approach to circumvent this problem is through the use of a slow release delivery platform which has been previously shown to induce the longevity of germinal center reactions, allowing for the generation of higher affinity antibodies and more evolved B cells. Here we show that vaccination with the slow-delivery platform, Vaccine Self-Assembling Immune Matrix (VacSIM), in combination with recombinant HA antigens, increases antibody titers against H1 and H3 HAs in vaccinated groups of mice. When mice immunized with VacSIM/H1 HA were challenged with a lethal H1N1 dose, a 25% increase in survival was seen compared to the non-VacSIM group and 100% increase against mock immunized. Similarly, when immunized with VacSIM/H3 HA and challenged with H3N2, a 1.8 log reduction in lung viral titers was seen. Further analysis of the vaccine-elicited antibodies showed that vaccination with VacSIM and HA increases HAI activity against multiple strains of influenza within both H1N1 and H3N2 subtypes. We anticipate that use of VacSIM will provide further insight into how consistent exposure through a slow delivery platform can shape the host immune response to increase protection against influenza infection.

Supported by grants from CIVIC
ISCOMs/MPLA adjuvanted SDAD protein nanoparticles induce improved immune responses and cross-protection in mice
Authors: Zhu Wandi , Park Jaeyoung , Dong Chunhong , Wei Lai , Pho Thomas , Champion Julie , Wang Baozhong
Published: August 2023

The development of a universal influenza vaccine to protect against influenza variants will be the priority goal in combating influenza virus infection. In this study, we exploited a novel type of protein nanoparticles consisting of influenza NP as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by using an SDAD heterobifunctional crosslinker. Intramuscular immunization of the NP/NA-M2e (core/shell) nanoparticles induced increased humoral and cellular immune responses. ISCOMs/MPLA as adjuvants further improved the nanoparticle immunogenicity and elicited stronger immune reactions conferring protection against different influenza viral challenges. The nanoparticles were delivered through intranasal route to determine the application as a mucosal vaccine. While the nanoparticles alone barely induced immune response after intranasal immunization, ISCOMs/MPLA adjuvanted nanoparticles induced strengthened antigens-specific antibodies responses, cellular responses and higher levels of mucosal IgA. Meanwhile, increased lung Trm, Brm, and alveolar macrophages, were observed from ISCOMs/MPLA adjuvanted nanoparticle group. Only the mice intranasally vaccinated with ISCOMs/MPLA adjuvanted nanoparticles fully survived during the infections, and better protection efficacy was observed when compared with other adjuvants. Our results emphasized the importance of supplementing of appropriate adjuvants to improve the immunogenicity and mucosal immune responses of vaccines in mucosal immunization. In conclusion, the ISCOMs/MPLA adjuvant combination could significantly improve the immune responses and protective efficiency of protein nanoparticles in different immunization routes.
Pandemic preparedness: Addition of TLR agonists CpG+MPLA to emulsified H5 confers protection against H5N1 challenge after a single dose
Authors: Davies Jenny E , Hwang Michael , Dollinger Emmanuel , Felgner Jiin , Jain Aarti , Nie Qing , Othy Shivashankar , Pone Egest , Davies D Huw
Published: August 2023

H5N1 vaccines adjuvanted in the oil-in-water emulsion MF59 are maintained in the Strategic National Stockpile for pre-pandemic preparedness. The US population are currently naïve to H5 and a prime/boost regimen will likely be needed to engender a protective response. Ideally, a rapid response to pandemic influenza would benefit from being able to induce protection from a single dose of vaccine. Here, we have used a systems immunology approach in mice to examine the effect of formulating recombinant H5 in AddaVax alone or AddaVax with CpG+MPLA. AddaVax emulsion alone does not stimulate the innate immune system, although the oil droplets do act as a nanocarrier for antigen and enhance B cell activation, presumably by BCR cross-linking. In vitro, germinal center reactions show inclusion of CpG+MPLA to the emulsion (a combination adjuvant we call IVAX-1) synergize to enhance B cell maturation and class-switching. Similarly, in vivo, the combination of H5/IVAX-1 enhances both H5-specific B and T cell responses compared to individual components. Single cell mRNA sequencing of spleen cells after a boost indicate responses to H5/CpG+MPLA and H5/IVAX-1 are more similar to each other but distinct from H5/AddaVax or H5/PBS. Unexpectedly, H5/AddaVax, H5/CpG+MPLA and H5/IVAX-1 all engendered 100% efficacy against lethal intranasal challenge of H5N1 virus, although only H5/IVAX-1 engendered 100% protection after a single dose. Our data indicate the potential for certain combination adjuvants to enhance the immunogenicity of influenza vaccines and achieve efficacy from single dose vaccination. Supported by AI160397.

