bluuebunny/crossref_metadata_2025 huggingface dataset:
The human antibody 3E1 targets a novel epitope of the HA stem region to neutralize the H1N1 and H5N6 influenza A viruses
Authors: Sun Bing , wang Wen Shuai , Sun Xiao Yu , Li Yan bing , Chen Hua Lan , Ding Jian Ping
Published: January 2023

As influenza A viruses remain a major threat to human health worldwide, the discovery of broadly neutralizing monoclonal antibodies that recognize conserved epitopes would facilitate the development of antibody-based therapeutic strategies. In this study, we show that a novel VH4-4-encoded human mAb named 3E1 could neutralize H1 and H5 subtype viruses in vitro and protect mice against the H1N1 and H5N6 viruses by inhibiting the low pH-induced conformational rearrangement of hemagglutinin (HA), hence blocking membrane fusion. The crystal structures of 3E1 Fab in complex with HA of two H1N1 strains reveal that 3E1, with both heavy and light chains, binds to a novel epitope of the conserved HA stem region, comprising parts of the fusion peptide, the F subdomain and the outermost β-strand preceding helix A. Together, these data suggest the potential of 3E1 as a therapeutic drug and new strategies for the development of antiviral drugs and universal vaccines against H1 and H5 subtype viruses.
Macrophage selenoproteins restrict intracellular replication of Francisella tularensis
Authors: Markley R L. , Williamson David R. , Katkere Bhuvana , Dewan Kalyan K. , Shay Ashley E. , Sumner Sarah E. , Prabhu K. Sandeep , Kirimanjeswara Girish S
Published: January 2023

There has been a growing appreciation for the influence of dietary nutrients on the microbiome as well as host response to infectious disease. The micronutrient selenium (Se) is known to regulate immune functions via selenoproteins, a class of proteins that contain the 21st amino acid selenocysteine. However, the mechanisms by which selenoproteins regulate immune functions during an acute infection are not clear. Therefore, we investigated the role of macrophage (Mac) selenoproteins during an acute bacterial infection. Francisella tularensis (Ft.), the causative agent of tularemia, is a gram-negative intracellular bacterium. Since Ft. infects and replicates primarily in Macs, we measured the bacterial replication in Macs derived from TrspM mice that are unable to synthesize selenoproteins. TrspM Macs had uncontrolled replication of Ft. compared to wild-type (WT) Macs. Moreover, TrspM mice were more susceptible to Ft. infection and harbored significantly higher levels of bacteria in their livers and spleens as compared to WT mice, which suggests that Mac selenoproteins are essential for restricting bacterial replication and promoting host survival. Since Ft. replication is closely associated with autophagy, we investigated if the Mac selenoproteins had an effect on autophagy. In fact, our data indicates that selenoproteins inhibit autophagy, which may limit the availability of nutrients to the bacteria. Future studies will seek to establish the influence of selenoproteins on the microbiome and how these changes impact the infectious disease process. Our studies demonstrate that nutrients affect the outcome of an infection by influencing host immune response, and reveal potential novel targets for antibacterial therapies.
Protective effects of Δ9-Tetrahydrocannabinol (THC) results from alteration in lung dysbiosis mediated by SEB-induced acute lung injury
Authors: Mohammed Amira K. , Alghetaa Hasan F , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Inhalation of Staphylococcal Enterotoxin B (SEB) is known to induce acute lung injury (ALI) and studies from our laboratory have shown that THC, a psychoactive ingredient found in Cannabis sativa, can attenuate the ALI. In the current study, we investigated the role played by lung microbiota in ALI with or without THC treatment. A dual-dose of SEB was given to C3H/HeJ mice, which were then treated either with vehicle or THC. SEB-administration caused ALI and 100% mortality while all THC-treated mice survived and suppressed the inflammation in the lungs. Furthermore, lung microbiota was collected and 16S rRNA sequencing was performed. The data were analyzed to determine the alpha and beta diversity. The major phylum was Proteobacteria, class Alphaproteobacteria and orders were Caulobacterles and Rhodobacteralesin in the lungs of vehicle-treated SEB group. Moreover, THC treatment led to elevated Firmicutes phylum due to significant increase in the beneficial genus, Lactobacillus. Lipopolysaccharide (LPS), gram negative bacterial endotoxin was found at significantly higher concentration in the BALF of vehicle-treated group in comparison with THC-treated mice. Beneficial metabolome levels were significantly higher in the colonic flush of THC-treated mice when compared to vehicle-treated SEB group, specifically butyric, propionic and acetic acids. Together, our data suggest that THC attenuates SEB-induced mortality and ALI by altering the microbiota in the lungs (Supported by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755, P20GM103641, R01AI129788 to PN and MN and MoHESR fellowship for AKM).
House dust mite exposure upregulates C3a receptor expression in pulmonary innate immune cells during the sensitization phase of allergic asthma by an IL-33-dependent mechanism
Authors: Laumonnier Yves , Quell Katharina , Wiese Anna Valeska , Vollbrandt Tillman , Köhl Jörg
Published: January 2023

