bluuebunny/crossref_metadata_2025 huggingface dataset:
Nontypeable Haemophilus influenzae specific memory TH17 cells protect against secondary bacterial pneumonia following Influenza A virus infection
Authors: Yang Ying
Published: August 2023

Nontypeable Haemophilus influenzae(NTHi) is one of the most common bacteria that cause secondary bacterial pneumonia following influenza A virus (IAV) infection, leading to hospitalization and death associated with IAV infection worldwide. Current efforts to develop vaccines against NTHi infection focus on inducing antibodies but are hindered by antigenic diversity among NTHi strains. Therefore, we interrogated the contribution of the memory T-helper type 17 (TH17) response in protective immunity against IAV/NTHi co-infection. We observed that co-infected mice exhibited prolonged NTHi infection in the lungs even at mild IAV infection and impaired NTHi-specific TH17 responses compared to NTHi mono-infected mice. However, immune mice that recovered from a prior NTHi infection were protected from homologous and heterologous secondary NTHi strains following IAV infection. Our data further revealed that memory NTHi-specific TH17 cells overcame IAV-driven impairments of anti-NTHi TH17 responses and were cross-reactive with different NTHi strains. Last, mice immunized with a NTHi protein that induced a strong TH17 memory response were broadly protected against diverse NTHi strains following challenge with IAV/NTHi co-infection. These results indicate that NTHi-specific memory TH17 cells are capable of providing serotype-independent protection against co-infection and demonstrate the advantage of developing broadly protective TH17-inducing vaccines against secondary bacterial pneumonia.
Mechanistic investigations of granzyme A adjuvant effects on γ9δ2 T cells
Authors: Phelps Kathleen , Rasi Valerio , Xia Mei , Hoft Daniel
Published: August 2023

γδ T cells are ideal effector cells against cancer and pathogens for numerous reasons: they are found in blood, mucosa, and lymphoid organs, develop memory responses independent of MHC restriction, present antigen, produce cytokines, and are cytolytic. An obstacle for the development of biomedical interventions targeting γδ T cells is the need for an adjuvant that promotes their expansion and is suitable for human use. The adjuvant routinely used, IL-2, has multiple drawbacks limiting its use for vaccination. Our lab has shown that memory γ9δ2 T cells, induced by BCG vaccination, are expanded in vitro by M. tuberculosis, M.bovis (BCG), and our lab’s vaccine candidate 6-O-methylglucoselipopolysaccharide (mGLP), in the presence of IL-2. Additionally, the expanded γ9δ2 T cells inhibit intracellular mycobacterial growth. This inhibition is dependent on granzyme A (GzmA) production by γ9δ2 T cells, and GzmA alone can inhibit intracellular mycobacterial growth.

We recently identified that GzmA can also promote expansion of γδ T cells, without exogenous IL-2, in response to mGLP and HMBPP, a phosphoantigen that stimulates all γ9δ2 T cells. To identify γδ T cell adjuvants, in vitro expansion assays were performed using human peripheral blood mononuclear cells, dendritic cells, antigen, and adjuvant. The absolute numbers of expanded effector γδ T cells were calculated using flow cytometry after 7 days of culture. We identified that the ability of GzmA to promote γδ T cell expansion is dependent upon TLR4, CD14, and GzmA homodimerization, but independent of GzmA enzymatic activity. This new information has broad implications for vaccine design, promoting γδ T cell responses, and understanding the role of GzmA in infectious disease and cancer.

Supported by grants from NIH (R01 AI048391) and Bill & Melinda Gates Foundation (OP1118659)
Pre-exposure to mRNA-LNP inhibits adaptive immune responses and alters innate immune fitness in an inheritable fashion
Authors: Qin Zhen , Bouteau Aurélie , Herbst Christopher , Igyártó Botond Z
Published: August 2023

During the ongoing SARS-CoV-2 pandemic, hundreds of millions of people have been exposed to the experimental mRNA-LNP-based COVID vaccines, of which long-term immune effects are unknown. We previously showed that this vaccine platform is highly inflammatory. Its synthetic ionizable lipid component, which has a long in vivo half-life, is responsible for the induction of inflammation and adaptive immune responses. The inflammatory component’s long in vivo half-life could be a reminiscence of a chronic infection, which is known to lead to immune exhaustion and non-responsiveness. Therefore, here, we sought to determine the effects of pre-exposure to the mRNA-LNP on adaptive immune responses and innate immune fitness.

We found that pre-exposure to mRNA-LNPs or LNP alone led to long-term inhibition of the adaptive immune response, which could be overcome using standard adjuvants. On the other hand, we report that after pre-exposure to mRNA-LNPs, the resistance of mice to heterologous infections with influenza virus increased while resistance to Candida albicans decreased. The diminished resistance to Candida albicans correlated with a general decrease in blood neutrophil percentages. Interestingly, mice pre-exposed to the mRNA-LNP platform can pass down the acquired immune traits to their offspring, providing better protection against influenza.

In summary, the mRNA-LNP vaccine platform induces long-term unexpected immunological changes affecting both adaptive immune responses and heterologous protection against infections. Thus, our studies highlight the need for more research to determine this platform’s true impact on human health.