NIAID Grant AI160397
Single cell and genomic profiling of PBMC-expanded NK cells in first-in-dog clinical trial of allogeneic adoptive NK cell therapy for dogs with cancer
Authors: Razmara Aryana , Farley Lauren , Harris Rayna , Judge Sean , Lammers Marshall , Dunai Cordelia , Murphy William J , Rebhun Roberty , Brown Titus , Kent Michael , Canter Robert
Published: August 2023

Natural killer (NK) cells are innate cytotoxic cells with a crucial role in anti-tumor responses, making them a promising candidate for adoptive immunotherapy (IT). CD5 depletion was previously used to isolate CD5dim-expressing NK cells as a source of expanded NK cells for IT, but this technique can limit the yield of the final NK product. Here, we used bulk and single cell (SC) sequencing to compare ex vivo culture conditions using standard CD5 depletion versus unmanipulated PBMCs as the source of expanded NK cells comparing transcriptomic profiles from 5 matched healthy beagle donors using both techniques and subsequently from 5 dogs with naturally occurring malignant melanoma who received allogeneic PBMC-expanded NK cells in a first-in-dog clinical trial. Notably, beagle-derived PBMC-expanded cells had upregulation of NK pathways related to activation and function compared to CD5 depleted cells, suggesting bulk PBMCs may yield a superior product. Clinically, there were no serious adverse events using PBMC-expanded allogeneic NK cells, and one dog survived 445 days post-treatment, suggesting possible clinical benefit. SC analysis of PBMCs from this dog before, during, and after treatment showed a robust NK cell population across timepoints with high expression of cytotoxicity-related genes, such as GZMB and NCR3, lasting 14 days post-NK transfer. SC data also suggested a previously uncharacterized concordance of NK and CD8 T cell gene expression profiles in canine peripheral blood. Overall, allogeneic NK transfer using PBMC-expanded NK cells appears promising in dogs with cancer, and pre-clinical and clinical data support the use of SC analysis as a valuable technique for biomarker identification and optimizing NK IT in dogs.

This study was funded by the National Institutes of Health/National Cancer Institute grant U01 CA224166-01, the 3U01CA224166-02S1 and 5R03CA252793-02 grants, and the V Foundation Victory over Cancer through the Canter Laboratory. Student support was provided by the AVMA/AVMF 2nd Opportunity Summer Research Scholarship.
A Novel Tissue Explant Culture Model For Testing of Candidate Treatments for Inflammation of the Uterus in Pigs
Authors: Giles Joanna L , Walton Robert , Wonfor Ruth , Nash Debbie
Published: August 2023

Background

Endometritis is an inflammatory condition of the uterus leading to complications with reproductive organs, miscarriage and infertility.

The first-line treatment of endometritis in pigs are antibiotics, which do not always resolve inflammation and contribute to antibiotic resistance. There is an urgent need for the development of alternative treatments for endometritis. Here we propose and validate the first porcine tissue explant model for uterine inflammation and test naturally derived compounds for their effectiveness as anti-inflammatory therapies in porcine endometrial tissue.

