bluuebunny/crossref_metadata_2025 huggingface dataset:
Rational design of TGFbRII dominant negative armored GPC3 CAR-T cells for the treatment of hepatocellular carcinoma
Authors: Merlino Ashley , Tu Eric , Overstreet Michael , Gilbreth Ryan , Clarke Lori , Cobbold Mark , Moody Gordon , Giardino Letizia , Chu Nina
Published: August 2023

Chimeric antigen receptor (CAR)-T therapy has yielded impressive clinical results in hematological malignancies and it is a promising approach for solid tumor treatment. However, toxicity is a concern hampering its broader use. In selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we employed a strategy to manage the toxicity profile by lead selecting a single-chain variable fragment with reduced affinity characterized by a high dissociation rate. Compared to a high-affinity construct, the low-affinity CAR maintained cytotoxic function but showed reduced toxicity in a mouse model of hepatocellular carcinoma (HCC). The HCC microenvironment is rich of the immunosuppressive cytokine TGFβ, prompting us to armor the low affinity GPC3 CAR-T cells with a dominant-negative TGFβRII (TGFβRIIDN). In vitro, expression of TGFβRIIDN nearly abolished TGFβ-induced SMAD2/3 phosphorylation and reduced suppression of IL-2 and IFNg production. In a xenograft model of a human TGFβhigh HCC, armored CAR-T cells had 10/10 complete responders (CR) while the unarmored CAR-T had 4/10 CRs. In four TGFβhigh patient-derived xenograft models armored CAR-T cells achieved 60–90% tumor growth inhibition (TGI) while the unarmored CAR-T achieved no significant TGI. The armored CAR-T cells infiltrated TGFβhigh HCC tumors more abundantly than their unarmored counterparts and expressed lower levels of immune checkpoint markers PD1 and LAG3. In line with these observations, we detected significantly more IFNg at peak response, confirming in vivo functional superiority of TGFβRIIDN armored CAR-T therapy. These data support the clinical development of AZD5851, GPC3 CAR-T armored with TGFβRIIDN.
Single cell transcriptomic analysis of the ovarian immune microenvironment reveals hormone-dependent changes
Authors: Brodeur Tia Y , Krementsov Dimitry
Published: August 2023

The ovary is a dynamic organ, where, driven by the estrous cycle, cells undergo continuous waves of apoptosis, proliferation, and differentiation. These changes parallel fluctuations in ovarian hormones such as estradiol (E2), however, the immune microenvironment during high-E2 and low-E2 states has not been described. We induced a high-E2 state by superovulation with i.p. injection of gonadotropins (GN) into 10 week old C57BL/6 mice. Flow cytometry and single-cell RNA sequencing revealed abundant NK cells, B cells, CD8+ T cells, CD4+ T cells, CD3+TCRβ+CD4−CD8− “double negative” T cells, and several distinct myeloid subsets. The frequency of NK cells in the CD45+ compartment was 14% in GN-treated mice and 7% in controls, suggesting increased proliferation or recruitment of NK cells in response to E2. Conversely, mature B cells represented 8% of CD45+ cells in GN-treated mice and 18% in controls. To determine whether CD45+ cells were recruited from circulation or ovarian resident cells, we injected fluorescently-labelled α-CD45 i.v. into live mice and performed flow cytometry on ovaries, spleen, and blood. The majority (88–96%) of ovarian CD45+ cells were unlabeled, suggesting that they were ovary-resident. To assess population dynamics during the naturally occurring low-E2 state of reproductive aging, we performed flow cytometry on ovaries of 40 week old mice. Interestingly, we identified significant increases in the number of macrophages and double-negative T cells in aged ovaries. Our findings reveal a diverse resident immune landscape in the ovary that responds robustly to hormonal changes. These findings may have implications for immune-mediated causes of infertility such as primary ovarian insufficiency and endometriosis.

Support by a grant from the NIH 3P30GM118228-05S3
Maternal high fat diet exposure results in MyD88 dependent alteration in placental and offspring intestinal fatty acid transporters
Authors: Jaleel Sana , Niu Xinying , Lu Dongmei , Mirpuri Julie
Published: August 2023

Introduction

We know that offspring born to parents with obesity are more likely to be obese. We hypothesized that the maternal high fat diet (mHFD) exposure may increase placental and offspring small intestinal transport of fatty acids. We hypothesized both these processes would be dependent on the presence of MyD88.

Methods

Wild-type (WT) and MyD88 deficient mice on either 60% mHFD or regular diet (RD) were profiled. The baseline expression of fatty acid transport proteins FATP1 and FATP4, fatty acid binding proteins FABP1, FABP4, and CD36 were profiled by qRT-PCR from placenta after C-section at E20, and offspring small intestine 2-weeks after birth. Serum from 0-day old RD and HFD WT mice were collected and analyzed using high-throughput, non-targeted mass spectroscopy based metabolic analysis.

Results

In WT mice, we found FATP1 was increased 32-fold and FABP was increased 7-fold in the mHFD placenta. FATP2 was decreased 2-fold and FATP4 was decreased 2.5-fold in the mHFD offspring small intestine. In MyD88 KO mice, we found no difference in any of the above fatty acid transporters. Serum of neonatal pups showed unique clustering of metabolites by PCA analysis in HFD and RD mice, with increased butanoate and propanoate metabolites in mHFD offspring.