National Institute of Allergy and Infectious Diseases (https://www.niaid.nih.gov) R01AI146420 and R01AI146101
Correlates of COVID-19 mRNA vaccine humoral immune responses in men living with HIV-1
Authors: Tuttle Dylan J , Castanha Priscila M S , Nasser Amro , Wilkins Maris S , Cuff Deirdre E , Chhibbar Prabal , Sluis-Cremer Nicolas P , Das Jishnu , Mailliard Robbie B , Rinaldo Charles R , Marques Ernesto T A
Published: August 2023

It is important that people with HIV-1 (PWH) receive an FDA-approved vaccine for COVID-19. Because PWH are known to elicit less effective immune responses to other licensed vaccines, we assessed the humoral immune response to the novel COVID-19 mRNA vaccines in 139 fully vaccinated male participants (45 with HIV-1 [MWH] and 94 without HIV-1 [MWOH]) of the MACS/WIHS Combined Cohort Study. Levels of anti-nucleocapsid (N) and anti-spike 1 (S1) IgG and IgA were determined using ELISA and the MSD electro-chemiluminescence multiplex assay platform, to classify natural infection and vaccination status, and quantify binding IgG. Functional characterization of antibodies (Ab) was determined by measuring neutralizing Ab levels against WA, Delta, and Omicron strains, and immune complex complement activation capacity by complement component 3 (C3) deposition. We found that MWH who received two standard doses of mRNA vaccines had lower levels of vaccine-induced anti-S1 IgG and neutralizing Ab against the Washington, Delta, Omicron variants compared to MWOH, and these Ab titers were boosted in the context of natural SARS-CoV-2 infection for MWH. Interestingly, Ab in MWH promoted more potent C3 deposition. In all participants the Moderna mRNA-1273 vaccine induced significantly higher levels of binding IgG and neutralizing Abs against the variants. Importantly, preexisting immunity to the endemic human coronaviruses, participant age, and blood biochemical measurements correlated with these vaccine responses. Our data indicate that COVID-19 mRNA vaccine-induced humoral responses are impaired in MWH. Further studies are needed to define the long-term effects of this impairment on prevention and control of SARS-CoV-2 infection in MWH.

Supported by grants from NIH: Pitt AIDS Research Training (PART) (NRSA 5T32AI065380-15) to DJT, NIH (NHLBI 5U01HL146208) and OAR/NHLBI Supplement (U01HL146208-03S2/S3), MWCCS Concepts X21033 and X20124, and NIH NIAID (R01-AI167711).
Development of broadly protective COVID-19 vaccine against up-to-date variants based on adenovirus type 5/35 vector platform
Authors: Kang Chang-Yuil , Chang Soojeong , Shin Kwang-Soo , Jung In Kyung , Park Hyemin , Oh Jonghan , Park Seowoo , Shin Jieun , Kim Jong Heon
Published: August 2023

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron strain has evolved into a highly divergent variant with several sub-lineages. These new emerging variants could impair the protective efficacy of COVID-19 vaccines based on the ancestral spike, causing breakthrough infections and re-infections. Therefore, it is imperative to develop new multivalent vaccines that can provide broad protection against currently prevalent new variants. We previously developed AdCLD-CoV19-1 OMI, an Ad5/35 platform based non-replicating recombinant adenoviral vector that encodes the spike protein of BA.1, and currently performing clinical studies. We also constructed vaccines against emerging subvariants such as BA.5, BA.2.75, XBB, BQ.1.1, and BN.1 using the Ad5/35 platform and evaluated their immunogenicity by sera from vaccinated mice. To develop the bivalent vaccine, we selected a combination of BA.5 and BA.2.75 vaccines by antigenic cartography map based on cross-neutralizing activities. We found that a bivalent vaccine could induce broadly neutralizing antibodies against the predominant circulating strains and improved cross-neutralization capacity. These data suggest that the bivalent vaccine broadens immunity against current prevalent omicron subvariants. We further investigate combinations of next-generation multivalent vaccines to confer broad protection against new subvariants. Based on the results of preclinical studies, clinical trials of the polyvalent vaccines will be conducted.

Supported by grant from the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (2020M3A9I2107463)
Targeting antigens to phosphatidylserine triggers rapid T- and B-cell responses
Authors: Hu Wenbo , Flaskamp Lavinia , Kranich Jan , Brocker Thomas
Published: August 2023

Phosphatidylserine (PS) is a phospholipid that is present in each cell membrane. In intact cells, PS is kept intracellularly, in dying cells, extracellular vesicles (EVs) and activated platelets, PS flips outside where it can be accessed by PS-binding reagents, such as Milk fat globule-EGF factor 8 (MFG-E8). In previous studies, we have established in vivo use of fluorescent MFG-E8 to label and characterize EVs. Here, we measured the antigen-specific immune responses elicited by targeting antigen (Ag) to PS+ structures, presumably EVs via MFG-E8 as a novel vaccination strategy. In contrast to non-targeted conventional Ag, PS-targeting by fusing Ag to MFG-E8 caused accumulation in the marginal zone of lymphoid follicles soon after injection. Hours later, Ag-MFG-E8 was deposited on the follicular dendritic cell network without the need of previous primary immunization. Mice immunized with PS-targeted Ag/Alum induced long-lasting germinal centers (GCs) and significantly more GC B cells. Furthermore, it resulted in an approx. 13,000-fold increase in Ag-specific IgG-levels and strongly accelerated antibody class switch recombination as compared to immunization with conventional Ag/Alum. Ag targeted to PS also induced more efficient CD8 T cell activation and IFN-gamma production post immunization. Therefore, targeting Ag to EVs by harnessing the PS-binding properties of MFG-E8 might turn out to be of advantage for the induction of very rapid and long-lasting immune responses.