Methods

Uterine endometrial explants were harvested from uteri of sows sent to slaughter. Tissue biopsies were collected at follicular and luteal phases of the oestrous cycle. Biopsies were washed and these tissue ‘explants’ were cultured. Explants were treated at 24 hr with oxytocin (to stimulate PGF2α production) or LPS (to mimic infection and induce inflammation. Explants were retained for RT-PCR analysis. Supernatants were harvested and concentrations of markers were measured by ELISA and Luminex.

Results

The data generated thus far show a dose dependent response of IL-6 secretion to LPS stimulation, which demonstrates the validity of our model. Inflammation can be stimulated in an in vitro setting to mimic porcine endometritis and we present data to show the change in cytokine levels in response to treatment with naturally derived compounds.

Conclusions

This data shows that inflammation can be stimulated in an in vitro setting to mimic porcine endometritis This model is a tool for testing of novel compounds and their potential anti-inflammatory effects on the porcine endometrium.

Supported by an Innovate UK Grant (Project Reference: 98015) (https://gtr.ukri.org/projects?ref=98015)
Benefits of Being Big: How a T cell response can occur as rapidly in a human as a mouse
Authors: Cannon Judy L , Moses Melanie E , Ferdous Jannatul , Fricke Matthew
Published: August 2023

The adaptive immune response must be fast in order to protect against exponentially growing novel pathogens. A remarkable property of the immune system is that despite a human which is approximately 2500 times larger than a mouse, the human immune response is initiated as fast as that of mice. This is in contrast to most biological processes slowing systematically with body mass. We have now collected data comparing the sizes of immune organs including spleen and lymph nodes across a large number of species including mice, rat, rabbit, macques, human, pigs, camels, cattle, and elephants. We mathematically demonstrate that while spleens scale similarly to other organs such as the hearts and kidneys, the size and number of lymph nodes scale differently than typical organ size. Using a novel computational modeling approach to predict the time that the first T cell is likely to encounter a dendritic cell presenting cognate antigen after infection, we show that the size and number of lymph nodes determine the timing of the adaptive immune response and enable even very large animals to respond quickly to pathogen infection. Combined with empirical scaling of lymph node volume, this suggests that the initiation of T cell adaptive immunity in lymph nodes occurs faster in larger animals like humans, compared to small laboratory animals which are the focus of many immunological and vaccine studies.

UNM Center for Advanced Research Computing, supported in part by NSF, for high performance computing resources, as well as the James S. McDonnell Foundation and NSF awards 2030037 and 2020247 for funding.
Computational prediction of highly conserved CD8+ and CD4+ T cell epitopes in bluetongue virus: A promising data for developing broad-spectrum bluetongue vaccine
Authors: Umeshappa Channakeshava Sokke , Kolla Harish Babu , Singh Karam Pal , Karakach Tobias , Nanjundappa Roopa Hebbandi , Mertens Peter Paul Clement
Published: August 2023

Bluetongue (BT) is an economically important arboviral disease of sheep, cattle, goats, and wild ruminants, particularly in America and Europe. However, it has remained uncontrolled due to the evolution of >32 serologically distinct BT virus (BTV) serotypes and the lack of broad-spectrum vaccines. While outer VP2 and VP5 proteins, involved in cell penetration, are less conserved, certain core BTV proteins NS1, NS2, NS3, and VP7 have higher amino acid conservancy. Here, using in silico epitope mapping and multiple sequence alignment, we analyzed all the antigenic BTV proteins for the presence of conserved T cell epitopes recognized by different mammals. We find that mouse Major Histocompatibility Complex-I (MHC-I) conserved epitopes are present in NS1, NS3, and VP7, and MHC-II-conserved epitopes in NS1, NS2, NS3, and VP7; and in bovines, Bovine Leukocyte antigen class-I and II-(BoLA-I & II)-conserved epitopes present in NS1, NS2, NS3, and VP7 proteins. The presence of these conserved epitopes from NS1, NS2, NS3, and VP7 proteins of BTV in the selected mammals, including bovine that is closely related to sheep, we believe they are also likely to be presented by MHCs of the primary natural host sheep. Thus, harnessing this knowledge in developing a pan-BTV vaccine would confer broad-spectrum protection against all the BTV serotypes in sheep and bovines.