Conclusion

Fatty acid transport regulators in the placenta and offspring small intestine were modified by exposure mHFD. In both the placenta and small intestine, this outcome was dependent on the presence of MyD88. Fatty acid metabolites were increased in the serum of neonatal pups exposed to mHFD. These findings suggest that mHFD exposure can modulate fetal metabolism and may be an opportunity for future development of therapeutics that target placental and intestinal transporters.
Regulation of myeloid cells and inflammation marks increased susceptibility in a genetic model of acute viral infection-induced tissue damage
Authors: Annis Jessica L , Crittenden Rowena B , Brown Michael G
Published: August 2023

M.H2k/b mice are identical to MA/My parent strain aside from a C57L-derived region within the MHC, which causes these mice to develop significant splenic tissue damage during acute murine cytomegalovirus (MCMV) infection. To understand how MCMV infection leads to the induction of tissue damage, we probed immunological differences between the M.H2k/b and MA/My mice over time. Using tissue imaging and flow cytometry, we observed increased infiltration of neutrophils to the marginal zones of the M.H2k/b mice aligning with the appearance of necrosis at 2 days post infection (dpi). While TUNEL staining identified increased cell death within these clusters, apoptosis was reduced, suggesting an inflammatory death pathway in the M.H2k/b neutrophils. In line with this hypothesis, multiplex ELISA identified increased IL-6 and TGF-β in the spleens of M.H2k/b mice at this time. Intriguingly, depletion of neutrophils prior to infection did not alleviate M.H2k/b histopathology or inflammation. Seeing the differential regulation of neutrophils, we assessed the state of other myeloid populations in the spleen during acute MCMV infection. We observed significant loss of marginal zone and red pulp macrophages (MZMs, RPMs) beginning at 2dpi and worsening over time in the M.H2k/b mice. Further exploration into how the loss of these populations may contribute to tissue damage identified buildup of toxic lipid peroxidation byproduct 4HNE early in the MZMs and progressing through the RPMs alongside the development of histopathology. In conclusion, we have identified increased inflammation, oxidative stress, and macrophage loss in M.H2k/b spleens during acute MCMV infection that tracks with localization and temporal appearance of histopathology.

NIH NIAID R01AI50072, Department of Medicine/Nephrology, Center for Immunity Inflammation and Regenerative Medicine, Beirne B Carter Center for Immunology Research.
Lupus-like phenotypes emerge from loss of macrophage derived Dnase1L3
Authors: Infante Angelica M , Engavale Minal B , Keyel Peter A
Published: August 2023

One leading cause of death in women is autoimmunity. One prominent autoimmune disease is Systemic Lupus Erythematosus (lupus). During lupus, inflammation is driven by autoantibodies that target cell-free DNA. Cell-free DNA is degraded by two serum endonucleases, Dnase1 and Dnase1L3. The loss of Dnase1L3 causes lupus in humans and mice, but the amount of Dnase1L3 necessary to prevent lupus onset is unknown. Since DnaselL3 is secreted by macrophages and dendritic cells, we hypothesized that loss of macrophage-derived DnaselL3 is sufficient to cause lupus-like phenotypes in mice. We generated conditional knockout (cKO) mice lacking DnaselL3 expression in macrophages. Sera was collected weekly from these mice until 50 weeks of age. To measure autoantibody induction, total IgG, total IgM, anti-dsDNA and anti-nuclear antibody levels were analyzed at 4, 20, 30 and 50 weeks. Sandwich ELISA was used to quantitate antibody levels in control and cKO mice. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgG, anti-ds DNA antibody levels increased in cKO mice. We conclude that SLE-like phenotypes arise in mice due to the loss of macrophage Dnase1L3. This suggest that macrophage derived DnaselL3 is critical to controlling lupus.

Plains Bridges to the Baccalaureate Program (PBB) (NIH 2R25GM83730-B)
Syk-Dependent Responses and Innate Cytosolic Defenses in Orientia tsutsugamushi-infected Macrophages
Authors: Soong Lynn , Fisher James R , Gonzales Casey , Liang Yuejin
Published: August 2023

Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. Human studies and animal models of scrub typhus have shown robust type 1-skewed proinflammatory responses during severe infection. Macrophages play a critical role in initiating such responses, yet mechanisms of innate recognition for O. tsutsugamushi remain unclear. In this study, we investigated whether Syk-dependent C-type lectin receptors (CLRs) contribute to innate immune recognition and the generation of proinflammatory responses. To validate a role of CLRs in scrub typhus, we infected murine primary macrophages with O. tsutsugamushi in the presence of selective Syk inhibitors and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that Mincle/Clec4a and Clec5a transcription was significantly abrogated upon Syk inhibition at 6 hours of infection. The effect of Syk inhibition on Mincle protein expression was validated via western blot. Syk-inhibited MF had diminished expression of type 1 cytokines and chemokines (Il12p40, Tnf, Il27p28, Cxcl1) during infection. Additionally, expression of innate immune cytosolic sensors (Mx1 and Oas 1–3) was highly induced in the brain of lethally infected mice. We established that Mx1 and Oas1 expression was reduced in Syk-inhibited MF, while Oas2, Oas3, and MerTK were not sensitive to Syk inhibition. This study reveals that Syk-dependent CLRs contribute to inflammatory responses to O. tsutsugamushi. It also provides the first evidence for Syk-dependent activation of intracellular defenses during infection, suggesting a role of pattern recognition receptor crosstalk in orchestrating macrophage-mediated responses to this poorly studied bacterium.

