bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinguished role of MerTk and Axl receptors-mediated efferocytosis in alveolar macrophages on pulmonary mucosal regulation
Authors: Pinto Kamila Guimaraes , Ferreira Antonia Correa , Leandro Monique , Rodrigues Leticia , da Silva Rigoni Thaís , Palermo Ester , Ferreira Jesuino Rafael Machado , Lopes Marcela , Vazquez Estefania , He Jianping , Kelsall Brian , Filardy Alessandra D’Almeida
Published: August 2023

Efferocytosis is paramount to regulating homeostasis and inflammation. The TAM receptor family (Tyro3, Axl and MerTk) mediates efferocytosis and simultaneously inhibits pro-inflammatory pathways. Here we investigated how MerTk and Axl regulate pulmonary mucosa homeostasis and inflammation, using silicosis as a study model. During homeostasis, lung cells and alveolar macrophages (AMs) from wild-type (WT) mice showed high expression of Axl, MerTk, and Gas6. Although we have found increased numbers of total airway cells, lower levels of TGF-b and IL-10, and higher levels of nitric oxide in the BALFs from both Axl−/− and MerTk−/− compared to WT mice, enhanced numbers of lung total cells, higher numbers of AMs, monocytes, and neutrophils were found in BALFs and lungs of MerTk−/− compared to Axl−/− or WT mice. We also found increased expression of CXCL1, CXCL2, TNF-a, and IL-6, and enhanced numbers of MHCII+AMs in the lungs of MerTk−/− compared to Axl−/− or WT mice. Furthermore, AMs from Axl−/− mice upregulated the expression of MerTk receptor. During silicosis, we found decreased pulmonary function, increased levels of TGF-b and CXCL1, as well as increased number of AMs and neutrophils in silicotic (SIL)-Axl mice compared to the SIL-MerTk or SIL-WT groups. In addition, SIL-Axl showed a reduced number of CD206+ AMs and an enhanced number of apoptotic, late apoptotic, and necrotic AMs compared to SIL-MerTk or SIL-WT groups. Finally, we found that both SIL-WT and SIL-MerTk upregulated mRNA levels for Axl compared to its PBS groups. Collectively, our data suggest that MerTk and Axl receptor-mediated efferocytosis are dedicated to regulating homeostasis and controlling inflammation in the lungs, respectively.

CNPq, CAPES and FAPERJ
Ionizing radiation induces lasting changes to the tissue resident memory T cell compartment
Authors: Hassert Mariah , Heidarian Mohammad , Badovinac Vladimir P , Harty John T
Published: August 2023

Exposure to sublethal ionizing radiation can occur through various mechanisms, including purposeful medical exposure, nuclear accidents, and acts of nuclear terrorism, making the development of effective medical countermeasures a high public health priority. The induction of DNA damage through reactive oxygen species makes rapidly dividing cells, such as naïve T cells highly sensitive to radiation induced depletion. A previous study demonstrated that broadly, circulating memory T cells are more resistant to numerical loss following radiation exposure compared to naïve T cells. However, the effects of radiation on the more recently described memory T cell subset, tissue resident memory T cells (TRM) remain unknown. TRM have been identified as a critical T cell subset in protection from a number of infections including influenza, respiratory syncytial virus and malaria. In particular, lung TRM are critical for strain-transcending heterosubtypic immunity to influenza virus. In a murine model of influenza infection (IAV), we have found that sublethal thorax targeted radiation resulted in the rapid numerical decline of lung TRM without major alterations in circulating IAV-specific memory CD8+ T cells. The depletion of anti-IAV lung TRM was long-lasting, and resulted in the loss of heterosubtypic immunity to IAV. Interestingly, any surviving TRM display elevated granzyme B expression, indicating functional consequences for TRM following radiation exposure, which could have implications for immune-mediated pathology. Designing effective vaccination strategies to reinvigorate this critical T cell subset will be an important step in combating the immunological effects of radiation exposure.

1F32AI174382-01
Maternal beta-glucosylceramide induces the generation of IRF4+ dendritic cells in offspring of allergic mothers
Authors: Bloodworth Jeffrey C , Hoji Aki , Blankenship Ross , Cook-Mills Joan
Published: August 2023

Background

β-glucosylceramides (βGlcCers) are elevated in allergic mouse dams and are necessary and sufficient to induce allergy predisposition in offspring. βGlcCers increase dendritic cell (DC) numbers in the fetal liver and pup lungs. We hypothesize that these lung DCs predispose offspring to allergic lung responses. We also hypothesize that βGlcCers induce elevated pro-allergy IRF4+ DC numbers during fetal hematopoiesis.

Methods

Pregnant mice were injected with a mixture of βGlcCers known to predispose offspring to allergic asthma. DCs were assayed by flow cytometry. In another approach, lung DCs from pups of non-allergic dams or allergy predisposed pups born to allergic dams were transferred to lungs of pups of non-allergic dams. Recipient pups received suboptimal sensitization and airway allergen challenge.

Results

An increase in number of IRF4+ DCs was observed in fetal livers when dams received βGlcCers compared to fetal livers from vehicle-injected dams. Higher numbers of IRF4+ DCs were in the lungs of pups born to allergic dams compared to pups born to non-allergic dams. Transfer of CD45.1 lung DCs from pups born to allergic dams to lungs of CD45.2 pups born to non-allergic dams induced allergy predisposition in the recipients compared to lung DC transfers from pups born to non-allergic dams. Furthermore, elevated numbers of endogenous CD45.2 IRF4+ lung DCs were observed in allergen-challenged pups that received lung DCs from pups born to allergic dams.