This project was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research foundation) – Project-ID 210592381 - SFB 1054 B03 to Thomas Brocker and Z02 to Jan Kranich.
Stabilization of N-terminal cysteines in HPLCHRMS quality control of peptide pools
Authors: Kreuzer Oliver J , Weller Michael G , Bosc-Bierne Gaby
Published: August 2023

Synthetic peptide pools are used in experimental and clinical immunology, for example, cancer immunotherapy. The analytical characterization is challenging due to the similarity of the peptides, usually only a pre-characterization of the single peptides is performed. However, regular quality control of the peptide mix would be highly desirable. Therefore, a high performance liquid chromatography-high resolution mass spectrometry (HPLCHRMS) method for quality control of a peptide pool was developed. Peptides were synthesized and purified to > 90% each. The lyophilized single peptides were combined to a peptide pool containing 32 peptides and analyzed by HPLC-HRMS. Quantification is carried out UV detection, and identification is obtained by high resolution mass spectrometry. Before separation on a capillary reversed-phase column, alkylation of thiols was performed. Best results were obtained with a linear gradient from 96% of solvent A (water with 0.05% TFA), 4% of solvent B (acetonitrile with 0.04% TFA) to 56% of solvent A, 44% of solvent B in 100 minutes. Without sample preparation, peptides with N-terminal cysteine will yield almost no UV signal. This surprising effect could be eliminated using iodoacetic acid (IAC) as an alkylation reagent. The amount of IAC and reductant (Tris(2-carboxyethyl)phosphine (TCEP)) was carefully optimized to avoid overalkylation. By a combination of capillary HPLC and HRMS, a method could be developed for the quality control of complex synthetic peptide pools. In contrast to the analysis of single peptides, analyzing complex peptide pools requires the stabilization of cysteine-containing peptides. Good results were obtained using iodoacetic acid as an alkylation reagent.
Peptide-pulsed MHC class II mutant dendritic cell vaccine has superior efficacy in a murine tumor model
Authors: Seishima Noriko , Olkhanud Purevdorj B , Becker William , Maeng Hoyoung , Lopez-Lago Miguel , Wiseman Charles , Williams William V , Berzofsky Jay A
Published: August 2023

Autologous dendritic cell (DC) vaccines with tumor antigens have been used clinically with limited therapeutic effects. Semi-allogeneic DC-based immunotherapy is still controversial, but it can be an alternative source and more attractive than autologous DC vaccines because the “off-the-shelf” DCs can be used for multiple patients without lengthy individual manufacturing time and may provide additional “allogeneic help”. This study aims to compare efficacy of syngeneic DC vaccines and semi-allogeneic DC vaccines and determine whether a therapeutic semi-allogeneic DC vaccine is more efficacious in tumor suppression. Female C57BL/6 mice were inoculated subcutaneously with human papillomavirus E6 and E7-expressing TC-1 cells. Syngeneic bone marrow dendritic cells (BMDCs) were generated from C57BL/6 and semi-allogeneic BMDCs were generated from two mouse strains, B6.C-H2-Kbm1/ByJ and B6(C)-H2-Ab1bm12/KhEgJ which had limited point mutations in the MHC class I H2-Kb allele or H2-IAb MHC class II allele, respectively. Each BMDC was pulsed with H-2Db-restricted E743–77 peptide and matured before injection. The mice received 4 intradermal injections of syngeneic or one of the semi-allogeneic E7-pulsed BMDC vaccines starting 8–9 days after the TC-1 implantation. Compared with saline control, the MHC class I mutant BMDC vaccine had efficacy similar to the syngeneic BMDC vaccine in suppressing TC-1 tumor growth. However, the MHC class II mutant BMDC vaccine had efficacy significantly superior to that of the other BMDC vaccines. Thus, MHC class II semi-allogeneic BMDCs may be more effective than syngeneic DC-based cancer vaccines, presumably because the class II alloantigens induce additional T cell help for anti-tumor immunity.
Elucidating the cellular mechanisms underlying adoptive cell transfer in models of glioma
Authors: Figg John William , Francis Connor , Ji Dan , Dean Bayli DiVita , Reid Alexandra , Falceto-Font Laura , Hilferty David , Mitchell Duane , Flores Catherine
Published: August 2023

Despite advances in solid tumor biology and oncology, pediatric and adult primary brain cancer and metastasis to the brain harbor dismal prognosis and poor outcomes. One avenue to overcome these clinical challenges is through developing robust immunotherapy to better target the tumor. Our group has previously shown that adoptive cellular therapy (ACT) provides therapeutic benefit against central nervous system (CNS) malignancies, namely medulloblastoma, brain stem glioma and glioblastoma. In this study, we characterized cell cycling differences of immune cellular sub-populations in peripheral lymphoid and tumor tissue compartments in vivo. ACT was conducted in orthotopic KR158 luciferase tumor-bearing mice, a syngeneic murine glioma model. Following completion of ACT, bromodeoxyuridine (BRDU) was administered to mice 12-hours prior to tissue collection and flow cytometry was utilized to measure the level of BRDU incorporation. 7-aminoactinomycin D was used in conjunction with BRDU to provide insight into all phases of the cell cycle. We observed significant differences in cycling populations of myeloid derived suppressor cells (MDSC) and T cells. In mice receiving ACT, MDSCs found in secondary lymphoid tissues were significantly more proliferative. In the tumor microenvironment (TME), proliferating MDSCs were significantly reduced compared to mice that did not receive treatment. Frequencies of proliferating CD4+ and CD8+ T-cells were significantly increased in cervical lymph node and TME, but reduced in spleen. These findings suggest that ACT changes immune cycling and migration dynamics in lymphoid tissue and TME to potentially promote antitumor T-cell responses and minimize tumor-mediated immunosuppression.