Supported by the NIAID grants (R21 AI156536, R01 AI132674, and T32 AI007526). JF was the recipient of a NIAID Emerging and Tropical Infectious Diseases T32 fellowship.
Neutrophils use a novel NLRP3 inflammasome-independent mechanism to release bioactive IL-1β in response to lysosomal disruption
Authors: Miller Brandon A , Pearlman Eric , Dubyak George R
Published: August 2023

IL-1β is an inflammatory cytokine mainly secreted by myeloid cells in response to infection or sterile tissue damage. Non-canonical secretion of IL-1β from macrophages downstream of activated NLRP3/caspase-1 inflammasomes is the best-characterized model; this is mediated by caspase-1 cleavage of GSDMD allowing N-GSDMD to form pores in the plasma membrane that act as conduits for IL-1β release and inducers of pyroptosis as a lytic cell death. The NLRP3 initiator acts as a sensor of perturbed cellular homeostasis including decreased cytosolic [K+]. In macrophages, this K+ efflux mediated NLRP3 activation is also triggered by agents that disrupt lysosomal integrity. While neutrophils also assemble competent NLRP3 inflammasomes and release bioactive IL-1β via GSDMD-dependent mechanisms, they resist the formation of plasma membrane N-GSDMD pores and progression to pyroptosis. We tested whether lysosome disrupting stimuli in neutrophils would phenocopy macrophage mechanisms to secrete IL-1β in an NLRP3 and GSDMD-dependent manner. Surprisingly, our data indicate that lysosome-disrupting stimuli induce neutrophils to release mature IL-1β and undergo rapid lytic cell death independently of NLRP3 inflammasome assembly and caspase-1 activity with only partial dependence on GSDMD. Ongoing studies testing effects of serine protease inhibitors support a mechanism in which cytosolic accumulation of the multiple serine proteases stored in the lysosome-like azurophilic granules coordinately disrupt canonical NLRP3 inflammasome assembly and directly cleave proIL-1β and GSDMD as part of a novel neutrophil-specific signaling mechanism for lysosomal disruption-induced processing and export of IL-1β.

P01-AI141350 R01-EY014362 R21-EY032662
Myeloid Plasticity of Neutrophil Precursors Contributes to Eosinophil Emergency Hematopoiesis
Authors: Haruna Nana-Fatima D , Politanska Yuliya , Abdala-Valencia Hiam , Berdnikovs Sergejs
Published: August 2023

Eosinophils and neutrophils are heterogenous cells that play diverse roles in homeostasis and inflammation. Tissue eosinophil origins are poorly understood, posing challenges in the functional interpretation of heterogeneous phenotypes. New evidence from our lab shows unexpected hematopoietic plasticity of Ly6G(+)IL-5Rα(+) band-stage neutrophils with potential to contribute to eosinophil lineage. Using flow cytometry and multi-omics approaches in mouse and human tissue samples derived from healthy and allergic conditions, we tested the hypothesis that neutrophil plasticity contributes to eosinophil diversity in inflammation. We found at least two distinct populations of neutrophils at a healthy baseline, one of which expressed classical neutrophils markers (S100A8/9) while the other expressed both neutrophil (MPO, ELANE) and eosinophil-specific (EPX, PRG2) granule proteins, indicating an intermediate hematopoietic state. In human and mouse neutrophils treated with IL-5/G-CSF in vitro, a subset with mixed characteristics acquired eosinophil-specific granular proteins and eosinophil cell surface markers (Siglec8, CCR3). In a mouse asthma model, using scRNA-seq, we found overlap in neutrophils and eosinophils with tissue remodeling functions. We also identified activated neutrophils and eosinophils playing unique roles in inflammation. Our findings implicate eosinophil emergency hematopoiesis driven by neutrophil plasticity as an overlooked process driving persistence of tissue eosinophils in inflammation.

Supported by grants from GSK ISS and NIH R01
Epithelial TLR9 upregulation by respiratory viruses impairs pulmonary antibacterial immunity
Authors: Rich Helen Eva Adams , Bhutia Simran , Martínez-Colón Giovanny , Warheit-Niemi Helen , Bame Monica , Moore Bethany
Published: August 2023

While much is known about signaling downstream of the dsDNA sensor TLR9, regulation of its expression is underexplored and has significant consequences for innate immune responses to pathogens. Mice lacking TLR9 surprisingly fare better upon coinfection with influenza and Staphylococcus aureus, and other groups have shown protection from S. aureus alone as well. These TLR9KO mice display lower inflammatory cytokine production, reduced cellular infiltration into airspaces, and increased survival. While macrophages taken from influenza-infected TLR9KO mice display increased bacterial phagocytosis and killing, inhibition of TLR9 on wild-type murine macrophages ex vivo surprisingly decreased their ability to phagocytize and kill S. aureus. Bone marrow chimera experiments revealed that lack of TLR9 in non-hematopoietic cells was responsible for the protection from bacterial super-infection seen in whole-body TLR9KO mice. To assess which structural cell types were important for this protection, we bred epithelial- and endothelial-specific TLR9KO mice, which revealed a role for epithelial cells but not endothelium. Interestingly, we also found that influenza infection caused upregulation of TLR9 in epithelial cells. Moreover, infection with two other respiratory viruses also caused epithelial cells to increase TLR9 expression, as did treatment with the antiviral mediator interferon beta (IFNB). IFNB has long been known to regulate susceptibility to bacterial coinfection during influenza, and these results suggest that it does so in part by upregulating TLR9. Together, these data show a role for epithelial TLR9 upregulation, likely driven by IFNB, in the pathogenesis of respiratory viral/bacterial coinfection.