Conclusions

These results suggest that βGlcCer induces increased numbers of pro-allergy IRF4+ DCs during fetal hematopoiesis. These results further suggest that lung DCs from pups born to allergic dams are induce allergen responsiveness and higher lung IRF4+ DC numbers.

Supported by grants from NIH (R01AI127695 and U01AI131337)
Iteratively stimulated T cells as tools to understand mechanisms of nonlymphoid recirculation
Authors: Künzli Marco , Soerens Andrew G , O’flanagan Stephen D , Joag Vineet , Quarnstrom Clare F , Scott Milcah , Masopust David
Published: August 2023

Memory T cell migration facilitates whole-body immunosurveillance and protection against recurrent infections and cancer. In the absence of inflammation, tissue resident memory T cells (TRM) patrol non-lymphoid tissues (NLT) while effector and central memory T cells (TEM and TCM) are mostly restricted to the blood and the blood/lymph system respectively. Memory T cells that retain the ability to recirculate through all compartments are rare and poorly characterized. Thus, the function of these cells as well as their non-lymphoid tissue entry and egress requirements under homeostatic conditions are not well defined.

Using a heterologous prime-boost immunization scheme and adoptive transfers, we have generated an abundant murine memory T cell population that has been exposed to >50 viral infections over the last 10 years. We termed these cells iteratively stimulated T cells (ISTCs). Parabiosis experiments revealed that ISTCs acquired pan-migration properties: they can be found in blood, non-lymphoid tissues, lymph nodes, and thoracic duct lymph (TDL) of the naïve parabiont. ISTCs persist in NLTs like the lung which are usually associated with short-lived TRM populations, and mice harboring recirculating ISTCs are protected from lethal Influenza challenge. These data indicate that establishing recirculating memory T cells might provide a new strategy to generate long-lasting immunity in tissues like the lung. Single cell RNA-sequencing revealed tissue entry and egress target molecules that will be tested in in vivo CRISPR-Cas9 screens to elucidate mechanisms of T cell recirculation.

Supported by a grant from SNSF P2B-SP3_200187
Ablation of PD-1 Reduces the Stability and Lipid Metabolism of Regulatory T Cells in the Tumor Microenvironment
Authors: Kim Myeong Joon , Kim Kyungsoo , Park Hyo Jin , Kim Gil-Ran , Hong Kyeong Hee , Oh Ji Hoon , Son Jimin , Park Dong Jin , Kim Dahae , Choi Je-Min , Lee Insuk , Ha Sang-Jun
Published: August 2023

Regulatory T cells (Treg cells) are responsible for immune homeostasis and highly express PD-1 in the tumor microenvironment (TME). However, the role of PD-1 in tumor-infiltrating (TI) Treg cells remains controversial. Indeed, PD-1 expression in Treg cells was even higher than in CD8+ T cells and conventional CD4+ T cells. Here, we identified that conditional deletion of PD-1 in Treg cells delayed tumor growth by reducing TI Treg cell pool and amplifying the functionality of TI CD8+ and CD4+ T cells. In Pdcd1fl/flFoxp3eGFP-Cre-ERT2(+/−) mice, in which both PD-1WT and PD-1KO Treg cells coexisted, TI PD-1KO Treg cells exhibited the impaired proliferative and suppressive capacity compared to TI PD-1WT Treg cells. Additionally, exTreg cells, which lost their Foxp3 expression, were more abundant in PD-1KO Treg cells than PD-1WT Treg cells. In TC-1 lung cancer, PD-1 antibody therapy was effective in reducing TI Treg cell pool. Single-cell analysis identified that PD-1 signaling promoted various pathways related to lipid metabolism, proliferation, and suppression in TI Treg cells. Single-cell TCR sequencing revealed that the clonal expansion of TI Treg cells was enriched in PD-1WT Treg cells compared to PD-1KO Treg cells. We also showed that conditional deletion of PD-1 in Treg cells reduced lipid uptake and mitochondrial mass of Treg cells in TME. These results suggest that PD-1 ablation or blockade can enhance antitumor immunity by exacerbating Treg cell stability and metabolic fitness in the TME.

This study was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (2017R1A5A1014560, 2019M3A9B6065221 to S-.J.H.; 2018R1A5A2025079, 2019M3A9B6065192 to I.L.). This study was also supported by the Korean Health Technology R&D Project (HV20C0144, HN21C1410 to S-.J.H.) through the Korean Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Improving anti-tumor CD8+ T cell function through manipulating glutamine metabolism
Authors: Fisher Emilie L , Chi Channing , Suguira Ayaka , Madden Matthew , Rathmell Jeffrey C
Published: August 2023

Immunotherapies that bolster the anti-tumor effects of cytotoxic CD8 T lymphocytes (CTLs) have improved outcomes for many, yet most patients fail to achieve durable responses. Investigations into strategies to further enhance the cancer killing capacity of these cells are crucial in the effort to improve immunotherapies. T cells radically alter their metabolism upon activation, and prior studies have shown pan-inhibition of glutamine metabolism (glutaminolysis) using the inhibitor 6-Diazo-5-oxo-norleucine (DON) enhances the function of CTLs in the tumor microenvironment. However, DON’s toxicity combined with this drug’s lack of specificity demonstrate a need for a more directed glutaminolysis targeting approach. I utilized a targeted glutaminolysis CRISPR library to investigate the role of individual enzymes targeted by DON. To interrogate the importance of these enzymes specifically in anti-tumor CTLs, I developed a model in which CRISPR-library edited antigen-specific CTLs were adoptively transferred into tumor-bearing mice. After 7 days of in vivo selection, next generation sequencing was performed on isolated CTLs to evaluate the survival advantage or disadvantage of these gene knockouts. While multiple DON targets resulted in decreased CTL fitness, deletion of the gene encoding for glutamine synthetase (GS) – which converts glutamate to glutamine – conferred an advantage to CTLs. Upon inhibition of GS, cells may increase cellular levels of glutamate, and preliminary data suggest this results in a decrease in glycolysis and increase in mitochondrial respiration capacity. Lastly, in vitro data demonstrate increased inflammatory cytokine production of CTLs upon genetic knockout or pharmacologic inhibition of GS.