The anaphylatoxin C3a exerts its biologic functions through the activation of its cognate G-protein-coupled receptor C3a receptor (C3aR). C3aR mRNA expression has been described both in human and mouse lungs. However, no systematic analysis of C3aR expression at steady state and under pro-inflammatory conditions in the different pulmonary cell subsets has been done so far. Using a newly developed Tandem-dye (td)-Tomato-C3aR reporter knock-in mouse, we found C3aR expression in pulmonary eosinophils, resident CD11b+ conventional dendritic cells (cDC), and monocyte-derived DCs (moDC) at steady state. In contrast, pulmonary CD103+ cDCs and plasmacytoid DCs stained negative. Surprisingly, lung and bronchoalveolar alveolar macrophages (AMs), as well as neutrophils, lacked td-Tomato-C3aR. C3aR antibody staining revealed that only moDCs express the receptor at the cell surface.

House dust mite (HDM) is a potent allergen that drives the development of allergic asthma in humans. Using the td-Tomato-C3aR reporter mouse, we found that HDM exposure resulted in the upregulation of C3aR in AMs, eosinophils, CD11b+ cDCs, and moDCs during the early phase of HDM-induced allergic asthma. Further, C3aR translocated to the cell surface in eosinophils and AMs following HDM exposure. Importantly, intra-tracheal administration of IL-33, an alarmin released by epithelial cells upon allergen contact, recapitulated these results. In summary, our findings demonstrate that allergen contact triggers the upregulation of C3aR and its surface expression in previously negative innate immune cells from the lung by an IL-33 dependent mechanism. C3aR-mediated activation of innate immune cells may play a critical role for early asthma development.
Chronic stimulation results in NK cell function impairment mediated by NKG2D
Authors: Alvarez Maite , Baker Jeanette , Wenokur Arielle , Morrisson Alyssa R. , Pierini Antonio , Simonetta Federico , Negrin Robert S.
Published: January 2023

NK cell exhaustion (NCE) has been described as a defect of NK cell function due to sustained proliferation resulting in downregulation of eomesodermin and T-bet. Using murine models of chronic stimulation, we have identified here a molecular signature of NCE characterized by up-regulation of the terminal differentiation marker KLRG1 and down-regulation of eomesodermin and of the activating receptor NKG2D. We show that chronic stimulation of mice lacking NKG2D resulted in a minimized NCE compared to control mice, thus pointing to NKG2D as a crucial player in NCE. Interestingly, in vitro studies revealed that MULT1 and Rae1, stress molecules known as NKG2D ligands, are up-regulated during activation, leading to NKG2D cis-binding and to its internalization, and thus reducing NKG2D accessibility. In order to minimize the genotoxic stress caused by prolonged stimulation and prevent NCE, NK cells were treated with the ATM/ATR DNA damage pathway inhibitor KU55933 (KU). KU treatment during NK cell activation prevented NK cell exhaustion as revealed by preserved cell function, improved expression of activation markers and of genes involved in cell survival, and sustained expression of NKG2D. Importantly, the adoptive transfer of NK cells expanded ex vivo in the presence of KU resulted in better survival in A20 and B16F10 mouse models of cancer that can be eliminated in a NKG2D-dependent or -independent manner respectively. Collectively, these data demonstrate that NCE is indeed an intrinsic regulatory mechanism of NK cell activation regulated, at least in part, by NKG2D and that prevention of NCE is a promising strategy to improve NK cell-based immunotherapy.
Aging induced changes in the microbiome affect the crosstalk and activity of monocytes and B1a cells
Authors: Bodogai Monica , Moritoh Kanakoh , Kim Ki , Wu Jiejun , Mattison Julie , Biragyn Arya
Published: January 2023