Funding for this research was made possible in part by the Michigan Postdoctoral Pioneer Program at the University of Michigan Medical School, and by NIH grants NHLBI T32HL007517 (Helen Rich) and NHLBI R35HL144481 (Beth Moore).
Defining how Antibiotic Treatments shape Host Immunity
Authors: Gross Julia , Basu Rahul , Bradfield Clinton , Sun Jing , Moyer Tom , Smelkinson Margery , Antonioli-Schmit Sophia , Fischer Elizabeth , Dekker John , Weiss David , Fraser Iain D C
Published: August 2023

Although antibiotics are clinically crucial, little is known about how they impact a patient’s immune response to infection. We address that gap by investigating how macrophages respond to bacteria treated with different classes of antibiotics. We treated a broadly susceptible E. coli strain with 8 separate antibiotics, infected macrophages, and compared how much TNF the various antibiotic-treated bacteria elicited from macrophages. We found that bacteria treated with cidal antibiotics (that kill bacteria) elicited ~2–3 fold more TNF than macrophages infected with equivalent amounts of bacteria treated with bacteriostatic (growth limiting) antibiotics. A panel of 12 clinical isolates from patients (4 each of E.coli, Enterobacter and Klebsiella) showed a similar pattern of antibiotic class-dependent inflammatory cytokine output. We then evaluated an in vivo IP injection model with antibiotic pre-treated bacteria, heat-killed bacteria, or vehicle alone. At two hr post infection, we saw ~35–200% increases in serum levels of pro-inflammatory cytokines induced by cidal-treated bacteria compared to static-treated bacteria. Consistent with prior data, both the vehicle and the killed bacteria elicited no inflammatory cytokines. Preliminary imaging data suggest that treatment with the cidal drugs causes most of the dead bacteria to form large aggregates, while treatment with the static drugs does not. We now aim to clarify the role of these aggregates in driving this inflammatory outcome. This may identify additional patterns of immune responses to antibiotic treated bacteria that could guide the development of improved antimicrobial treatment strategies. This work is supported by the Intramural Research Program of NIAID, NIH.

Supported by Intramural Research Program of NIAID, NIH.
Deep phenotypic and functional characterization of NK cells during NK-mediated cytotoxicity
Authors: Nickle Rebecca , Chou Lisa , Bhattacharya Sam , Tam Miguel , Wiethe Carsen
Published: August 2023

Natural Killer (NK) cell-mediated cytotoxicity is a critical mechanism of the immune system to respond against harmful stimuli. The development of emerging cell therapies, such as CAR-NK cells, requires a deeper characterization of cultured NK cells. Here we show a phenotyping of ex vivo and in vitro NK cells, and demonstrate how high quality and reliable reagents from BioLegend ensure a more in depth cytotoxic analysis. NK cells were isolated from fresh human PBMCs, using our MojoSort™ Human NK Cell Isolation Kit, and incubated at different ratios with Tag-it Violet™ stained K562 target cells in IMDM + BioLegend’s Cell-Vive™ T-NK Xeno-Free Serum Substitute, in the presence of CD107a-PE. Cells were then harvested for surface phenotyping and the supernatants were collected for cytokine analysis using LEGENDplex™ Human CD8/NK Panel. Tag-it Violet™ and Apotracker™ Green staining of K562 cells demonstrate significant decrease in proliferation and significant increase in apoptosis, respectively. Surface staining of CD107a reveals increased degranulation of both CD56bright CD16− and CD5 + CD16+ NK cells that correlates with increased apoptosis of K562 cells. Supernatant cytokine analysis revealed significant increases in Granzyme A and B, Perforin, and Granulysin, among others. Taken together, these results show a more complete picture of the phenotypical and functional state of NK cells by measuring target cell killing as well as characterizing NK surface markers and secreted molecules involved in NK-mediated cytotoxicity. BioLegend reagents are designed to address ever increasing needs for deeper understanding of immunological processes and support the demand to develop sophisticated and consistent cell-based therapies.
Longitudinal analysis reveals age-dependent immune responses to SARS-CoV-2 infection in a mouse model
Authors: Qian Wei , Wei Xiaoqin , Mann Barbara J , Sun Jie
Published: August 2023

Aging is a prominent risk factor for COVID-19 disease severity and mortality in humans. To gain insight into the age-associated alterations of host responses, we performed flow cytometry and single-cell RNA sequencing (scRNA-seq) on lungs and bronchoalveolar lavage (BAL) from young and aged wildtype C57BL/6 mice at days 0, 3, 10, and 21 after infection with the mouse-adapted SARS-CoV-2 strain MA10. We revealed that disease severity and infection kinetics were age-related, as reported in humans, with increased morbidity and mortality in aged mice. During the acute phase of infection, aged animals had a higher local inflammatory response than young mice, as evidenced by the rapid accumulation of neutrophils and inflammatory monocytes in the lung parenchyma and BAL. During the post-acute phase of disease, these features further divided, with aged mice displaying prolonged proinflammatory responses, as well as deficits in cellular and humoral responses. Using scRNA-seq, we identified 33 clusters in the lung. Differential gene expression revealed several age-dependent and dynamic changes within various immune, epithelial, and endothelial cell types. Cell-cell interactions following infection are also altered in the lung by aging. Taken together, our findings suggest that advanced age is associated with a prolonged inflammatory state, poorly induced adaptive immune responses, and altered cell-cell interactions following SARS-CoV-2 infection.
SARS-CoV-2 Delta requires human ACE2 but not human TMPRSS2 to infect mice and elicits greater lung injury and adaptive immune response than Omicron in human ACE2 knock-in mice
Authors: Verma Shailendra Kumar , Ana-Sosa-Batiz Fernanda , Shafee Norazizah , Timis Julia , Maule Erin , Miller Robyn , Pinto Paolla D A , Valentine Kristen , Conner Chris , Webb Devid , Jarnagin Kurt , Kim Kenneth , Shresta Sujan
Published: August 2023