Supported by grants from NIH (T32 GM007347, R01 CA217987)
Modulating T Cell Metabolism to Improve CAR-T Cell Therapy success
Authors: Wu Meng-Han , McCalab Megan , Pham-Danis Catherine , Kohler Mark Eric , Rincon Mercedes
Published: August 2023

Chimeric antigen receptor (CAR)-T cell therapy is a promising treatment for lymphoblastic leukemia and lymphoma. Despite initial response to CAR-T cell therapy, about 40% of patients relapse. Modulating CAR-T cell metabolism (immunometabolism) is being considering as a novel approach to improve CAR-T cell therapy efficacy. We have identified MCJ (DnaJC15) as a novel target to enhance CAR-T cell mitochondrial metabolism. MCJ is an endogenous negative regulator of Complex I of electron transport chain. The absence of MCJ in MCJ KO CD8 cells increases Complex I activity, mitochondrial membrane potential, and mitochondrial respiration without affecting glycolysis or ROS production. Lack of MCJ also enhances ATP-dependent effector functions of CD8 cells, including cytokine secretion and cytotoxicity. Using the B16 melanoma cell model, we have shown that antigen-specific MCJ KO CD8 cells are superior in eliminating melanoma cells through enhanced effector function in vitro and in vivo. Importantly, we have generated MCJ-deficient CD8 CAR-T cells against CD19+ B cell leukemia and shown that they exhibit superior killing capacity through their increase cytotoxicity activity in vitro. MCJ KO CAR-T cells have higher mitochondrial maximal respiration and spare respiratory capacity. Furthermore, in vivo MCJ KO CAR-T cells have superior efficacy than WT CAR-T cells, and increase overall survival of the leukemia-bearing mice. Together, these data demonstrate MCJ as a novel target to enhance CAR-T cell therapy efficacy. To show the translational relevance of our studies and moving forward to the clinic, we are currently investigating whether silencing MCJ in human CD8 CAR-T cells also improve their efficacy.

R21 AI110016 R21 AI149187 R56 AI148434 R01 CA260909 R21 CA223389
Caloric restriction induces macrophage reprogramming and improved epithelial ovarian cancer survival
Authors: Singh Harshit , Udumula Mary Priyanka , Hijaz Miriana , Munkarah Adnan , Giri Shailendra , Rattan Ramandeep
Published: August 2023

Calorie restriction (CR) without malnutrition is a dietary intervention where energy intake is reduced by 15 – 40%. The benefits of CR in improving various diseases and extending lifespan has led its investigation in cancer. We have reported 30% CR to limit epithelial ovarian cancer (EOC) progression. Here, we highlight the ability of CR to induce immuno-metabolic reprogramming of tumor associated macrophages and improve EOC survival. Female C57/B6 mice were injected intraperitoneally with 8 × 106 mouse EOC ID8 (p53+/+, p53−/−, or ID8 p53−/−,BRCA1−/−) cells and fed ad libitum (RD) or subjected to 30% CR. Immune profiling was done by flow cytometry. Metabolic phenotype was determined by XF seahorse analyzer. Macrophage marker were measured by qPCR and western blot. Mice on 30% CR exhibited reduced tumor progression and increased overall survival compared to RD mice in all ID8 models as indicated by median survival; ID8p53+/+ (65 vs 95 days), ID8p53−/− (50.5 vs 76.5 days), and ID8p53−/−,BRCA1−/− (44 vs 53 days). Immune profiling showed increase in antitumor associated pro-inflammatory macrophages (F4/80+CD38+) and decrease in protumor anti-inflammatory (F4/80+CD206+) macrophages, increasing the pro / anti-inflammatory ratio. Pro-inflammatory macrophages showed increased intracellular iNOS. The phenotype change in CR mice was accompanied with reprogrammed energy metabolism indicated by increased glycolysis, reflective of pro-inflammatory macrophages. Decrease in anti-inflammatory macrophages resulted in robust anti-tumor T cell response. A 30% CR reprograms macrophage metabolism and promotes their pro-inflammatory anti-tumor phenotype, restores antitumor immunity resulting in reduced EOC growth and improved survival

R01 B11209 to RR Henry Ford Cancer institute Post Doctorate Fellowship to HS
L-fucose enhances anti-tumor immunity through modulation of monocyte-derived dendritic cell signaling
Authors: Burton Chase , Lester Daniel , Lau Eric
Published: August 2023

Although immunotherapies have emerged as standard of care for severals cancer types, their efficacy remains limited to ~30% of patients. Limited efficacy has in part been attributed to a lack of infiltrating tumor-suppressive immune cells and overabundance of immunosuppressive cell populations. To combat this problem, extensive research efforts have focused on modulating intratumoral T cells to overcome the immunosuppressive tumor microenvironment (TME). While these treatment modalities show promise in preventing intratumoral T cell exhaustion, they fail to provide robust and durable anti-tumor immune responses. For this reason, the targeting of other intratumoral immune subpopulations—particularly dendritic cells (DC)—has become a subject of increased study. The dual activation of anti-tumor and suppression of pro-tumor immune populations is expected to significantly enhance immunotherapeutic outcomes.