Aging is thought to mostly dysregulate conventional B2 cells while leaving unaffected innate B1 cells. We recently questioned this assumption by providing evidence that upon aging, in humans, macaques and mice, B1a cells become converted into 4-1BBL+mTNFa+MHC class-IHiCD86Hi B cells (4BL cells) 1,2. Here we report that this conversion is induced by changes in the gut microbiota. Metabolites of the aging gut microbiota initiate monocyte infiltration in the omentum, where they convert B1a cells into 4BL cells. By utilizing 4-1BBL-41BB and mTNFa-TNFRII axes, 4BL cells further induce granzyme B (GrB)-expressing cytolytic CD8+T cells. Thus, for the first time, we provide a mechanistic proof that innate B1a cells also become dysregulated in aging of mammals, resulting in the loss of their immunosuppressive function and the gain of a new function – ability to induce potentially antitumor and autoimmune CD8+T cells. Our data shows the role of the microbiome/monocyte/B-cell/CD8-cell network in several aging-associated diseases.

This work was supported by the Intramural Research Program, NIA/NIH, and CRADA with Janssen Research & Development, LLC, USA
Adenosine generation limits radiation-induced tumor immunogenicity by abrogating recruitment and activation of CD103+ DCs
Authors: Wennerberg Erik , Cronstein Bruce , Formenti Silvia C , Demaria Sandra
Published: January 2023

Radiation therapy (RT) induces release of ATP in the tumor micro environment (TME) triggering recruitment and activation of dendritic cells (DCs), including CD103+ DCs, identified as the key DC subsetresponsible for cross-presentation of tumor antigens to CD8+ T cells. However, CD39 and CD73-expressing cells in the TME hydrolyze ATP into adenosine that potently inhibits DCs and CD8+ T cells. Here, we tested the hypothesis that adenosine generation limits the ability of RT totrigger anti-tumor immunity.

Wild type (WT) or BATF3−/− mice (CD103+DC-deficient) were inoculated s.c. with TSA tumor cells (day 0) and assigned to treatment with: (1) control Ab; (2) anti-CD73 Ab (100 μg) (3) RT (20 Gy); (4) RT + anti-CD73 Ab. Antibodies were administered i.p. on day 11, 14, 17 and 20. RT was given locally as single 20 Gy dose on day 12. On day 18, tumors were analyzed by flow cytometry for DC and T cell infiltration. Mice were monitored for tumor progression. DCs generated in vitro by culture of bone marrow from WT mice with Flt3L (>90% CD103+) were labeled with CFSE and intravenously injected in BATF3−/− recipient mice. Tumors were harvested after 48h and analyzed by flow cytometry for infiltration of CFSE+ DCs.

In tumors of RT- but not sham-treated mice, anti-CD73 mAb increased infiltration of CD103+DCs and enhanced CD8+T cell/Treg ratio. Importantly, CD73 blockade had no anti-tumor effect by itself but significantly improved RT-induced tumor control. The therapeutic effect of RT + CD73 blockade was abrogated in BATF3−/− mice. Moreover, CD73 blockade increased recruitment of adoptively transferred CD103+DCs to irradiated tumors in BATF3−/− mice. Overall, data suggest that adenosine is a critical regulator of RT-induced immunogenicity.
Phagocytic plasticity of macrophage towards healthy self cells: inflammatory activation elicit self-attacking phenotype in macrophages lacking SIRPα-CD47 restraint
Authors: Bian Zhen , Shi Lei , Liu Yuan
Published: January 2023