SARS-CoV-2 pathogenesis remains poorly understood in large part due to lack of knowledge about the mechanisms of entry and behaviors of SARS-CoV-2 variants in vivo. We generated single and double knock-in (KI) mice expressing human ACE2 (hACE2) and/or human TMPRSS2 (hTMPRSS2) under endogenous promoters in place of murine ACE2 (mACE2) and TMPRSS2 on the C57BL/6 and BALAB/c genetic backgrounds to evaluate Delta vs Omicron BA.1 infection, disease, and immune response. Delta replication was observed in lungs of mice expressing hACE2 but not hTMPRSS2 or mACE2 following intranasal inoculation, and similar levels of Delta replication were observed in lungs of hACE2-KI and hACE2xhTMPRSS2-KI mice. Thus, Delta requires hACE2 but not hTMPRSS2 to infect mice. In contrast, BA.1 established similar levels of replication in lungs of single and double KI mice, demonstrating that BA.1 requires neither hACE2 nor hTMPRSS2 to infect mice. Although no significant differences in viral burden were observed in hACE2-KI mice infected with Delta vs BA.1, Delta-infected hACE2-KI mice exhibited increased histopathologic lung injury and higher SARS-CoV-2-specific CD4+ and CD8+ T cell responses (spleen) and anti-SARS-CoV-2 spike IgG titers (serum). Additionally, hACE2-KI mice on the C57BL/6 background showed more severe lung disease and stronger Th1 response than BALB/c. These results associate the severity of lung disease with the magnitude of Th1-dominant immune responses, and set a foundation for dissecting mechanisms of COVID-19 pathogenesis in hACE2-KI mice representing Th1- vs Th2-dominant genetic backgrounds.
Multi-omic spatial profiling in COVID-19 lung revels temporal evolution of the disease and novel collagen VI biomarker for outcome
Authors: Pugh Matthew R , Fennel Éanna , Leahy Ciara , Ross Aisling M , Reynolds Gary , Perry Tracey , Youd Ester , Leopold Gareth , Taylor Graham S , Murray Paul G
Published: August 2023

Whilst the peripheral immune response to COVID-19 infection is well characterised, less is known about the tissue response in the lungs of affected patients. We conducted a multi-omic investigation in post-mortem lung tissue from fatal COVID-19 infection comprising bulk Quantseq RNA sequencing, spatial transcriptomic profiling on the Nanostring GeoMX, multiplex immunohistochemistry on the Lunaphore COMET and in-situ transcriptomcics with RNAscope. We found the SARS-CoV-2 virus was progressively cleared in more temporally advanced disease. Furthermore, collagen VI was found to be transcriptionally up-regulated by bulk sequencing and within the interstitium on spatial transcriptomics. These findings were supported by multiplex immunohistochemistry at a proteomic level which showed fibrillar deposition of collagen VI in the interstitum. To validate these findings clinically, we looked at blood markers for collagen VI synthesis and degradation by ELISA in a cohort of severe and mildly infected COVID-19 patients. Blood markers of collagen VI synthesis (PRO-C6) was significantly predictive for mortality in hospitalised (p = 0.0065) and intensive care (p = 0.028) COVID-19 patients. Overall, blood PRO-C6 levels, based on increase interstitial lung deposition, could help identify high-risk of patients enabling better treatment stratification. The study also demonstrates the power of post-mortem investigation and spatial profiling in elucidating clinically relevant insights into complex disease phenomena.
Anamnestic expansion of Omicron-reactive CD8+ T cells after booster SARS-CoV-2 mRNA vaccination across different immunocompromised states
Authors: Müller Thomas R , Sekine Takuya , Trubach Darya , Niessl Julia , Olofsson Anna , Gao Yu , Cai Curtis , Nowak Piotr , Blennow Ola , Hansson Lotta , Mielke Stephan , Bergman Peter , Ljunggren Hans-Gustaf , Karlsson Annika C , Saini Sunil K , Aleman Soo , Buggert Marcus
Published: August 2023

Restricted immune responses to SARS-CoV-2 mRNA vaccination have been observed frequently in individuals suffering from various immunodeficiencies. With increased antibody evasion properties of rising Omicron subvariants, there is a need to assess if other components of adaptive immunity generate resilient responses against SARS-CoV-2 across immunodeficient states. Here we assessed T cell responses of n=279 individuals covering five different immunodeficiencies and healthy controls before and after booster vaccination and - in a subset of patients - additional Omicron infection. We observed significantly increased Omicron-reactive T cell responses upon booster vaccination, particularly in initial poor responders, across all patient groups. Overall T cell composition shifted towards an increased CD8/CD4-ratio, which was accompanied by a more pronounced cytotoxic profile. Moreover, Omicron-reactive T cell responses showed signs of longevity represented by a novel TEMRA subpopulation with stem-cell-like characteristics and increased proliferative capacity. Regardless of underlying immunodeficiency, booster-vaccinated and Omicron-infected individuals were protected against severe disease and exhibited ameliorated and diversified T cell responses directed explicitly against Omicron, despite a solid immunological imprint of multiple wildtype Wuhan-based vaccine doses.