Recently, our group found that the dietary sugar L-fucose (L-fuc) reduces melanoma tumor growth via a CD4+ T cell-dependent mechanism. Interestingly, analysis of immune cells in the tumors of L-fuc-treated mice revealed dynamic changes in the DC compartment, predominantly in the monocyte-derived DC subset (moDC). Additionally, myeloid cells treated ± L-fucex vivo exhibited enhanced T cell stimulation, increased neutral fluorescent bait uptake, and altered cytokine production. qPCR-RT analysis revealed that L-fuc affects downstream CD209 signaling, leading to a shift towards an immunostimulatory signature. Together, these data suggest a mechanism to enhance anti-tumor immunity by increasing abundance and immunostimulatory activity of moDCs and supports the use of L-fucose as an immunotherapeutic agent.

Supported by funding from Moffitt Cancer Center Skin Cancer Sport Project
Iron-overload affects anti-tumor immune response in pancreatic cancer
Authors: Cappello Paola , Scagliotti Alessandro , Tiberi Giorgia , Autino Federica , Greco Luana , Rubbino Federica , Connor James , Laghi Luigi
Published: August 2023

HFE is the most frequently mutated gene in hereditary hemochromatosis, a disease in which iron accumulates in organs leading to toxicities. Worldwide, two main polymorphisms in HFE protein have been observed, namely C282Y and H63D. Even if associated to milder iron accumulations, H63D polymorphism increases the risk of cancer development and aggressiveness. As observed in pancreatic cancer patients, a great percentage of those that underwent to surgery also present H63D polymorphism but displayed a worse survival.

To better characterize how the iron accumulation affects anti-tumor response in the context of the pancreatic ductal adenocarcinoma (PDA), we crossed genetically engineered mice (GEM) that spontaneously develop PDA with mice engineered to express H67D mutation (HfeH67D), the orthologue of H63D in human. Histological analysis of GEM showed that H67D polymorphism accelerated PDA progression, increased the number of metastases, and altered the composition of infiltrating immune cells. Accordingly, these mouse models also displayed a reduced overall survival compared to controls. By exploiting OVA as a well-known antigen, we injected OVA-expressing PDA cells subcutaneously in mice carrying or not the H67D mutation to assess their ability to mount a normal memory response. OVA-expressing PDA cells grew faster in the presence of H67D polymorphism, but immunized mice displayed a reduction in tumor volume similar to the WT counterpart. HfeH67D mice displayed an increased production of anti-OVA IgG antibodies, especially IgE, but a decreased in OVA-specific IFNγ-secreting T cells.

Overall, H67D polymorphism impacts the activation of anti-tumoral immune response, which consequently worsens PDA progression.

Supported by grants from AIRC (ID22234; ID26341)
Adjuvant skewing of host immune response to vaccination and subsequent infection with influenza virus
Authors: Noureddine Moataz , Chang Lauren A , Warang Prajakta , Jangra Sonia , De Geest Bruno , García-Sastre Adolfo , Schotsaert Michael
Published: August 2023

Despite being a vaccine-preventable disease, seasonal influenza is responsible for thousands of hospitalizations and deaths yearly. Currently licensed quadrivalent inactivated influenza virus vaccines (QIV) aim to induce neutralizing antibodies but are poor inducers of T-cell responses, which could be potentially overcome with adjuvants. We evaluated adjuvant skewing of host-immune responses after vaccination and its persistence after infection. Hereto, we compared two squalene-based adjuvants (AddaVax, and AddaS03) and IMDQ-PC, a lymph node-draining TLR7/8 agonist. All adjuvants resulted in enhanced antibody titers and protection against challenge with H1N1 influenza virus. Lung cytokine/chemokine profiling at 5 days post-infection showed that AddaVax results in higher levels of type 2 immunity-stimulating cytokines IL-4, -5, and -13 in the lungs, which is also reflected by a higher IgG1:IgG2a ratio. Meanwhile, IMDQ-PC showed higher serological IgG2a titers after vaccination and higher levels of IFNγ in the lungs after challenge. To investigate the role of the myeloid compartment in adjuvant skewing of host responses, we examined adjuvant-mediated skewing of bone-derived macrophages in-vitro using Arginase I and iNOS as M2 or M1 markers, respectively. We also pre-licensed macrophages with IL-4 and IFNγ to evaluate adjuvant-intrinsic skewing effects. Licensing was required to induce M1 or M2 phenotypes by squalene-based adjuvants, whereas IMDQ-PC skewed towards M1 phenotype irrespective of licensing. Correlating with the observed type I immune host response, IMDQ-PC induced iNOS more robustly with IFNγ than the other adjuvants. And, AddaS03 induced the highest levels of Arginase I in the IL-4 group.

This study was also partly funded by CRIPT (Center for Research on Inuenza Pathogenesis and Transmission), a NIH NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract number 75N93021C00014), by the NIAID funded SEM-CIVIC consortium to improve inuenza vaccines (contract number 75N93019C00051), to AG-S. By NIH/NIAID HHSN272201400008C (L.C.), and NIH/NIAID R21AI151229 and R01AI160706 (M.S.). B.G.D.G. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant N 817938).
Modulating vaccine inflammation with thermophobic trehalose glycolipid adjuvant polymers
Authors: Mancini Rock , Hendricksen Aaron , Ryan Austin , Aguilar-Carreno Hector , Ezzatpour Shahrzad
Published: August 2023