5R01NS112315-04
CAR+ and CAR− T cells differentiate into an NK-like subset that is associated with increased inflammatory cytokines following infusion
Authors: Luciani Fabio , Louie Raymond Y , Cai Curtis , Singh Mandeep , Deveson Ira , Ferguson James , Amos Timothy , McGuire Helen , Samir Jerome , Gowrishankar Kavitha , Adikari Thiruni , Balderas Robert , Bishop David , Gottileb David , Blyth Emily , Micklethwaite Kenneth
Published: August 2023

Chimeric antigen receptor (CAR) T cells have demonstrable efficacy in treating B-cell malignancies. Factors such as product composition, lymphodepletion and immune reconstitution are known to influence CAR+ T cell survival and persistence, however the determinants of CAR+ T cells’ fate and differentiation and how the patients’ reconstituting immune system influence therapy outcomes remain poorly understood.

We applied single cell multi-omics analysis of RNA, protein and clonal profiles of CAR+ T and PBMC in patients receiving donor-derived products from a piggybac CAR19 clinical trial (N=8 patients). We used these data to reconstruct a differentiation trajectory, which explained the observed phenotype and fate of CAR+ and CAR− T cells within the first 30 days of post-infusion. Following initial lympho-depletion, endogenous CAR− CD8+, γδ T cells and CAR+ T cells clonally expanded, and differentiated across heterogenous phenotypes. They transitioned from a dominant resting or proliferating state into precursor of exhausted T cells, and finally into a terminal NK-like phenotype. The subset of terminal NK-like CAR+ T cells was associated with increased serum concentrations of proinflammatory cytokines including IL-12 and IL-18. Validation on a published cohort (61 patients) showed that this subset was associated with clinical outcome at 6 months. Finally, we analysed in detail one patient that developed CAR T cell induced lymphoma and discussed the impact of early phase therapy in determining this unexpected outcome.

These results demonstrate that CAR- and CAR+ T cells share a differentiation profile which may be determined by non-CAR derived signals and which may influence patients’ immune-recovery and outcome.

National Health And Medical Research Funding (Australia) (APP1121643, APP1128416) BD Bioscience
Combinatory analysis of immune cell subsets and tumor-specific genetic variants can predict clinical response to PD-1 blockade for non-small cell lung cancer patients
Authors: Dutta Nikita , Rohlin Anna , Eklund Ella Äng , Magnusson Maria , Nilsson Frida , Akyürek Levent , Torstensson Per , Sayin Volkan , Lundgren Anna , Hallqvist Andreas , Raghavan Sukanya
Published: August 2023

Blockade of PD-1/PDL1 (PD-1 blockade) with antibodies has revolutionized treatment options for non-small cell lung cancer (NSCLC) patients with tumors without targetable mutations. The benefit of PD-1 blockade is limited only to a subset of the patients. This study aims to identify biological markers of clinical response in a discovery cohort (n=15) of patients early during treatment to distinguish responders from nonresponders using immunological and genetic markers. Immune subsets identified in the blood are characterized to establish a panel of immune markers that could distinguish the two. Blood drawn from NSCLC patients before and after PD-1 blockade was analyzed by clinical flow cytometric assay for changes in the frequencies and concentration of immune cell populations. Next generation sequencing was performed on the extracted DNA from archival tumor biopsies from the same patients. irRECIST criteria was used to evaluate the clinical responses with cut-off at 10 months. Responders had a significantly increased frequency of activated effector memory CD4 & CD8 T cells & decreased frequency of central memory CD4 & CD8 T cells in circulation post-treatment compared to pre-treatment. In a separate cohort we validate initial findings in a cohort of n=29 & observe that effector memory CD8 T cells in responders produced high TNFα at pretreatment. Pathogenic & likely-pathogenic mutations in TP53, KRAS, KEAP1, NOTCH-1 and STK11 in the patients that responded to PD-1 blockade are observed. A multivariate analysis combining immune and genetic parameters could discriminate responders from non-responders. We report that combined analyses of selected immune cell & genetic parameters could predict early clinical response to PD-1 blockade.
Dendritic cell expansion and activation enhances the adaptive immune response against TNBC brain metastases
Authors: Conarroe Claire Ann , Bullock Timothy N
Published: August 2023

Despite initial control by chemotherapy, Triple Negative Breast Cancer (TNBC) quickly acquires resistance and metastasizes to distal organs, including the brain. Median survival following diagnosis of TNBC brain metastases is a meager 7.3 months—indicative of a substantial unmet clinical need, with only a small subpopulation responding to checkpoint immunotherapy. As suboptimal antigen presentation can contribute to the weak T lymphocyte response to intracranial tumors, we hypothesized that enhancing dendritic cell (DC) presence in tumors may support intracranial T cell responses to tumors. Using a murine model of intracranial metastasis, we assessed immune cell frequency and function and in vivo cytokine staining for flow cytometry to test whether Flt3L can be used to promote DC presence within tumors, and whether the addition of polyIC further enhances subsequent T cell responses.