Supported by grants from SciLifeLab National COVID-19 Research Program and financed by the Knut and Alice Wallenberg Foundation, the Swedish Research Council, Nordstjernan AB, Region Stockholm (Clinical research position-, ALF-, and CIMEDgrants). TRM is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, project: 496946670).
Identifying Epitope Sites on the Receptor Binding Domain (RBD) of SARS-CoV-2
Authors: Jiang Jiansheng , Boughter Christopher T , Ahmad Javeed , Byod Lisa F , Natarajan Kannan , Meier-Schellersheim Martin , Margulies David H
Published: August 2023

COVID-19 and SARS-CoV-2 variants continue to threaten human health and life worldwide. Thousands of structures related to SARS-CoV-2 have been rapidly determined, either by X-ray crystallography or CryoEM and deposited in the Protein Data Bank (PDB) since 2020. Here, we systematically investigated the structures of 302 antibodies and 78 nanobodies in complex with spike protein or the receptor binding domain (RBD) of SARS-CoV-2. We identified 23 common epitopic sites (ES) on the RBD surface and revealed the vital role of the complementarity-determining Region (CDR) loops in recognizing epitopes. The 23 ES are characterized according to the secondary structure feature and accessible surface area. About 75% of the total surface of RBD areas could access by the elicited antibodies, while the CDR3 loops occupied 50% of the contact surface. The analysis of paratope-epitope (antibody-antigen) interaction based on these epitope sites revealed the features of potent neutralizing the virus and the unique usage of amino acids of antibody. The clustering analysis of the antibody biophysical properties and surface area availabilities on the RBD found many binding motifs. Remarkably, most variants of concern (VOC) escape mutations, including Omicron occur within these 23 ES. This analysis not only explains the differential ability of antibodies to recognize epitope sites on the RBD surface but also offers predictive guidelines for understanding the role that accumulated SARS mutations might play in escape from antibodies elicited by immunogens. This structural characterization of the epitope-paratope interactions provides insights for structure-based vaccine design and therapeutic strategies and drugs against the future virus.
Inflammatory and immune response signatures associated with COVID-19 severity
Authors: Gibson David S , McLaughlin Joseph , McDaid Darren , Lynch Seodhna , Okane Maurice , Kelly Martin , Bhavsar Manav , McGilligan Victoria , Shukla Priyank , Zhang Shu-Dong , Murray Elaine , Rai Taranjit Singh , Walsh Colum , Bjourson Anthony J
Published: August 2023

Severe COVID-19 is characterised by an overactive pro-inflammatory response of the immune system which is associated with new persistent symptoms. Differences in the persistence of symptoms, plasma inflammatory proteins and antibodies between hospitalised and non-hospitalised cases were investigated in the current study.

n=120 participants were recruited with informed consent, of whom n=60 were hospitalised with severe disease. Blood samples and detailed symptom data were collected 3 months after a positive PCR test for SARS-CoV-2. Plasma samples were analysed by OLINK Explore 384 inflammation panel and ACE2 neutralisation and IgG assays. R software was used to identify statistically significant differences in protein concentration in hospitalised cases, relative to non-hospitalised cases. Differences in anti-SARS-CoV-2 IgG antibody concentration, ACE2 neutralisation and symptoms were also analysed between hospitalisation subgroups.

14 proteins including angiopoietin-like protein 2 and C-X-C motif chemokine 17 were significantly elevated (by greater than +/− 0.20 log2 fold change; p < 0.05) at 3 months in the hospitalised study group, relative to non-hospitalised cases. The concentrations of BA.2 and BA.3 specific anti-SARS-CoV-2 IgG and spike receptor binding domain neutralising antibodies were more frequently detected in non-hospitalised cases. The hospitalised cohort more frequently reported persistence of muscle pain, headache, fatigue and shortness of breath.

Characteristic changes in the plasma proteome and symptom clusters that persist 3 months after infection have been observed in hospitalised COVID-19 cases. This data could help inform clinical care requirements for longer term COVID-19 recovery.

Supported by grants from UKRI, SFI and HSC R&D Office
RORα plays an important role in generation and maintenance of memory B cells
Authors: Xu Yijiang , Moroney Justin , Zhou Yulai , Zan Hong , Casali Paolo
Published: August 2023

Memory B cells (MBCs) are antigen-specific and long-lived. Upon reactivation, they can differentiate into plasma cells to secrete large amounts of high-affinity mostly class-switched antibodies. Prompted by our integrative analysis of the human MBC transcriptome and chromatin landscape which redundantly identified the transcriptional factor RORα as central to the identity of human class-switched (sw)MBCs, we investigated the contribution of RORα to the generation and maintenance of MBCs. We found that RORα is selectively expressed at a high level in both human and mouse swMBCs. We then constructed genetically modified AicdacreRorafl/fl mice, in which RORα is specifically deleted in activated B cells undergoing class switching. Upon immunization with NP-CGG, AicdacreRorafl/fl mice significantly reduced the generation of NP-specific swMBCs as part of a primary response otherwise without perturbation of germinal center formation, class-switch DNA recombination, somatic hypermutation or plasma cell differentiation, but leading to a defective anamnestic anti-NP-antibody response. To define the role of RORα in MBC maintenance, we generated tamoxifen-inducible B cell RORα conditional knockout CD19cre/ERT2Rorafl/fl mice, in which RORα could be ablated in swMBCs already generated in response to immunization with NP-CGG. RORα ablation in swMBCs led to loss of swMBCs and the anamnestic anti-NP-response. Thus, B cell RORα plays an important role in not only the generation but also the maintenance of swMBCs. Further mechanistic insight in swMBC generation and maintenance will be provided by Cut&Tag and RNA-Seq experiments to identify sites of RORα enrichment across the genome and transcriptional targets of RORα in swMBCs.