Adjuvants drive the efficacy of many vaccines, however, they also elicit undesirable inflammation which is commonly pyrogenic resulting in local or systemic increases in temperature. Based on this, we envisioned that inversely coupling adjuvant potency to small (~2 °C) temperature changes could be a uniquely effective approach to balance efficacy while maintaining safety and tolerability. Here we report the first Thermophobic Adjuvant Polymers (TAPs) that reversibly attenuate potency in response to temperature. TAPs consisted of co-polymers containing both trehalose glycolipid adjuvant and thermoresponsive N-isopropylacrylamide (NIPAM) subunits. In an Influenza A/California/04/2009 virus vaccine mouse model, TAPs were effective adjuvants. Mice immunized with TAPs adjuvanted vaccine exhibited increased neuraminidase neutralizing antibodies, lymph CD4+ T cells, and CD4+/44+/62L+ lung central memory T cells. Protection after viral rechallenge was comparable between TAPs and alum adjuvanted vaccines. At 37 °C in-vitro, TAPs elicited cytokine profiles comparable to other glycolipid adjuvants in the JAWSII cell line and primary CD11c+ BMDCs. Relative to 37 °C, inflammation (TNF-α and IL-6) was enhanced at 35 °C and abrogated at 39 °C. DLS and NOESY-NMR suggest that the temperature-dependent activity is due to intramolecular hydrophobic shielding between the adjuvant and NIPAM which is enhanced beyond the lower critical solution temperature. Overall, this study demonstrates TAPs are an effective influenza vaccine adjuvant and elicit inflammation which is inversely linked to temperature. We envision this approach could be extended to many other adjuvants to enhance efficacy while maintaining safety and tolerability.

Supported by grants from NIH (1R01CA234115), AAI (Careers in immunology fellowship), and the Washington Research Foundation.
SARS-CoV-2 Ancestral and Variant of Concern Breakthrough infections boost both cross-reactive cellular and humoral immune responses in mRNA-vaccinated Hamsters
Authors: Schotsaert Michael , Diego Juan Garcia-Bernalt , Singh Gagandeep , Jangra Sonia , Garcia-Sastre Adolfo , Laporte Manon , Chang Lauren A , Handrejk Kim , Rathnasinghe Raveen , Zahed Sara El , Chang Max W , Warang Prajakta , Aslam Sadaf , Benner Christopher , Krammer Florian , Mena Ignacio
Published: August 2023

When mRNA vaccines were rolled out under EUA, it was observed that a single vaccination, already protected vaccinees from severe disease, even before neutralizing antibodies appearded. To better understand the underlying mechanism of protection, we vaccinated hamsters once with a suboptimal dose of Pfizer/BioNTech COVID-19 mRNA vaccine (5mg/animal). Four weeks post vaccination, animals were challenged with different variants of SARS-CoV-2. Vaccination resulted in antibody ELISA titers against SARS-CoV-2 spike protein, but antibody titers were too low for efficient neutralization of antigenically matching USA-WA1/2020 (WA1) or drifted variants of concern (Alpha, Beta, Delta and Mu). Nevertheless, suboptimal vaccination resulted in reduced morbidity for WA1 and Alpha-challenged animals and complete control of lung virus titers for WA1, Alpha and Delta but with breakthrough infection for the antigenically more distant Beta and Mu. Moreover, T cell responses were higher in vaccinated animals compared to unvaccinated ones at 5 days post infection, suggesting a recall of vaccine-primed T cells. Infection with different SARS-CoV-2 variants also back-boosted neutralizing antibody responses against the challenge virus and in the case of the variants of concern, against the antigenically distant but vaccine-matched WA1. Transcriptomic analysis of host immune responses to infection reflects both vaccination status and disease course, is further compared with lung pathology and suggests a role for interstitial lung macrophages in vaccine-mediated protection. In summary, protection after suboptimal vaccination against SARS-CoV-2 challenge in hamsters correlates with recall of both vaccine-induced B- and T-cell responses.

This study was also partly funded by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIH NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract number 75N93021C00014) to A.G.S and M.S. and by R01 AI160706 to M.S.
The T cell receptor repertoire reflects the early dynamics of the immune response to vaccination
Authors: Sachidanandam Ravi
Published: August 2023

Early ascertainment of the immune response to vaccinations is important. The T cell receptor (TCR) is a promising target to achieve this, with the complete TCR repertoire in PBMCs reflecting the T cells present in an individual. To this end, we developed Tseek, an unbiased and accurate method for profiling the TCR repertoire by sequencing the TCR α and β chains and developing a suite of tools for repertoire analysis. We explored the T cell response to both the COVID-19 mRNA vaccine (n=9) and the seasonal inactivated Influenza vaccine (n=5) at several time points. Neutralizing antibody titers were also measured in the covid vaccine samples. The COVID-19 vaccine elicited a broad T cell response involving multiple expanded clones, whereas the Influenza vaccine elicited a narrower response involving fewer clones. Distinct T cell clones responded at different time points over a month, providing temporal details lacking in the antibody measurements. The physical symptoms following administration of the vaccinations were not indicative of the TCR/antibody responses while the TCR responses broadly presaged the antibody responses. Additionally, the TCR repertoire is akin to a fingerprint: donors of blood samples taken years apart could be identified from the TCR repertoire. TCR repertoire profiling shows great promise as an early and sensitive measure of the adaptive immune response to vaccination, which can help improve immunogen selection and optimize vaccine dosage and spacing between doses.
A rationally designed combined intranasal adjuvant drives robust and broad humoral, cellular, and mucosal immune responses to SARS-CoV-2
Authors: Wong Pamela T , Jangra Sonia , Landers Jeffrey J , Laghlali Gabriel , Rathnasinghe Raveen , O’Konek Jessica J , Janczak Katarzyna W , Garcia-Sastre Adolfo , Baker James R , Schotsaert Michael
Published: August 2023

Multiple FDA-approved SARS-CoV-2 vaccines have provided excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Furthermore, while these parenteral vaccines have provided substantial protection, they have been less effective in inducing mucosal immune responses which are critical for effectively blocking viral transmission in vaccinated individuals. As mucosal immune responses are most effectively induced through infection or mucosal immunization, we have developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). Through activating this array of receptors, we have shown that the NE/IVT adjuvant better recapitulates the innate immune activation events that occur during natural viral infection, subsequently leading to more tailored and effective downstream adaptive immune responses. The combination adjuvant with various spike protein antigens elicited robust protective humoral, mucosal, and cellular responses to SARS-CoV-2 in mice, with markedly enhanced TH1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous and antigenically distant drift variants of SARS-CoV-2. These results highlight the potential of this adjuvant to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates for use mucosally.