The interaction between macrophage signal-regulatory protein alpha (SIRPα) and its ligand CD47 serves as a critical self-recognition mechanism, in which their interaction triggers inhibitory signaling via SIRPα cytoplasmic ITIMs and prevent macrophage phagocytosis toward self-cells. Despite this mechanism being suggestively imperative, CD47−/− mice demonstrate no, or only mild, macrophage phagocytosis toward self-cells, suggesting additional activation and inhibitory mechanisms controlling macrophage behavior. Studying our recently established SIRPα −/− mice, as well as CD47−/− mice, we identify that macrophage phagocytosis toward self cells requires activation by inflammatory cytokines/factors. Ex vivo phagocytosis assays demonstrate that macrophages from SIRPα −/− or CD47−/−, and even WT mice, are generally incompetent to attack self-cells, which bear no ‘eat-me’ signal and thus do not activate macrophage phagocytosis. However, treating these macrophages with IL-17, IL-6, IL-1β, TNFα and TLR agonists, but not IFNγ, dramatically initiates potent phagocytosis toward self-cells, for which only the CD47-SIRPα interaction restrains. Mechanistic studies suggest that a PKC-Syk-mediated signaling pathway, to which IL-10 conversely inhibits, is required for activating macrophages toward self cells. Application of activated SIRPα −/− macrophages in targeting CD47-expressing cancer cells also display potent eradication of B16, LLC, MC38, and EL4 cancer cells. These findings significantly expand our knowledge of macrophage phagocytic plasticity under different conditions and also provide insights into strategies for enhancing transplantation tolerance and macrophage-based cancer eradication.
VAT-associated ST2+Tregs from omentum support tolerance to peripheral tumors
Authors: Meza-Perez Selene , Silva-Sanchez Aaron , de la Luz Garcia-Hernandez Maria , Mudunuru Uma , Simpler Thomas S. , Randall Troy D.
Published: January 2023

Tumor metastasis to theomentum is associated with poor clinical outcomes. Although the omentum hasimmune function due to the activities of milky spots, the role of this organ inanti-tumor immunity has not been completely described.

By implanting tumor cells in mice we found that peritoneal tumors are not rejected and cause an increasein visceral adipose tissue (VAT)-associated regulatory T cells in the omentumas well as low frequency of tumor specific CD8 T cells. Additionally, peritoneal tumors impaired the immunity to secondary dermal tumors by limiting the expansion of tumor specific CD8 T cells that mediate tumor rejection.

To better understand this phenomenon, we studied the VAT-associated Tregs, which can be divided in ST2+ and ST2- (ST2 a component of the IL-33R). Particularly, VAT-associated ST2+ Tregs are not found in conventional lymphoid organs, nonetheless we find that following peritoneal tumor inoculation ST2+ Tregs are mobilized and home to secondary dermal tumors. To test whether the ST2+ Tregs were responsible for preventing secondary dermal tumors, we inoculate mice lacking ST2 (ST2−/−) with peritoneal tumors and challenged them with dermal tumors; interestingly, we found that secondary dermal tumors where rejected in ST2−/− mice whereas tumors in wild type mice continue to grow. Finally, to corroborate our findings we used a mouse strain that develops spontaneous ovarian tumors that metastasize to the omentum. By treating these mice with anti-ST2 we observed a reduction in tumor weight, decrease in ST2+Tregs and an increase in tumor specific CD8+ cells in omentum and ovaries. Together these data suggest that omental ST2+ Tregs have an important role promoting and maintaining tolerance in tumor metastasis.
Follicular CD8 T cells in autoantibody-mediated disease.
Authors: Valentine Kristen M , Davini Dan , Al-Kuhlani Mufadhal , Gravano David M , Sindi Suzanne , Hoyer Katrina K
Published: January 2023