Flt3L treatment effectively increases the number of DC within the brains and meninges of mice, however these newly expanded populations exhibit lower proportions of the costimulatory molecule CD86 than the untreated control group, indicative of an immature phenotype. 24 hours after polyIC treatment, a higher percentage of DCs in brain tumors express CD86 but do not produce IL-12. Despite the lack of intratumoral IL-12, the combination of Flt3L and polyIC treatments led to a higher presence of T cells within tumors and their production of IFN-γ in the TME than either treatment alone. In conclusion, simply increasing the presence of DC within intracranial tumors is insufficient to promote T cell immunity; rather, deliberate activation of de novo DC is required for this full effect.

Supported by grants from NIH (2T32CA009109-46 and 1R21CA226607-01A1) and the Schiff Foundation
Androgen signaling shapes the sexual differences of skin immunity by regulating ILC2 and dendritic cells
Authors: Chi Liang , Gribonika Inta , Corral Dan , Gschwend Julia , Han Seong-Ji , Lim Ai Ing , Link Verena , Wells Alex , Collins Nicholas , Bouladoux Nicolas , Schneider Christoph , Belkaid Yasmine
Published: August 2023

The skin immune system mediates the skin health status, but the sex differences of the skin immune system are still unclear. This study aims to investigate sex differences in the skin immune system and decipher the underlying regulatory factors. We found that female mice have a higher magnitude of adaptive immune responses during commensal bacteria association and pathogen infection. Correspondingly, females have a higher level of skin dendritic cells (DCs), which play a fundamental role in commensal-induced adaptive immune responses. These sex differences were regulated by male sex hormones, that castration of males normalized the sex differences in the level of skin immune cells as well as the adaptive immune responses to bacteria. However, our scRNA-seq data reveals that androgen receptor is not expressed in DCs, but highly expressed in skin ILC2. Females have a significantly higher level of ILC2 than males and ILC2 from females also have a more activating gene expression signature than ILC2 from males. Females also have a significantly higher level of ILC2-produced IL-13 and GM-CSF than male mice. In addition, our data reveal that IL-13 and GM-CSF play a critical role to maintain the level of skin DCs, that IL-13/GM-CSF deficient mice have a remarkable decrease of skin DCs, and the sex differences of DCs are also largely impaired. Therefore, based on these findings, we suggest that androgen signaling negatively regulates the level of skin ILC2 and DCs, thereby shaping sex-specific skin immunity to commensals, pathogens, and/or other stimuli. Collectively our work proposes a mechanism for the heighten immunity observed in female and have important implication for our understanding of tissue immunity in the context of infection and cancer.
Impaired mTOR Signaling Causes Immune Suppression in Diabetes
Authors: Genito Christopher J , Darwitz Benjamin , Thurlow Lance R
Published: August 2023

It is estimated that over 11% of the U.S. population has diabetes and that 38% of the adult population is pre-diabetic. Diabetes carries increased risk and severity of many infections, which has been attributed to defects in the innate immune system. Despite the high impact of diabetes on infection, the underlying mechanisms of immune dysfunction in the diabetic host have yet to be fully elucidated. We have recently shown that innate immune cells in a diabetic host environment cannot undergo respiratory burst of reactive oxygen species and nitric oxide in response to infection with Staphylococcus aureus, the most common pathogen associated with frequently severe and invasive skin and soft tissue infection (SSTI) in patients with diabetes. The inhibition of respiratory burst was due to defects in the activation-dependent expression of the glucose transporter GLUT1, which is required for respiratory burst in neutrophils and macrophages. We therefore sought to further elucidate the specific mechanisms by which phagocytes in a diabetic environment are impaired in their ability to control S. aureus SSTI. We observed that mTOR signaling is impaired in a diabetic mouse model of S. aureus SSTI, which is upstream of both respiratory burst and GLUT-1 expression. Inhibition of mTOR signaling with rapamycin phenocopies the higher bacterial burden during diabetic infection, as well as the inhibition of respiratory burst and GLUT-1 expression. We further observed that mTOR activity upon macrophage activation is impaired in the absence of insulin and growth factor signaling. These findings begin to elucidate a role for mTOR signaling in the mechanism of immune suppression caused by diabetes.
CD4 co-receptor regulates sexual dimorphic NK cell responses to T. gondii infection
Authors: Roy Tathagato
Published: August 2023

Immunity to Toxoplasma gondii is sexually dimorphic in humans and mice with females having higher morbidity and mortality during immune dysfunction and HIV/AIDS. The mechanisms underlying these sex differences are unclear. Since CD4+ T cells are very low during HIV/AIDS and females are more susceptible to Toxoplasmic encephalitis (TE) when they have AIDS, we investigated how a lack of CD4 co-receptor expression (CD4KO) and CD4+ T cells (MHCIIKO) impacted T. gondii survival and innate immune responses in mice. Female CD4KO mice succumbed to T. gondii much faster than male CD4KO mice. To dissect why female CD4KO mice died faster we tested their NK cell responses to acute T. gondii infection compared to male CD4KO mice. Although in wild type (WT) animals both sexes had similar increases in NK cell numbers and IFNg production, infected CD4KO female mice had 50% fewer IFNg+ NK cells than infected WT female mice. However, infected male CD4KO had the same number of NK cells producing IFNg compared to WT male mice. Since CD4 co-receptor deficient mice still have functional helper T cells that are CD4-we next tested survival and NK cell responses in female and male MHCII deficient (MHCIIKO) animals, which have very few helper CD4+T cells. To our surprise survival, NK cell numbers, and IFNg+ NK cells were not significantly different between WT or MHCIIKO female and male mice. These results suggest only in female mice and not in male mice, CD4 co-receptor expression is required for survival via optimal NK cell responses during acute T. gondii infection. Our findings reveal an unappreciated sexual dimorphic role of CD4 co-receptor expression in regulation of NK cell responses to acute T. gondii infection.