This research was supported by R01-AI160706 to P.W. and M.S, and in part by R01-DK130425 to M.S.
A SARS-CoV-2 vaccine designed for manufacturability results in unexpected potency and non-waning humoral response
Authors: Anderson Stephen , Campbell Elliot , Dobkin Julie , Osorio Louis , Kolloli Afsal , Ramasamy Santhamani , Kumar Ranjeet , Sant’Angelo Derek B , Subbian Selvakumar , Denzin Lisa
Published: August 2023

Despite the availability of a number of COVID-19 vaccines, there still exists a large unmet need for vaccines which provide durable immunity and protection against emerging SARS-CoV-2 variants, and for vaccines with lower manufacturing costs and reduced logistical requirements for distribution. We have developed a novel protein component vaccine candidate, MT-001, based on a fragment of the SARS-CoV-2 spike protein surrounding the receptor binding domain (RBD). Mice vaccinated with a 2-dose prime-boost regimen of MT-001 adjuvanted with alum and CpG exhibited peak anti-spike IgG ELISA titers comparable to those reported for mRNA vaccines from Pfizer/BioNTech (BNT162b2) and Moderna (mRNA-1273), and, in contrast to the mRNA vaccines, these titers were found to be durable with midpoint GMTs in the range of 105 – 106 up to 12 months post-vaccination. Syrian golden hamsters vaccinated with a similar 2-dose regimen of MT-001 exhibited undetectable levels of SARS-CoV-2 in lung tissue four days after intranasal challenge with SARS-CoV-2/US-WA1. In mice, the results showed an increased breadth of the response, with significant neutralization titers against the Omicron BA.1 variant at this timepoint.

These attributes make MT-001 a compelling candidate for a next-generation COVID-19 vaccine. MT-001 could be particularly valuable as an annual booster for individuals with diverse histories of vaccination, SARS-CoV-2 infection, and/or predispositions resulting in an immunocompromised state. MT-001 was designed for manufacturability and ease of distribution, and we demonstrate that these attributes are not inconsistent with a highly immunogenic vaccine that confers durable and broad immunity to SARS-CoV-2 and its emerging variants.

Supported by grants from Rutgers University CCRP2 and the New Jersey Health Foundation. In-kind support from CEPI is also gratefully acknowledged.
MEF2C is a critical regulator of human NK cell metabolism
Authors: Li Joey , Senthilkumar Vignesh , Shah Siya , Padilla Eddie , Riggan Luke , Ball Andrea , Matulionis Nedas , Divakaruni Ajit , Christofk Heather , O’Sullivan Timothy
Published: August 2023

Natural killer (NK) cells are critical for the first line of defense against viral infection and malignancy. While the transcriptional and epigenetic regulation of activated NK cells is well characterized in mice, these processes are not yet fully understood in human NK cells. We performed a targeted CRISPR/Cas9 ribonucleoprotein (RNP) transcription factor screen in mature primary human NK cells to identify putative transcription factors required for optimal effector function. The transcription factor myocyte enhancer factor 2C (MEF2C) emerged as a previously unidentified regulator of NK cell homeostasis, cytokine production, and cytotoxicity. MEF2C-deficient NK cells activated with IL-2 and IL-15 displayed impaired proliferation, degranulation, and production of granzyme B. CRISPR-mediated deletion of MEF2C resulted in disrupted glycolysis and oxidative phosphorylation using both SCENITH and Seahorse extracellular flux analysis. Liquid chromatography/mass spectrometry (LC/MS)-based metabolomics of MEF2C-deficient NK cells revealed reductions in the metabolites glyceraldehyde-3-phosphate and a-ketoglutarate/succinate, accompanied by increased accumulation of lipid metabolism products including phosphorylethanolamine and saturated long-chain fatty acids. In vivo, CRISPR/Cas9 RNP-mediated disruption of MEF2C expression in mouse NK cells resulted in decreased expansion during mouse cytomegalovirus infection. Together, these studies reveal MEF2C as a novel regulator of NK cell effector function through control of multiple critical cell-intrinsic metabolic pathways.
Macrophage recognition of non-self MHCI triggers signaling pathways directing differentiation and initiating tissue repair
Authors: Warunek Jordan , Mathews Lisa , Oberbarnscheidt Martin H , Lakkis Fadi G , Turnquist Heth R
Published: August 2023