The role of CD8 T cells, unlike CD4 T cells, in antibody-mediated autoimmune disease remains largely unexplored. CD8 T cells act both in a cytotoxic CD8 T cell role and helper-like role during autoimmunity. CD4 T follicular cells (Tfh), interact with B cells to promote proliferation, activation and antibody class switching. In autoimmunity, dysregulated self-reactive CD4 Tfh can promote autoantibody production. We have identified a novel population of CD8 T cells within the germinal center that resembles CD4 Tfh cells during systemic autoantibody-mediated disease. IL-2 deficient (IL-2-KO) mice develop systemic autoantibody-mediated disease including autoantibodies against red blood cells (RBC) resulting in autoimmune hemolytic anemia. We show that the absence of either CD4 or CD8 T cells prevents the development of anti-RBC antibodies and reduces lymphoproliferation in IL-2-KO mice. RNA-sequencing of IL-2-KO CD8 T cells revealed an increase in the expression of genes associated with CD4 Tfh lineages (Bcl-6, CXCR5, PD-1, ICOS and IL-21) compared with wild type (WT) CD8 T cells. Flow cytometry of expanded CXCR5+PD-1+ CD4 Tfh cells and CXCR5+PD-1+ CD8 T follicular (Tfc) cells confirms the protein expression of these Tfh markers and the production of IL-21 in IL-2-KO late stage disease (day 18–21). In vitro culture of IL-2-KO CD8 Tfc cells induced antibody class switching in WT B cells at levels similar to that induced by adjuvant-induced WT CD4 Tfh cells. In comparison to CXCR5- PD-1-CD8 T cells, CD8 Tfc cells expressed higher levels of CD40L. Our results indicate that CD8 T cells contribute to autoimmune disease in part through differentiation into follicular T cells that function to induce B cell differentiation and antibody class-switch.
Dual TCRα expression alters agonist selection, redirecting Treg-biased insulin-specific thymocytes into the conventional T cell lineage
Authors: Schuldt Nathan J , Auger Jennifer L. , Spanier Justin , Martinov Tijana , Breed Elise R. , Fife Brian T , Hogquist Kristin Ann , Binstadt Bryce Anthony
Published: January 2023

Despite accounting for ~10% of the T cell population in mice and humans, the role of dual TCR-expressing T cells in immunity remains poorly understood. It has been hypothesized that dual TCR T cells pose an “autoimmune hazard” by allowing self-reactive TCRs to escape negative selection. We revisited this hypothesis using the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). We bred NOD mice hemizygous at both TCRα and β (TCRα+/− β+/−) loci, rendering them incapable of producing dual TCR T cells. We found that NOD mice lacking dual TCRα expression were resistant to developing diabetes resulting from an increased insulin-specific Treg:Tconv ratio. Furthermore, early depletion of CD25-expressing cells was sufficient to induce diabetes in single TCR T cell NOD mice demonstrating that these mice are capable of developing diabetes and that Treg cells are responsible for their observed resistance to diabetes. Further investigation uncovered a previously-unrecognized impact of dual TCRα expression on agonist selection. Specifically, we found in single TCR T cell mice that single positive (SP) thymocytes had a higher Treg:CD4 ratio indicating increased Treg commitment, that insulin-specific thymocytes exhibited higher CD5 expression suggesting increased TCR signaling, and that deletion of SP thymocytes was reduced. Altogether these data demonstrate that dual TCRα expression perturbs agonist selection of the insulin-specific thymocyte subset, resulting in skewing of this population toward the diabetogenic conventional CD4+ T cell lineage and away from the regulatory T cell lineage. Thus, dual TCR expression can promote autoimmunity by reducing the ratio of protective Treg cells to pathogenic T cells.
CD27 co-stimulation increases regulatory T cell survival and limits progression of atherosclerosis.
Authors: Winkels Holger , Meiler Svenja , Bürger Christina , Reim Sigrid , Weber Christian , Borst Jannie , Ley Klaus , Lutgens Esther , Gerdes Norbert
Published: January 2023

Co-stimulatory signals are important shaping adaptive immunity which is pivotal to all stages of atherosclerosis. The interaction of the co-stimulatory molecules CD27 and CD70 modulates Treg development. Furthermore, T cell activation, proliferation, and also differentiation are affected by CD27-CD70 interactions. Thus, we hypothesized that the deficiency of CD27 will lead to exacerbated atherosclerosis.