We are grateful for start-up funding by the Department of Microbiology and Immunology, Faculty of Medicine, Dalhousie University. PPCM wishes to acknowledge support from EU H20:20 grant ‘PALE-Blu’ (project number 727393-2).
Effect of estrogen signaling in CD8 T cell mediated antiviral response
Authors: Mithila Farha J , Hasan Mohammad , Pacia Jessica , Beura Lalit
Published: August 2023

Estrogen, the major female sex hormone has been linked with immune response disparity between males and females. However, the exact mechanism by which estrogen impacts our ability to fight viral infection and development of immunologic memory remains unknown. CD8 T cells represent the primary protective mechanism against viruses and cancer. Here we aim to understand how CD8 T cell mediated antiviral immune response is regulated by estrogen. Systemic LCMV infection in ovariectomized female mice generated lower virus-specific memory CD8 T cells compared to sham surgery controls. To check if this reduction in memory response is CD8 T cell intrinsic, I generated a transgenic virus-specific estrogen receptor-α deficient CD8 mouse line (ESR1 KO). Using a co-adoptive transfer model, I compared the differentiation of ESR1 KO and control CD8 T cells in both systemic LCMV and local influenza infection. The lack of estrogen signaling resulted in reduced abundance of effector CD8 T cells in the context of both infections. ESR1 KO CD8 T cells also displayed reduced cytotoxic ability. In vitro studies revealed that the CD8 T cell expansion was primarily impacted by the lack of estrogen signaling, whereas activation and cellular apoptosis remain unchanged. Altogether, this suggests a crucial role of sex steroid estrogen in initial effector CD8 T cell differentiation, that has significant ramification for generation of protective immune responses in pathological or physiological estrogen deficiency.

Supported by grants from Searle Scholars Foundation, NIH 2P20GM109035, NIH P20GM121298, NIH T32HL134625, NIH T32GM136566
ASXL1 disruption preserves CD8 T cell exhaustion-progenitor epigenetic programming during chronic stimulation
Authors: Kang Tae Gun , Lan Xin , Alli Shanta , O’Connell Casey L , Gronbaek Kirsten , Jones Peter , Han Jin-Hwan , Spatz Marianne L , Youngblood Ben A , Zebley Caitlin C
Published: August 2023

Differentiation of CD8 T cells into a terminally exhausted state is a major hurdle limiting efficacy of both immune checkpoint blockade and adoptive cellular immunotherapy approaches. Prior studies describing disruption of the epigenetic regulators DNMT3A and TET2 in CD8 T cells have revealed a critical role of DNA methylation in repressing the proliferative capacity and effector potential of CD8 T cells during exhaustion. Disruption of these genes has also been noted to preserve the proliferative capacity of hematopoietic stem cells (HSCs), resulting in clonal hematopoiesis (CH) and establishing the broader concept that these epigenetic regulators control cellular developmental potential. In addition to DNMT3A and TET2, the histone modifier ASXL1 is a critical regulator controlling CH. Here we report that disruption of ASXL1 in CD8 T cells preserved a stem-like phenotype denoted by increased TCF1 expression, IFN-γ production during chronic infection. At the chronic stage of infection, ASXL1 KO CD8 T cells retained a heightened ability to undergo a proliferative burst during PD-L1 blockade. Genome-wide epigenetic profiling demonstrated that disruption of ASXL1 resulted in inhibition of DNA methylation accompanied by changes in chromatin accessibility. These data document crosstalk between histone modifications and DNA methylation in regulating the transition of stem-like progenitor T cells into terminally exhausted T cells. Collectively, these data demonstrate that ASXL1 is a novel epigenetic regulator controlling development of exhausted CD8 T cells and represents a novel immunotherapeutic target for improving responses to T cell-based cancer immunotherapy.
Pannexin-1 channels promotes CD8+ T cell effector and memory responses through distinct metabolic pathways
Authors: Banuelos Alma , Vardam-Kaur Trupti , Gabaldon-Parish Maria , Zhou Maggie , Wanhainen Kelsey Marie , van Dijk Sarah , de Gois Macedo Bruna , Salgado Caio Loureiro , Santiago-Carvalho Igor , Peng Changwei , Jameson Stephen , Da Silva Henrique Borges
Published: August 2023