Injury resolution requires a coordinated immune-mediated response executed by macrophages (MΦs). Yet, how MΦs convert local stimuli into effective repair is poorly understood. Furthermore, organ transplants (Tx) will fail over time due to fibrosis, despite immunosuppressants targeting adaptive immune cells. We hypothesize this is due to dysregulated MΦ-mediated tissue repair of grafts. Donor MHCI can be recognized by monocytes and MΦs via paired-immunoglobulin-like receptor-A (PIR-A), but if non-self MHCI recognition disrupts repair is an unanswered question. To test this, Wildtype (WT), Rag2−/−γc−/−, and Pira−/− C57BL/6 (H-2b) MΦ differentiation was assessed after exposure to allogeneic (BALB/c; H-2d; allo) or syngeneic (syn) materials using flow cytometry or phosphoflow. The function of syn- and allo-exposed MΦs were compared with live-cell imaging of wound healing assays. Allo materials generated Ly6chiCD86hi MΦs in both WT and Rag2−/−γc−/− recipients in vivo. Recombinant H2-Dd alone behaved similarly in vitro. Interestingly, Ly6chi CD86hi MΦ differentiation in response to non-self MHCI was not PIR-A mediated, but by Ly49d-triggered PLCγ2 activation of NF-κB and mTOR coupled with increased glycolysis. Moreover, MΦ allo exposure profoundly amplified MΦ repair in comparison to other well characterized MΦ subsets (i.e. LPS/IFNγ or IL-4 stimulated). We identify an unappreciated relationship between innate allorecognition and MΦ repair. Specifically, MΦs recognize non-self MHCI via Ly49d, and triggering this novel pathway mediates MΦ differentiation in phenotypically pro-inflammatory subsets with amplified repair functions. Such a mechanism would contribute to Tx graft fibrosis and related pathology over time.

Supported by grants from NIH (5T32AI074490-14, R01HL122489)
Bacterial blockade of host protein synthesis triggers cell death in dendritic cells to restrict Legionella pneumophila infection
Authors: Vazquez Marrero Victor R , Zhang Jenna , Doerner Jessica , Pollock Tzvi , Shin Sunny
Published: August 2023

Legionella pneumophila is a facultative intracellular bacterial pathogen that is responsible for a pneumonia called Legionnaire’s disease. Legionella invades cells by using a type IV secretion system that delivers effector proteins into the host cell cytosol to facilitate its intracellular survival and replication. Macrophages serve as the primary replicative niche for Legionella. In contrast, dendritic cells (DCs) have been shown to rapidly restrict Legionella infection by activating cell death. The host and bacterial factors that induce this response remain elusive. We found that DCs undergo caspase-dependent cell death and that this response is triggered by a subset of secreted effectors that block host protein synthesis. Specifically, we show that these effectors decrease the levels of pro-survival proteins in DCs, thus leaving cells poised to undergo cell death. Altogether, our data show that pro-survival molecules in DCs serve as guard proteins that detect pathogenic activities to limit bacterial replication.

Supported by a National Science Foundation Graduate Research Fellowship Program (DGE-2236662)
A cell reporter system to examine toll-like receptor ligands in channel catfish
Authors: Felch Kristianna Lee , Bengtén Eva , Wilson Melanie
Published: August 2023

Toll-like receptors (TLR) are a family of innate immune receptors that recognize microbe- or danger-associated molecular patterns (MAMPs or DAMPs). TLR ligand binding results in the recruitment of adaptor proteins, which initiate signaling cascades that lead to the activation of NF-κB and interferon regulatory factors (IRFs). Importantly, this signaling initiates both innate and adaptive immune responses. Channel catfish is an important aquacultural species, and the catfish genome encodes 20 TLRs. In contrast, mammalian genomes encode from 10 to 13 TLRs. Our hypothesis is that fish, due to their aquatic environment, express additional TLRs that provide protection from pathogens and pollutants. Our goal is to gain a functional understanding of catfish TLRs by identifying their ligands. To screen potential ligands, we use the HEK Dual-null cell line, which is engineered to not express endogenous TLRs. This cell line is stably transfected with a SEAP reporter gene under the control of NF-κB. Using this model, it was confirmed that TLR7 is activated by imiquimod, a synthetic ssRNA analog. In addition, we have identified the full repertoire of IFN genes in the catfish genome. This allows us to assess products downstream of TLR activation by qPCR. Taken together, this system permits us to study the TLR signaling pathways that drive catfish immune responses. In the future, once TLR signaling is better understood, specific ligands can be incorporated into vaccines as adjuvants to direct favorable immune responses and protect farmed and domesticated animal species.

Supported by pre-doctoral fellowship USDA-NIFA-AFRI 2022-67011-36580 and grant NSF-IOS 1656419
Low-input chromatin profiling reveals inter- and intra-lineage differences across immune cell types
Authors:   Immunological Genome Consortium, Bale Michael J , Ahimovic Dughan J , Lee Juliana , Marunde Matthew R , Lin Carolina P , Maryanski Danielle N , Keogh Michael-Christopher , Josefowicz Steven Z
Published: August 2023

Transcriptional regulation in cells is governed by myriad traits that allow specific responses to ever-changing environments. Inclusive of these regulatory factors are the histone proteins that compact the genome while additionally being decorated with diverse post-translational modifications (PTM) suggested to direct the cellular machinery to express / repress lineage- and context-specific loci. Our consortium recently performed a detailed analysis of chromatin accessibility (ATACseq) across multiple mouse immunocyte populations, which advanced our understanding of how cis-regulatory elements (CRE) control transcriptional programs in rarer immune cell types. Understanding the role of histone PTMs in this process has remained elusive due to the high cell-input demands of the gold-standard assay: ChIPseq. To this end, we developed a streamlined CUT&RUN approach to support robust profiling at 10K cells per target in ~100 sorted mouse immunologic populations. We have used this to map histone PTM states characteristic of active (H3K4me1/3, H3K27ac, H3K36me3) or repressed (H3K27me3) chromatin and architectural factors associated with genome organization (H3.3, CTCF), in tandem with RNA- and ATAC-seq. We further dissected the effect of genomic variation by utilizing a B6xCast/EiJ F1 hybrid mouse to understand how CRE variation at each allele independently impacts transcriptional regulation. Taken together, our data provides a deeper understanding of transcriptional regulation in the mouse immune system across differentiation and perturbation states.
Defining TLR4-independent molecular pathways activated during endotoxemia
Authors: Hunat Junela Cecille Serrano , Calkins Phoebe , Salgado Cristal , Omodia Glory , Awan Hiba , Aaron Anique , Harberts Erin M
Published: August 2023