Cd27−/− mice were crossed with Apoe−/− mice. Cd27−/−Apoe−/− and littermate controls (Cd27+/+Apoe−/−) were sacrificed at the age of 18 and 28 weeks. Bone marrow of Cd27−/−Apoe−/− and littermate controls was transplanted into Apoe−/− recipient mice. Lesion size, histology and cellular composition were analyzed in the ascending aorta.

Human and murine T cells co-localize with CD27 in atherosclerotic lesions. At early stages of atherosclerosis, Cd27−/−Apoe−/− mice displayed bigger and more complex atherosclerotic lesions. Flow cytometry revealed a significant decrease in abundance of splenic and aortic Tregs and increased apoptosis of Treg in the thymus of Cd27−/−Apoe−/− mice. In contrast, at later stages of atherosclerosis (28 weeks), splenic and lesional Treg content and atherosclerotic burden were similar. Transplantation of Cd27−/−Apoe−/− bone marrow, in contrast to control, induced increased atherosclerotic plaque size and a profound pro-inflammatory plaque phenotype accompanied by reduced frequency of aortic and splenic Tregs. Furthermore, the number of circulating leukocytes was increased.

Taken together, CD27 deficiency impairs thymic Treg development which exacerbates early atherogenesis. However, the role of CD27–CD70 interactions does not affect later stages of atherosclerosis.
A new approach to examine the ability to control HCMV in patients after haploidentical stem cell transplantation
Authors: Anikeeva Nadia , Grosso Dolores , Flomenberg Neal , Sykulev Yuri
Published: January 2023

We utilized the recently developed approach (1) to examine HCMV-specific responses of CD8 T cells in patients who underwent haploidentical stem cell transplantation (HSCT) by analyzing the pattern of Ca2+ signaling in individual T cells assembled into a continuous monolayer after stimulation with HCMV peptide epitopes. Three groups of responding CD8+ T cells were identified: (i) T cells that rapidly develop sustained Ca2+ signaling typical for T-cell responses to a strong agonist peptide, (ii) T cells developing delayed but sustained Ca2+ signaling that is characteristic for T-cell responses to a weak agonist peptide, and (iii) T cells that revealed oscillating Ca2+ signaling indicative of suboptimal T cell activation. T cells showing sporadic and transient Ca2+ spikes were considered unresponsive. In patients whose immune system was still rebooting after transplant, HCMV was reactivated and this was mirrored by a very low fraction of HCMV-specific T cells, which mount rapid and sustained Ca2+ signaling, while the majority of responding T cells exhibited delayed or oscillating Ca2+ signaling. In contrast, a significant fraction of HCMV-specific T cells from patients who control the virus revealed rapid and sustained Ca2+ signaling. The ability to monitor patients’ immune system response to HCMV enables us to tailor treatment to better keep CMV in check.
Improved single T cell mRNA and TCR α and β chain detection using optimized chemistry and dead cell quality control for RNA-seq on the Fluidigm C1 system
Authors: egidio camila , Ooi Aik , Gonzales Michael , Brockman Joel , Chen Shuwen , Durruthy-Durruthy Robert , Rogers Clare , Ray Manisha
Published: January 2023

The ability to link T cell clonality with the transcriptional profile at the single-cell level provides deep insight into antigen-specific responses and the dynamics of clonal expansion and plasticity. Recent reports show that while it is possible to obtain the transcriptome profile and paired α, β TCR chain information from RNA-seq data for a single T cell, the process is technically challenging. T cells have low RNA content and RNA rapidly degrades during cell preparation for experimental setup.