Sensing of extracellular metabolites controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin-1 (Panx1). Here, we report that T cell-specific Panx1 is needed for efficient CD8+ T cell responses to viral infection and cancer. Panx1 favors both the expansion of effector CD8+ T cells and the survival of memory CD8+ T cells. Our data suggests that, while memory CD8+ T cells require Panx1 for mitochondrial function, Panx1 promotes the glycolytic pathway in effector CD8+ T cells. Panx1 promotes memory CD8+ T cell survival together with the eATP sensor P2RX7. However, Panx1 induces effector CD8+ T cells independently of eATP. Rather, we found an unexpected link between Panx1, lactate export and the activation of effector CD8+ T cells. Therefore, Panx1 regulates effector and memory CD8+ T cells through export of distinct metabolites and by engaging different intracellular pathways.
CpG therapeutic efficacy in a murine model of metastatic Lymphangioleiomyomatosis (LAM) is mediated by plasmacytoid dendritic cell and T cell immune responses
Authors: Amosu Mayowa , McCright Jacob , Moore Kaitlyn , Maisel Katharina
Published: August 2023

Introduction

LAM is a slow growing, metastasizing neoplasm affecting women of reproductive age, marked by abnormal growth of smooth muscle-like cells leading to cystic lung destruction. LAM has hallmarks of cancer, like expression of immune checkpoint receptors. Effective cancer therapies promote robust T cell responses through activated dendritic cells (DCs). Anti-cancer strategies, like toll like receptor (TLR) activation and immune checkpoint inhibition could work as LAM therapy. In this study, we examine the effect of CpG, a TLR9 agonist, on the DC and T cell responses in murine LAM.

Methods

We used a mouse model of metastatic LAM to determine survival after biweekly intranasal CpG therapy (10μg/ 5μg) with/ without systemic α-PD-1, rapamycin, or α-CD317 therapy. We used ELISA to measure the cytokine profile and flow cytometry to quantify cell populations between CpG-treated and untreated LAM lungs.

Results

We found that CpG treatment enhances median survival from 32 to 60 days in murine LAM (p <0.0001). Efficacy of CpG treatment in LAM is facilitated by plasmacytoid DCs. pDC depletion in CpG-treated mice decreases survival from 60 to 52 days (p=0.028). CpG-treated LAM lungs also produce more IFN-α (p=0.031). CpG-treated LAM lungs have increased IFN-γ (p= 0.012) and IL-12 (p= 0.005), cytokines secreted by cytotoxic T cells.

CpG-treatment is synergistic with α-PD-1 checkpoint inhibition (p=0.004) and can be administered with rapamycin without adverse effects on median survival.

Conclusions

In LAM, CpG therapy is mediated by pDC dependent immune responses and cytotoxic T cells. This suggests that adjuvant immunotherapy, like CpG, may offer new treatment options for LAM compatible with the current standard of care, rapamycin.

The LAM Foundation Research Grant Award
Deciphering the crosstalk between skin commensals and the immune system
Authors: Bouladoux Nicolas , Chen Y Erin , Fischbach Michael A , Belkaid Yasmine
Published: August 2023