Sepsis is the leading cause of nosocomial mortality, accounting for almost 20% of annual global deaths. Patients who recover from septic shock maintain a significantly increased risk for mortality due to secondary infection. Toll-like receptor 4 (TLR4) recognizes Gram-negative bacterial lipopolysaccharides (LPS) and initiates robust immune activation that can be detrimental to the host. Towards the end of septicemia, cells self-regulate and remove surface TLR4 which contributes to immune ataxia. TLR4 deficient (TLR4−/−) C57BL6 mice are used as a model for the immune refractory period following septic shock and compared to wildtype (WT) mice. In the serum of both WT and TLR4−/− mice, 51 proteins were previously found to be dysregulated over the course of endotoxemia. We hypothesize that these 51 proteins represent TLR4 independent activation mechanisms. This study further investigates protein and mRNA distribution during endotoxemia. Liver, spleen, kidney, heart, and brain were collected at 0, 6, 12, and 18 hours post-intraperitoneal injection of 30 mg/kg E. coli LPS. Using qPCR and Western Blot to quantify gene and protein expression, we observed down regulation of genes involved with metabolism (Adsl, Mdh1) and erythrocyte cytoskeleton stabilization (Dmtn). In the same samples we observe activation of genes and proteins involved in T-cell function (Coro1a), and the complement cascades (Masp1, C1qb, Cfd, F5). These results further define TLR4 independent activation during endotoxemia as a model for immune ataxia that occurs after primary septic shock. This project could provide proof of principle to support the development of more targeted treatments to address immune activation during secondary infection after septic shock.

This project was supported by a Towson University FDRC grant to EMH and a Fisher College of Science and Mathematics undergraduate research award to JCH, HA, PC, and AA.
TGF-β promotes glycolysis through PFKL in activated macrophages and exacerbates sepsis by disrupting blood coagulation
Authors: Gauthier Thierry , Yao Chen , Jin Wenwen , Lim Yun-Ji , Patino Liliana C , Liu Na , Mitrovic Mladen , Tussiwand Roxane , O’Shea John J , Chen Wanjun
Published: August 2023

The metabolism of macrophages plays a key role in their immune responses, but the mechanisms by which the metabolism is regulated remain largely unknown. We show here that TGF-β induces a unique phenotype of macrophages and regulates the glycolysis of macrophages independently of inflammatory cytokine production. Specifically, TGF-β increased expression and activity of phosphofructokinase-1 liver type (PFKL) in macrophages and thus promoted their glycolysis during cell activation, yet paradoxically suppressed the production of proinflammatory cytokines in the same macrophages. The upregulation of glycolysis was mediated by an mTOR-c-MYC dependent pathway, whereas the inhibition of cytokines was ascribed to downregulated activation of the major pro-inflammatory transcription factors, namely AP-1, NFkB and STAT1. Importantly, we found that in LPS-induced sepsis model, TGF-β enhancement of macrophage glycolysis led to a decreased survival in mice by increasing blood coagulation. Analysis of cohorts of patients with sepsis and covid-19 revealed that the expression of PFKL, TGF-b receptors TGFBRI and coagulation factor F13A1 in monocytes positively correlated with the progression of the disease. Thus, TGF-β is emerging as a critical cytokine regulating macrophage metabolism and could serve as a therapeutic target in patients with sepsis and Covid-19.
AIM2 Promotes Epithelial Tuft Cell Development During Intestinal Type 2 Immune Responses
Authors: Wilson Justin E , Tribble Stephanie L , Verdugo Sydney A N , Reinartz Dakota M
Published: August 2023

The epithelium performs a growing list of immunologically relevant functions at mucosal sites. Specialized epithelial cells of the intestine interact with the microbiota and lamina propria immune cells to regulate host defense, yet we know little about the molecular regulators linking these interactions during different immunological insults. Here, we found that the DNA sensor Absent in Melanoma 2 (AIM2) promotes development of epithelial tuft cells, which initiate intestinal type 2 immunity. Aim2-deficient intestinal epithelial organoids displayed reduced tuft cell gene signatures and numbers during treatment with the tuft cell-promoting cytokines IL-4 and IL-13. Tuft cells respond to the microbial metabolite succinate, leading to IL-13 production by type 2 innate lymphoid cells (ILC2s) and tuft cell hyperplasia in vivo. Succinate-fed Aim2−/− mice displayed reduced tuft cells and IL13 expression compared to similarly treated wild type mice. Inflammasome-deficient Asc−/− mice and wild type mice displayed similar succinate-induced tuft cell profiles, suggesting AIM2 promotes tuft cell-ILC2 activation independently of its inflammasome function. RNA sequencing revealed Aim2−/− intestinal organoids displayed diminished expression of O-linked N-acetylglucosamine transferase (OGT), which is required for IL13/STAT6-induced tuft cell expansion. In line with these findings, Aim2−/− intestinal organoids and succinate-treated mice presented reduced OGT protein expression and STAT6 phosphorylation. These combined results reveal an novel role for AIM2 during type 2 intestinal inflammation in the intestine via promotion of tuft cell expansion, potentially through maintaining OGT-STAT6 signaling.

Supported by grants from NIH (K22 CA212030) and the AZ Dept. of Health Services (AZBRC New Investigator Award).