National Institute of General Medical Sciences (P20GM103432)
Brief IL-12, IL-15, IL-18 activation drives the acquisition of two distinct memory-like NK cell states
Authors: Foltz Jennifer Ann , Tran Jennifer , Fisk Bryan , Wong Pamela , Khan Saad M , Berrien-Elliott Melissa M , Becker-Hapak Michelle , Russler-Germain David A , Marsala Lynne , Bednarski Jeffrey J , Zimmerman Clare , Cubitt Celia C , Marin Nancy D , Zhou Alice Y , Jacobs Miriam T , Foster Mark , Schappe Timothy , Pence Patrick , Cashen Amanda , Griffith Obi L , Griffith Malachi , Petti Allegra A , Fehniger Todd A
Published: August 2023

IL-12/15/18 activation induces natural killer (NK) cell differentiation into cytokine-induced memory-like (ML) NK cells with enhanced IFNγ and killing of tumor targets that is antigen independent, rendering them distinct from conventional (cNK) and CMV-induced adaptive NK cells. In leukemia patients, ML NK cells have an excellent safety profile and 47% CR rate (PMID: 32826231). Despite their clinical promise, little is known about the biology underlying ML NK cells. Based on individual variability of human ML NK cell function, we hypothesized that ML NK cells manifest molecular heterogeneity. To this end, we applied Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) to profile RNA and protein expression on the single-cell level, and evaluated NK cell function.

Overnight IL-12/15/18 stimulation induced broad activation with distinct transcriptional programs evident in CD56bright compared to CD56dim NK cells. In contrast, following in vitro differentiation for 7 days, IL-12/15/18 activated NK cells acquired a unique transcriptional and protein signature, and manifest one of two ML NK cell states (ML-1, ML-2), or remained similar to cNK cells. ML-1 was marked by increased activating receptor expression (NKp46, NKp30, NKp44) while ML-2 had increased expression of inhibitory receptors, CD25, HLA-DR, MKI67, and IFNG. In functional assays, ML-2 cells produced more IFNγ following cytokine stimulation than ML-1 cells. Further, ML-1 and ML-2 had divergent transcription factor profiles suggesting distinct mechanisms of regulation. Collectively, our findings reveal molecular and functional heterogeneity of ML NK cells, and investigations into ML subsets in patients receiving ML NK cell therapy are ongoing.

AAI Intersect Fellowship for Computational Scientists & Immunologists (JAF, TAF, AAP), T32GM139799 (JAF), P50CA171963 (TAF, MMB-E), R01CA205239 (TAF), P30CA91842 (TAF)
Mycobacterium tuberculosis induces immune evasion in macrophages through RNA-binding protein tristetraprolin
Authors: Liu Jianguo , Wei Jiawei , Huan Ning , Ramos-Espinosa Octavio , Eickhoff Christopher , Liu Ethan , Hoft Daniel , Liu Jianguo
Published: August 2023

Immune evasion of Mycobacterium tuberculosis (Mtb) facilitates intracellular bacterial growth. The mechanisms of immune evasion, however, are still not fully understood. In this study, we reveal that tristetraprolin (TTP), one of the best characterized RNA-binding proteins controlling stability of the target mRNAs, mediates innate immune evasion of mycobacteria. We found that TTP knockout mice displayed reduced bacterial burden in the early stage after Mtb aerosol challenge. Macrophages deficient in TTP also showed an inhibition in mycobacterial growth. Live mycobacteria induced TTP protein expression in macrophages, which was blocked by mTOR inhibitor rapamycin. Rapamycin promoted TTP protein degradation. Proteasome inhibitor MG-132 recovered the shortened TTP protein half-life induced by rapamycin. Ubiquitination mediated TTP protein degradation triggered by rapamycin in infected macrophages. TTP induction suppressed the expression of iNOS/TNF-α/IL-12/IL-23, and weakened the bactericidal effects in infected macrophages, whereas blocking mTOR pathway with rapamycin enhanced the bactericidal effects of infected macrophages through TTP inhibition. Overall, this study reveals a novel role for RNA-binding protein TTP in Mtb immune evasion mechanisms and provides a potential target for host-directed therapy against TB.

R21AI171734 & R21AI151936
Hyperglycemia facilitates dengue virus infection and host immune alteration
Authors: Lin Chiou-Feng , Shen Ting-Jing , Chen Chia-Ling
Published: August 2023

Patients with diabetes mellitus are highly comorbid with severe dengue diseases caused by the dengue virus (DENV). In search of possible host factors involved in DENV infection under hyperglycemic stress, cells and murine treated with high glucose (HG) increase viral protein expression and virion release but have no effects on antiviral interferon responses as well as viral early infectious stage. Following HG stimulation, a cellular replicon-based assay displays an increased viral translation while the glucose uptake inhibitor phloretin treatment blocks such an effect. Treating HG causes increases in the protein and gene expression of translational factor poly(A)-binding protein (PABP) in a glucose transporter-associated PI3K/AKT-regulated manner. PABP silence further causes a significant decrease in HG-increased virion production. In addition, HG treatment enhances the formation of the PABP-eukaryotic translation initiation factor 4G complex, regulated by protein-disulfide isomerase. Furthermore, streptozotocin-treated hyperglycemic mice reveal increased mortality, brain viral protein expression, and viral loads under DENV infection. This study demonstrates that hyperglycemic stress facilitates DENV infection by strengthening PABP-mediated viral translation.