The mechanisms underlying the crosstalk between the microbiota and the immune system remains largely unknown. We previously established that skin colonization with Staphylococcus epidermidis results in the induction of T cell responses that control tissue function in a way that both enhances cutaneous immunity and promotes tissue repair. To uncover the molecular determinants of S. epidermidis that stimulate T cells, we engineered a library of S. epidermidis mutants with defined cell envelope alterations. Under physiological cutaneous colonization, we found that S. epidermidis cell envelope mutants elicit marked changes in the composition and function of the T cell populations in the skin. The removal of cell envelope lipoproteins and D-alanylation of teichoic acids almost completely abolished CD8+ T cell induction without significant effect on CD4+ T cell stimulation. Deletion of Tlr2 gene also strongly reduced the CD8+ T cell response after S. epidermidis colonization, while the CD4+ T cell response remained unchanged, a phenotype similar to the elimination of cell envelope lipoproteins and D-alanylation of teichoic acid in S. epidermidis. Furthermore, suprabasal keratinocytes express the highest level of TLR2 among keratinocyte subsets. Together, these results support the notion that the ability of S. epidermidis to induce CD8+ T cells is partially dependent on the host sensing of S. epidermidis wall teichoic acids and lipoprotein through TLR2. Using complementary strategies including novel mice models and imaging, current studies are focusing on deciphering on which cell population(s) TLR2 sensing of S. epidermidis molecular determinants is required for the induction of CD8+ T cell responses.
The differential influence of commensal and pathogenic flagella on intestinal barrier function
Authors: Patry Robert T , Kemter Andrea , Mimee Mark , Arnold Jack , Hesser Lauren A , Ionescu Edward , Nagler Cathryn
Published: August 2023

It is well known that accumulated changes in microbiota composition over several decades have contributed to an increase in non-communicable chronic diseases. While much effort has been devoted toward achieving a consensus on the microbes, and their products, driving this increase, inconsistent results suggest more research is required to understand the disparity in observed outcomes. Previous work from our lab identified a consortium of commensal Clostridia that alone was sufficient to preserve intestinal barrier integrity and prevent sensitization to food allergens in mice by inducing IL-22. Further investigation determined that members of this consortium display flagella. When isolated, these commensal flagella (Fla-C) exhibited TLR5-dependent IL-22 induction at a magnitude comparable to flagellin from SalmonellaTyphimurium (Fla-ST). However, mice treated with Fla-C maintained intestinal barrier function significantly more effectively than Fla-ST or negative control PBS-treated mice as measured by a serum FITC-dextran permeability assay. This was accompanied by a striking difference in the expression of Reg3 antimicrobial peptides and IL-17; all of which were significantly increased by treatment with Fla-ST when compared to Fla-C. Sequencing of Clostridia consortium-derived flagellated isolates revealed flagellin genes with unique hypervariable regions when compared to fliC from S. Typhimurium. These results highlight the distinct impacts of commensal and pathogenic flagella and suggest an important role for flagellated commensal bacteria in the maintenance of intestinal epithelial barrier function.

Supported by a grant from National Institutes of Health (RO1 AI06302)
Microbial regulation of anti-tumor immunity in colorectal cancer
Authors: Chung Charlie , Habel Jill , Shen Zeli , Rubino Rachel , Westcott Peter , Fox James G , Beyaz Semir
Published: August 2023

Colorectal cancer (CRC) is one of the most common and deadly types of cancer. Although recent advances in early detection and revolutionary therapies such as checkpoint blockade immunotherapy resulted in improved outcomes in CRC, a large fraction of CRC patients, particularly with metastasis, do not respond well to existing immunotherapy modalities. This necessitates a better mechanistic understanding on tumor – immune cell interactions in the context of CRC. Microbiome influences multiple biological aspects of the host including immune responses against cancers. We recently defined a causal role for microbiome in regulating epithelial stem cell MHC-II expression and tumor initiation in the intestine. Here we expound on the significance of microbiome - MHC-II axis in CRC progression and metastasis. We found that colonization of microbes that correlate epithelial MHC-II expression in the intestine restricts CRC progression and improves overall survival in three different clinically-relevant orthotopic CRC models across mice strains. Notably, microbiome-induced anti-tumor effects are dependent on T cells. Finally, genetic inactivation of MHC-II in cancer cells diminished tumor preventing effects of microbes. These results underscore a causal role for microbe - MHC-II axis in regulating anti-tumor immunity during tumor progression. Microbes that boost MHC-II expression promote anti-tumor immunity in the colon, which may synergize with therapeutic approaches such as immunotherapy.