We investigated two key experimental parameters, chemistry and workflow, on the gene expression profile and the ability to detect paired TCR α, β chains for single human and mouse T cells, using the C1™ system (Fluidigm®). The sensitivity and overall performance of the Clontech® SMART-Seq® v4 kit were compared with those of the SMART-Seq v1 kit. Two workflows were used to process the cells on C1: a four-hour workflow that included live/dead cell staining and imaging to allow cell viability assessment and exclusion of dead cells from downstream steps, and a two-hour workflow that excluded those steps, decreasing processing time and potentially improving RNA preservation. For both mouse and human T cells, SMART-Seq v4 chemistry outperformed v1 for reads aligned to RefSeq, bases aligned to coding and intronic sequences, and reconstruction of paired α, β TCR chains. Additionally, we observed 73% detection of paired α, β TCR chains when dead cells were excluded through imaging analysis versus only 38% detection from the shorter workflow without dead cell exclusion. We conclude that the use of an improved chemistry, coupled with a C1 workflow that allows viability assessment of single cells, improves detection of α, β TCR chains in parallel with mRNA profiling.
MiR-155 and LP-BM5 retrovirus-augmented Monocytic myeloid derived suppressor cells, are players in a profound murine immunodeficiency-causing syndrome
Authors: Green Kathy A , Green William R
Published: January 2023

We have shown augmentation of monocytic MDSCs (M-MDSCs) during a profound immunodeficiency causing LP-BM5 retrovirus infection of B6 mice. Enriched splenic M-MDSCs preparations in suppression assays, inhibited T-, and the much less reported B-, cell responsiveness. This inhibition of T-cell proliferation and IFN-gamma production was almost completely iNOS/NO dependent, whereas suppression of B-cell responses was only ~50% iNOS/NO dependent. To explore additional mechanism(s) in this inhibition of B-cell responsiveness, we reported that VISTA, a negative checkpoint regulator, is involved in LP-BM5 M-MDSC suppression of ex vivo B-cell responses. In light of the reported involvement of miR-155 in B-cell and myeloid cell function, we are now also evaluating M-MDSCs from 5 week LP-BM5 infected miR-155 k.o.s. Our results show miR-155 k.o. M-MDSCs are substantially less (30–50%) suppressive compared to M-MDSCs from infected Wt. mice, and at 10 w.p.i. the k.o.s exhibit a different kind of immunodeficiency: 1) spleen cells are significantly more responsive to B-cell polyclonal activators, 2) significantly less splenomegaly and 3) have fewer resident splenic M-MDSCs. In an initial experiment, involving adoptive transfer of M-MDSCs from donor LP-BM5 infected Wt. B6 to infected miR-155 k.o.s, we were able to significantly augment miR-155 k.o.: 1) splenomegaly, 2) splenic B-and T-cell immunodeficiency, and 3) the number of residing splenic M-MDSCs, to levels almost comparable to those of Wt. infected mice. These results highlight involvement of multiple and unique suppressive pathways in the under-studied area of MDSC suppression of B-cell responses, and were compatible with a role for M-MDSC in LP-BM5-induced immunodeficiency.
RNA Virus hijacks TRIM29 to suppress the host innate immune response
Authors: Xing Junji , Zhang Zhiqiang
Published: January 2023

The host response to RNA virus infection relies on an intrinsic innate immune response to restrict viral replication. Antiviral signaling activation of innate immunity requires strict modulation to avoid damage to the host from exacerbated cytokines and inflammation. However, the precise mechanism of how antiviral signaling activation is strictly controlled still remains obscure. Here, we found the E3 ligase TRIM29 was specifically expressed in poly I:C induced human myeloid dendritic cells (mDCs). TRIM29 was highly induced in human mDCs in response to poly I:C and played negative role to type I interferon (IFN) production. The deficiency in TRIM29 resulted in enhanced type I interferon responses to poly I:C and reovirus infection. Importantly, challenge of wildtype mice with reovirus led to lethal infection. In contrast, deletion of TRIM29 protected the mice from this developing lethality. Additionally, the heart, intestine, spleen, liver and brain of TRIM29−/− mice exhibited attenuated viral loads of reovirus. Meanwhile, the heart of TRIM29−/− mice showed elevated type I IFN production. Mechanistically, TRIM29 was shown to interact with MAVS, the key adaptor in the RNA sensing pathway, and induce its ubiquitination and degradation. Taken together, RNA virus hijacks the E3 ligase TRIM29 to shut down the host innate immune response for immune evasion.
A robust microparticle platform for a STING-targeted adjuvant that enhances both humoral and cellular immunity during vaccination
Authors: Junkins Robert D. , Johnson Brandon M. , Gallovic Matthew D. , Collier Michael A. , Watkins-Schulz Rebekah , Cheng Ning , David Clement N , McGee Charles E. , Sempowski Gregory D , Shterev Ivo , McKinnon Karen , Bachelder Eric M. , Ainslie Kristy M. , Ting Jenny P-Y
Published: January 2023