Supported by NIH grants AI 079705, AI 105813, AI 167416 and LRA grant 641363 to PC.
The Ikaros zinc finger transcription factor Eos promotes TH2 differentiation and function by propagating IL-2/STAT5 signaling
Authors: Tuazon Jasmine , Read Kaitlin , Sreekumar Bharath , Yaeger Michael , Varikuti Sanjay , Jones Devin , Warren Robert , Powell Michael , Rasheed Mustafa , Duncan Elizabeth , Childs Lauren , Gowdy Kymberly , Oestreich Kenneth
Published: August 2023

The Ikaros zinc finger transcription factor Eos has largely been associated with chromatin regulation promoting immunosuppressive regulatory T cells. However, Eos’ role in the differentiation and function of pro-inflammatory T cells has remained unclear. Surprisingly, our work reveals that Eos is a positive regulator of CD4+ T helper 2 (TH2) cells—effector T cells that participate in anti-helminthic immunity but are also implicated in inducing allergic asthma. Using in vitro-generated TH2 cells and an in vivo allergic asthma mouse model, we found that Eos-deficient T cells had reduced gene and protein expression of critical TH2 transcription factors (including the lineage-defining transcription factor Gata3), effector cytokines, and differentiation receptors. Among the various TH2-polarizing pathways, the IL-2/STAT5 axis and its downstream TH2 gene targets emerged as one of the most significantly downregulated networks in Eos deficiency. Using in vitro-generated TH2 cells and overexpression of Eos zinc-finger-domain mutants, we discovered that Eos forms a novel complex with and promotes the tyrosine-phosphorylated activation of STAT5. Additionally, we showed that components of the IL-2/STAT5 pathway participate in a feed-forward loop to promote Eos expression in TH2 cells, further supporting Eos’ regulatory connection with the IL-2/STAT5 axis. Together, these data define a novel mechanism whereby Eos mediates IL-2/STAT5 activity to facilitate TH2 differentiation. This work is significant, as its findings reframe our understanding of Eos’ role in T cell differentiation.

K.J.O. is supported by a grant from The National Institutes of Health (NIH) AI134972, as well as from The Ohio State University College of Medicine and The Ohio State University Comprehensive Cancer Center. J.A.T. is supported by funding through the Susan Huntington Dean’s Distinguished University Fellowship and the NIH T32 “Interdisciplinary Program in Microbe-Host Biology” pre-doctoral fellowship administered through the OSU Infectious Diseases Institute and OSU Department of Microbial Infection and Immunity. K.M.G. is supported by NIH grant 1R01ES028829-01A1. L.M.C. and K.J.O. were supported in part by the Jeffress Trust Awards Program in Interdisciplinary Research. K.A.R. is supported by funding through The Ohio State University College of Medicine Advancing Research in Infection and Immunity Fellowship Program.
Exposure of autoimmune uveitis-prone germ-free mice to commensal microbiota promotes development of ocular inflammation
Authors: Murphy Caitlin Elizabeth , Horai Reiko , Zhang Amy , Jittayasothorn Yingyos , Nagarajan Vijayaraj , Caspi Rachel R
Published: August 2023

Autoimmune uveitis is a group of intraocular inflammatory diseases driven by retina-specific T cells that target the neuroretina and is one of the leading causes of blindness. Using a spontaneous mouse model of autoimmune uveitis (R161H), in which CD4 T cells express a transgenic retina-specific T cell receptor, we have shown that gut commensal microbiota are involved in activating retina-specific T cells and promoting uveitis. To study how a germ-free (GF) host responds to de novo microbiota exposure, R161H TCRa−/− mice, rederived into GF conditions and developing much milder disease than their SPF counterparts, were cohoused with SPF mice for 4 wks (exGF). Fecal samples were collected weekly to examine changes in the gut microbiome by 16S amplicon sequencing. Disease progression was monitored twice weekly by fundus examination. At the endpoint, eyes were collected for histopathology, and T cells from lymph nodes, eyes, and intestinal lamina propria were analyzed by flow cytometry and RNAseq for immunophenotype and gene expression.

Exposure to commensal microbiota strongly promoted disease development. After 2 wks of cohousing, exGF disease scores reached the severity of age-matched SPF mice and were significantly higher than those of control GF mice. Flow cytometry analyses revealed an increased percentage of CD4 T cells expressing the proinflammatory cytokines IL-17A and IFN-g in exGF mice. 16S analyses showed that by 1 wk of cohousing exGF mice had acquired gut microbiota of SPF mice, which we hypothesize to include microbes affecting disease outcomes. RNAseq data will provide insights into phenotypic differences among retina-specific T cells in different tissues and may uncover novel pathways involved in uveitis development.
Gut microbiome-derived isoflavone metabolites may ameliorate experimental autoimmune encephalomyelitis (EAE) through estrogen receptor signaling in CD8 T-cells
Authors: Peterson Stephanie R , Jensen Samantha , Mangalam Ashutosh K
Published: August 2023

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS which may be influenced by diet- and gut bacteria-produced metabolites. We have shown that phytoestrogen-metabolizing bacteria are reduced in MS patients compared to healthy individuals. The importance of phytoestrogen-metabolizing bacteria was validated in EAE, an animal model of MS, where lack of isoflavones (a type of phytoestrogen) in the diet and/or lack of phytoestrogen-metabolizing bacteria lead to increased disease severity. However, the cellular and molecular pathways through which isoflavones/gut microbiota modulate EAE is unknown. Cellular analysis of the gut and periphery showed that isoflavone-fed mice have an increased number of CD8 T-cell in the colon compared to mice on an isoflavone-free diet. Moreover, CD8 T-cell knockout mice lose isoflavone-mediated disease protection, suggesting an important role of CD8 T-cells in this process. Due to the structural similarity between isoflavone metabolites and endogenous estrogen, we hypothesize that isoflavone metabolites may induce regulatory CD8 T-cells via engagement of estrogen receptors (ERs). Flow cytometric analysis of colonic CD8 T-cells showed that isoflavone-fed mice had higher expression of both ERα and ERβ and produced lower levels of IFNγ, perforin, and IL10. We are utilizing conditional KO mice lacking ERα or ERβ exclusively in CD8 cells to determine the importance of ERs in the generation of diet-induced CD8 T-cells. In summary, our data suggest that bacterial metabolism of isoflavones may lead to EAE disease suppression through immunomodulation of CD8 T-cells via estrogen receptor signaling.