This work is supported by the grants from the National Science and Technology Council (NSTC109-2327-B-006-010 and 110-2320-B-038-064-MY3), Taipei, Taiwan.
Brd4 regulates metabolic reprogramming of macrophages upon H. pylori infection
Authors: Modi Nikita , Chen Yanheng , Chen Lin-feng
Published: August 2023

Metabolic reprogramming plays an important role in the regulation of several macrophage functions, including bacterial killing and cytokine production. Brd4 has emerged as a key innate immune response regulator by modulating the inflammatory cytokine expression in macrophages. However, the potential role of Brd4 in metabolic reprograming of macrophage activation remains unknown. Here, we showed that Brd4 facilitated a HIF-1α-dependent glycolytic shift for macrophage activation in response to H. pylori infection. H. pylori infection triggered Brd4-dependent polarization of macrophages to the M1 phenotype, which was associated with an increase in glycolysis and the upregulation of glycolytic genes, glucose transporter 1 (Glut1) and Hexokinase II (HK2). Mechanistically, Brd4 was recruited to the promoters of Glut1 and Hk2 by HIF-1α and regulated glycolysis by facilitating HIF-1α-dependent expression of Glut1 and Hk2. Furthermore, we found that Brd4-mediated glycolysis was essential for M1 macrophage activation since the expression of iNOS, a marker of M1 polarization, was down-regulated in Brd4-deficient macrophages and could be rescued by the addition of pyruvate. Consistently, the iNOS-mediated killing of H. pylori was decreased in Brd4-deficient macrophages and also in mice with myeloid-specific deletion of Brd4. Our study identified a novel function of Brd4 in metabolic reprograming of macrophages through glycolysis and iNOS expression in response to bacterial infection.

Supported by grant from NIH R03 AI163932
Inhibition of the mevalonate pathway causes accumulation of CD14 in the endoplasmic reticulum of macrophages
Authors: Frey Tiffany A , Heine Grace , Howe Andrew
Published: August 2023

Mevalonate kinase deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase enzyme, which acts early in the mevalonate pathway to produce sterol and non-sterol isoprenoid compounds. Patients with MKD have episodes of inflammatory symptoms accompanied by increased plasma levels of pro-inflammatory cytokines. CD14 is a receptor that binds to lipopolysaccharide (LPS) and induces the release of cytokines. Isoprenoid depletion was previously shown to increase cellular and decrease extracellular levels of CD14 in macrophages after LPS stimulation. The objective of this study is to characterize the cellular localization of CD14 following isoprenoid depletion. RAW 264.7 macrophages were treated with lovastatin to deplete isoprenoids and CD14 localization was examined using immunofluorescence and western blotting after LPS stimulation. CD14 co-localized with the endoplasmic reticulum (ER) protein calnexin following lovastatin treatment. To quantitate ER CD14, cell lysates were digested with endoglycosidase (Endo) H, which removes high mannose glycans found on ER proteins, but not complex glycans added in the Golgi apparatus. Western blot results showed that the level of Endo H sensitive CD14 was higher in lovastatin as compared to carrier-treated cells. Co-incubation of lovastatin-treated cells with mevalonate or geranylgeranyl pyrophosphate (GGPP), but not cholesterol, prevented the ER accumulation of CD14 indicating that GGPP depletion may cause this effect. Understanding how mevalonate pathway products contribute to protein trafficking in macrophages will expand our understanding of inflammatory diseases.
Evolutionary signatures of host-virus conflict reveal a family of micropeptides regulating mito-immune functions
Authors: Herrera Natalia E , Chang Tyron , Alemu Samrawit , Pinkham Chelsea , Hancks Dustin
Published: August 2023

Genomics has led to an expansive set of predicted genes; yet, functional annotation is rate-limiting. To drive discovery of immune functions, we exploit genomic scars of pathogen-conflict like rapid evolution and virologs. Virologs are host-coding sequences stolen and repurposed by viruses to rewire cells. Classic examples include master regulators SRC, IL-10, and IL-17. To identify novel immune activities, we screened all viral genomes in the database for factors displaying sequence similarity to cellular factors. Subsequent filtering using motif analysis and expression paired with our functional analysis of our top hit - a micropeptide named C15ORF48 - revealed a vertebrate specific MItochondrial STress Response (MISTR) circuit. MISTR consists of related micropeptides that interface with the electron transport chain (ETC). MISTR factors – MISTR1, MISTR Antiviral(AV)/C15ORF48, and MISTR (H)ypoxia are regulated by ultraconserved miRNAs and stress signals such as cytokines, infection, and hypoxia. MISTR factors are encoded by diverse viruses that infect a range of hosts from squirrels to algae; they are shown to be ancient as they are encoded by yeast but ill-defined in any system. We find that cellular MISTR factors shape chemical- and virus-triggered apoptosis, which is counteracted by a viral-MISTR. Emerging data link MISTR functions to inflammatory signaling via modulation of ETC Complex IV. We show that viral-MISTRs antagonize higher-order configurations of the ETC termed supercomplexes – whose precise function has remained of debate. Using viral systems to seed discovery, we identified new dimensions for textbook functions which implicate supercomplexes as an unappreciated battleground in immune defense.
Macrophage plasticity and activation states drive differential expression of murine macrophage galactose-type lectin (MGL) orthologs
Authors: Paez Reina N , Browning Sarah , Files Megan A , Varghese Alyssa A , Huante Matthew B , Strong Emily , Lee Sunhee , Endsley Mark A , Endsley Janice J
Published: August 2023