NIH (P30CA045508-33 to S.B.), the Oliver S. and Jennie R. Donaldson Charitable Trust (S.B.), The Harold and Leila Y. Mathers Charitable Foundation (S.B), The Mark Foundation for Cancer Research (20-028-EDV to S.B.) and STARR Cancer Consortium (I13-0052 to S.B.), Chan Zuckerberg Initiative / Silicon Valley Community Foundation (2021-239862)
Roles of the dynamic gut microbiota and their associated immunoregulatory effects for successful oral immunotherapy treatment
Authors: Goldberg Michael R , Appel Michael Y , Mor Hadar , Neriya Dafna Magid , Mazgal Faiga , Muller Efrat , Nachshon Liat , Borenstein Elchanan , Tamir Snait , Youngster Ilan , Louzon Yoram , Koren Omry , Elizur Arnon
Published: August 2023

Background

We previously demonstrated that patients with IgE-mediated food allergy (FA) have a unique gut microbial signature.

Objectives

To characterize the changes to the gut microbiome populations and putative immunoregulatory effects, associated with successful OIT.

Methods

Stool samples were collected at inception of treatment from 252 patients undergoing OIT, and from 58 non-allergic controls. Additional samples were collected at successful completion of treatment (n=66). Bacterial 16S rRNA gene sequences from extracted sample DNA were amplified by PCR, with microbial communities analyzed by QIIME2. Stool extracts were evaluated for allergen-specific IgA2, and for their induction capability of immunoregulatory genes in a colonic epithelial line (Caco-2).

Results

For all FA types, patients with higher initial allergen eliciting doses (ED) had microbiota populations significantly closer to that of controls, compared to more sensitive patients with lower ED (p<0.0001). The relative abundance of Prevotella and Bacteroides/Firmicutes ratio significantly varied during OIT progression, increasing towards controls. Post-OIT stool eluents induced mRNA expression of APRIL and ZO-1 in Caco-2 cells and allergen-specific IgA2 was significantly increased in post- vs pre-OIT eluents (p<0.01). In Transwell assays, heat killed Bacteroides fragilis decreased FITC-dextran4 leakage across the epithelial layer.

Conclusions

Gut microbiome populations are dynamic during OIT, possibly reflecting a recovery from a FA to a nonallergic state. Immunoregulatory changes and positive effects on epithelial cell integrity suggest causative roles for the gut microbiome in maintaining the desensitized state.

Supported by a grant from the Israeli Science Foundation (1527/21)
A vanishing family of commensal bacteria and its impact on host health and disease
Authors: Pepin Deanna M , Popple Sarah , Ghezzi Hans , Seo Jung Hee , Osborne Lisa , Tropini Carolina
Published: August 2023

The gut microbiome plays a critical role in intestinal structure, immune homeostasis, and inflammatory diseases. The rise of these diseases in industrialized countries has been correlated with the loss of microbial diversity in the gut. One bacterial family, Muribaculaceae (Mb), is becoming a lost member in industrialized human microbiomes and many studies have correlated low Mb abundance with different disease states. Our goal is to determine how Mb depletion may be connected to disease, if introducing Mb back can ameliorate the impact of disease, and uncover immune mechanisms necessary for colonization of Mb. To study this, we developed two mouse models with complex microbiomes; one containing Mb (Mb positive), and one without Mb (Mb naïve). We evaluated intestinal structure with confocal microscopy, measured the production of short-chain fatty acids (SCFAs), and compared these mice in a Citrobacter rodentium (C. rodentium) infection model. Mice without Mb lost colonic mucus striation and had a significant drop in the production of SCFAs. Additionally, Mb positive mice had reduced C. rodentium colonization compared to mice lacking Mb. Lastly, we introduced 8 characterized Mb isolates to the Mb naïve mice to quantify immune responses by flow cytometry. We found that Mb is capable of establishing in Mb naïve mice, resulting in robust germinal centre B cell responses, yet no subsequent plasmablast accumulation in the mLN or lamina propria. Determining the mechanisms involved in disease amelioration by Mb and its impact on host immune responses is a novel research direction that could increase our understanding of colonization dynamics and highlights the potential of this bacteria as a model for microbiota therapies.