Supported by grants from NIH (1R01AI137075-01) and VA (1I01CX002212)
Comparison of the effect of autoclaved and non-autoclaved soil exposure on mouse immune system
Authors: Gonzalez-Rodriguez Martin Ignacio , Kummola Lura , Salomaa Tanja , Nurminen Noora , Hiihtola Lotta , Marnila Pertti , Laitinen Olli , Sinkkonen Aki , Hyöty Heikki , Junttila Ilkka
Published: August 2023

The biodiversity hypothesis suggests that decreased interaction with microbes can lead to an imbalance in the human microbiota (referred to as dysbiosis), which in turn will contribute to the development of immune-mediated diseases including asthma, allergies, and autoimmune diseases such as type 1 diabetes. We have previously utilised a novel approach of bringing humans and mice into contact with microbially rich plant- and soil-based material. In humans, exposure to this material increased commensal microbial diversity in daycare children which also linked to higher levels of anti-inflammatory cytokines TGF-b and IL-10 in plasma. Here, we compared the effects of autoclaved and non-autoclaved, fresh soil powder on the mouse immune system, to understand if soil-derived material contains molecular structures that convey the immunoregulatory signals. These effects were evaluated phenotyping immune populations by flow cytometry, assessing cytokine profiles in serums and microbiome diversity by genetic sequencing. Our results indicate that exposure to non-autoclaved soil did not influence the health of mice but was associated with elevated serum IFN-g expression, and reduction of GATA-3 and Foxp3 expression in mLNs. The observed immunological effects warrant further studies with autoclaved and non-autoclaved soil exposure in immune-related diseases, such as allergy and inflammatory bowel disease models in mice.

Academy of Finland, Tampere Tuberculosis Foundation, State Research Funding (for Fimlab Laboratories), Finnish Medical foundation and Northern Finland Laboratory Centre (NordLab).
Myeloid-specific

ablation of Cblb is necessary to bolster defense against pulmonary fungal infection
Authors: Sharma Jaishree , Nanjappa Somashekarappa Gowda
Published: August 2023

Fungal pneumonia is a severe clinical problem, especially in patients with compromised immunity. The long-term therapeutic and prophylactic use of antifungal drugs in high-risk patients has promoted the emergence of multidrug-resistant fungi. Myeloid cells, mainly alveolar macrophages, are critical for orchestrating the immune responses, limiting pathogen growth, and resolving pathology during lung fungal infection. Thus, it is essential to understand mechanisms and uncover the host-fungus interface regulating early immune responses in the lung. Here, we studied the myeloid cell-specific role of the host element, CBLB, an E3 ubiquitin ligase, for antifungal immunity in the lungs. Using a mouse model of pulmonary blastomycosis, we found that targeted ablation of Cblb in myeloid cells (LysMcre/creCblbfl/fl), but not in all the cells, was essential to mediate protective immunity. Myeloid-specific Cblb deletion enhanced survival, reduced fungal burden, and reduced inflammation following lethal lung fungal infection. Mechanistically, myeloid-specific ablation of Cblb led to enhanced numbers of alveolar macrophages and increased activation and ROS production by neutrophils. Interestingly, we found an augmented type 17 innate lymphocytes in LysMcre/creCblbfl/fl mice than in controls, suggesting their potential crosstalk with Cblb-deficient myeloid cells. Our study suggests that myeloid-specific ablation of Cblb is necessary to enhance immunity against pulmonary fungal infections and proposes this as a potential therapeutic avenue.

Supported by grants from American Lung Association (IA-697123, to SGN), NIH (R01 AI53522, to SGN), and UIUC (Faculty Start-up Funds, to SGN)
Investigating the lipid-immune signaling axis in modulating the inflammatory response to Salmonella enterica serovar Typhimurium infections
Authors: Taddeo Jessica R , Beld Joris , Tukel Cagla , Tam Vincent
Published: August 2023

Salmonella enterica serovar Typhimurium (STM) is an intracellular pathogen that causes gastroenteritis and can disseminate systemically through survival in phagocytes. Eicosanoids-derived bioactive lipids play critical roles in both the induction and resolution of inflammation during bacterial infections or other insults. A subset of these lipids are endogenous ligands for nuclear receptors/transcription factors Peroxisome proliferator-activated receptors (PPAR), which play major roles in fatty acid metabolism and modulation of immune functions (including macrophage MΦ polarization). To elucidate the role of PPARα, we infected streptomycin-pretreated C57BL/6 (wildtype) and Ppara−/− mice with STM (colitis model). We determined that Ppara−/− mice have increased eicosanoid production, decreased inflammatory gene expression and histopathology scores in cecal tissue, and reduced systemic dissemination of bacteria, compared to C57BL/6 mice. These data suggest that the activation of PPARα results in a deleterious immune response favoring STM infection. When polarizing MΦ to various subsets, STM infection enhanced M2b and M2c polarizations (anti-inflammatory and immunoregulatory) in a PPARα-dependent manner. Using liquid chromatography-mass spectrometry (LC/MS), we also determined that STM infection alters the fatty acid profiles in polarized MΦ. Specifically, C57BL/6 M2b-polarized STM-infected MΦ have decreased ceramide levels compared to uninfected control, which would inhibit programmed cell death and enhance bacterial intracellular survival. These findings provide important insights into how the reprogramming of immune metabolism affects host-pathogen interactions during bacterial infections.

This work was supported by National Institute of Allergy and Infectious Disease R01 AI168550 (VT)