Macrophage plasticity and functional activation are critical to the broad array of macrophage-mediated functions that help determine disease outcomes. Macrophage galactose-type lectin (MGL), a type-2 C-type lectin receptor (CLR) primarily expressed on myeloid cells, has been shown to play an anti-inflammatory role in the innate response to some infectious agents. Our lab recently demonstrated that MGL-1 contributes an antimicrobial and anti-inflammatory role in a murine model of experimental tuberculosis. We further observe that MGL-1 and MGL-2 expression by RAW 264.7 macrophages are similarly activated by whole mycobacteria, but differentially regulated by signaling through toll-like receptors (TLR) as well as cytokine and glucocorticoid receptors. We find that synthetic agonists and microbial ligands of TLR2, TLR4, and TLR7 positively regulate MGL-2 expression, suggesting responsiveness to a broad repertoire of antigens compared to MGL-1. Additionally, activation of MGL-2 expression differs between ligands of multiple bacterial species, both pathogenic and non-pathogenic. Pathway inhibition experiments show that MGL-2 upregulation by microbial stimuli occurs through MyD88-dependent Toll-like receptor (TLR) signaling. Although both MGL-1 and MGL-2 are classically associated with alternatively activated macrophages, we show that MGL-2 expression is associated with both M1 and M2 polarized macrophages, while MGL-1 expression is restricted to M2. These results demonstrate a strong relationship between macrophage activation states and the differential activation of MGL signaling and indicate non-redundant roles of MGL-1 and MGL-2 in innate immunity of murine models of disease.

Supported by grants from NIH, NIAID (R61 AI138328, R33 AI138328)
Secondary cues alter ISG protein repertoires to unlock antiviral subprograms triggered by interferons
Authors: Hancks Dustin , Chang Tyron
Published: August 2023

Immune defenses are triggered by cytokines like interferon-α (IFN-α) and IFN-γ in diverse cells. Both IFNs induce a class of factors termed IFN-stimulated genes (ISGs). Many ISG proteins have antiviral functions. There is a growing appreciation that ISG repertoires are cell-type specific and that this bias impacts infection outcomes. However, the cues and mechanisms driving these variable responses are ill-defined. Given key roles for immunometabolism in immune cell activation, we hypothesized that local cues like metabolites shape tissue specific ISG defenses. To model the impact of metabolic cues on host defenses in non-immune cells, we infected human cells with two unrelated viruses: vaccinia virus, the prototypical poxvirus, and herpes simplex virus-1 (HSV-1). Unexpectedly, we find that cells favoring glycolysis, but not OXPHOS, catalyze potent IFN-γ restriction of vaccinia and HSV-1 that attenuate viral-gene and protein expression as well as infectious virus production. This response is specific as modulating IFN-α signaling by supplementation with either glycolysis or OXPHOS cues has no effect on viral replication. This interferon-induced subprogram is independent of differences in canonical IFN-γ signal transduction, occurs at low levels of IFN-γ, and induced by physiological levels of glucose. RNA-seq reveals no marked changes in the “usual suspects” (e.g. ISGs). Instead, we find secondary cues regulate ISG protein levels. Deletion of a single ISG protein, which is differentially stabilized by secondary cues, is sufficient to rescue vaccinia replication. These data indicate that secondary cues can alter infection outcomes in the same cell-type by unlocking subprograms embedded in interferon catalyzed responses.
DNA-PKcs regulates the cGAS-STING signaling axis during infection with intracellular bacterium Listeria monocytogenes
Authors: Morales Abigail , Dominguez Zuley A , Rodriguez Ricardo A , Morales Rosalie , Dilgen Teague C , Flores Melissa
Published: August 2023

The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS) and the ER-localized stimulator of interferon genes (STING) are critical components of innate immune signaling. Though the intracellular signaling pathway was first characterized as a critical host defense against viral infection, recent evidence has suggested that the pathway is also activated during infection with some types of bacteria, though the importance is complicated by the differential impact of type I interferon on the progression of distinct bacterial infections: in response to some species of bacteria, type I interferon production is detrimental, while it has a protective function during infection with others. DNA-PKcs is a critical kinase in the repair of and response to DNA double-strand breaks (DNA DSBs) in mammalian cells. Several recent reports have indicated that it also regulates the cGAS-STING signaling axis in virally-infected cells, suggesting that it may have previously unappreciated roles in regulating cytoplasmic signaling. Notably, we find that DNA-PKcs is required for optimal transcriptional induction of IFN-β and a number of other well-characterized interferon-stimulated genes (ISGs) in macrophages during infection with L. monocytogenes. Additionally, we find that phosphorylation of kinase TBK1 and activation of IRF3 are significantly diminished in Scid (DNA-PKcs−/−) macrophages relative to wildtype controls during infection with L. monocytogenes. As type I interferon is detrimental to effective Listeria clearance in murine infection models, these findings may provide an additional rational explanation as to why Scid mice clear Listeria more efficiently than their wildtype counterparts.