Supported by grants from The W. Garfield Weston Foundation
B cells regulate luminal bile acid composition in the small intestine
Authors: Mohammed Ahmed Dawood , Ball Ryan A , Nagarkatti Mitzi , Nagarkatti Prakash S , Kubinak Jason
Published: August 2023

Recent studies have demonstrated that bile acids (BA) influence immune cell homeostasis in the gut. However, whether and how, the host immune response can influence BA biochemistry is unknown. This is an important question to answer because small shifts in BA composition in the gut can have significant pathophysiological consequences. Previously, we have demonstrated that CD19−/− mice, which have a defect in B cell activation and consequently an aberrant humoral immune response, develop a small intestine (SI) enteropathy that is associated with abnormal luminal BA composition. However, CD19−/− mice are not B-cell-deficient and present with multiple aberrant immune phenotypes. Here, we used B cell-deficient JHminus;/minus; mice to more effectively define this interaction. Results of our experiments provide three key observations. First, JH−/− mice develop altered SI BA pools compared to their WT littermates. Second, adoptive transfer of WT B cells into neonatal JH−/− mice led to the development of normal BA pools and rescued mice from SI enteropathy. Third, adoptive transfer of B cells incapable of secreting IgA into neonatal JH−/− mice failed to restore normal bile acid composition and only moderately protected animals from developing SI enteropathy. Collectively, the results of our experiments clearly demonstrate that B cells regulate bile acid composition in the SI and that the synthesis of mucosal IgA may be particularly important for driving this effect.
Oral mucosa resident CD8+ T cell reactivation exacerbates murine periodontitis
Authors: Saavedra de Paula Flávia M , Herzberg Mark C , Masopust David , Stolley J Michael
Published: August 2023

Periodontitis is the world’s most common chronic inflammatory condition. Progressive forms of periodontitis are initiated by dysbiotic subgingival biofilms yet sustained by unresolved local inflammation. While gingival CD4+ T cells are thought to be major drivers of periodontal inflammation, the role of tissue resident memory (TRM) CD8+ T cells in periodontitis progression and severity remains unknown. We sought to determine whether local reactivation of oral CD8+ TRM of a defined antigen specificity would accelerate ligature-induced periodontitis (LIP), a well-established murine model of experimental periodontitis. Ovalbumin (OVA)-specific OT-I T cells were transferred and expanded though systemic viral infection in naïve hosts. Six-days later, OT-I T cells were ‘pulled’ into the oral mucosa via swabbing of the oral surfaces with OVA peptide in irritants. This approach, collectively referred to as Viral-Prime: Epitope-Pull (VPEP), durably augments oral OT-I TRM in the oral mucosa to manipulate and study. Utilizing ‘VPEP’ memory mice, oral CD8+ TRM were reactivated with OVA peptide concurrent with LIP. Control mice received oral swabbing with an irrelevant peptide. Alveolar bone loss and interstitial immune infiltrates were characterized using microCT imaging and flow cytometry, respectively. We found that oral TRM reactivation accelerated alveolar bone loss and enriched neutrophils, macrophages and CD8+ T cells within ligature-proximal gingiva. Bulk RNAseq analysis revealed several inflammatory and matrix-remodeling genes induced in maxillary and mandibular gingiva upon oral TRM reactivation. Collectively, we provide evidence that oral CD8+ TRM reactivation can exacerbate periodontitis.

This work is supported by NIH/NIDCR grants R90 DE023058 to F.M.S. and T90 DE 022732 & K99DE031014 to J.M.S