Most FDA-approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly-understood mechanisms. Stimulator of interferon genes (STING) is an exciting adjuvant target due to its role in anti-viral immunity; however, a major hindrance is STING’s cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but has only been assessed at doses that are likely cost-prohibitive to clinical translation for vaccines against infectious diseases. Hence, alternative platforms for the intracellular delivery of STING-agonists are needed. We evaluated the adjuvant activity of a potent STING agonist, encapsulated in acid-sensitive acetalated dextran (Ace- DEX) polymeric microparticles (MPs) which target antigen-presenting cells for intracellular release. This formulation was superior to all particle delivery systems tested, achieved protective immunity in vivo at doses of STING agonist 50-fold lower than previous reports, and elicited no observable toxicity in animals. Compared to soluble agonist, the STING agonist Ace-DEX MPs enhanced type-I interferon responses up to 1000-fold in vitro and 50-fold in vivo, caused up to 104-fold increases in antibody titers, enhanced Th1-associated responses, and expanded germinal center B cells and memory T cells. It also provided protection against a lethal influenza challenge. Thus, Ace-DEX MP-encapsulated STING agonist represents a novel and feasible vaccine adjuvant of humoral and cellular immunity.
Changes in immune gene transcription associated with phases of lactation in a model marsupial, Monodelphis domestica
Authors: Fehrenkamp Bethaney D , Miller Robert D
Published: January 2023

All mammalian neonates are highly dependent upon milk for nourishment and immune protection. This is especially true for marsupials, a lineage of mammals with limited placental development and an increased reliance on an extended lactation period. Most newborn marsupials do not receive passive maternal immunity in utero and therefore are entirely dependent upon factors within the milk for immune protection until capable of mounting their own responses. Early exposure to potential pathogens, prior to the development of a functional immune system, requires a complex strategy for providing immunological protection. Preliminary transcriptome sequencing results revealed two distinctive peaks in transcription of immune transfer related genes within the mammaries throughout the course of lactation. Additional time points were investigated using quantitative real-time PCR targeting specific immune markers. Lymphocyte markers (CD3E and CD79b) were elevated in a biphasic pattern of expression throughout lactation, suggestive of two waves of lymphocytes entering the mammaries throughout lactation. Immunohistochemistry analyses are underway to investigate the presence of lymphocytes within the mammaries throughout lactation and correlation with gene expression patterns. These investigations have the potential to impact the understanding of the role lactation plays in neonatal immune development from an evolutionary perspective.
Analysis of Cytokines and Chemokines Circulating in Plasma Obtained from a Phase I Prime-Boost Dengue Vaccine Trial
Authors: Fessehazion Arsiema , Currier Jeffrey R , Koren Michael , Keiser Paul , Lin Leyi , Jarman Richard G , Friberg Heather
Published: January 2023

A safe and efficacious vaccine for dengue is seen as the best rationale for decreasing the burden of morbidity and mortality associated with dengue infection. Identification of acute phase soluble factors as biomarkers of successful vaccination would has ten the development of a dengue vaccine. The Walter Reed Army Institute Research (WRAIR) is exploring the potential of a Prime-Boost vaccine strategy using purified inactivated virus (PIV) and live attenuated virus (LAV) tetravalent dengue vaccine candidates. A phase 1 clinical trial was performed testing different dosing schedules, PIV followed by LAV or vice versa at 0–1 or 0–6 months. Plasma samples were collected pre-vaccination and at multiple time points immediately post-vaccination to assess the kinetics of the acute phase soluble factor response to vaccination. We tested the plasma samples using Luminex-based magnetic bead kits measuring 59 different chemokines and cytokines. Our results reveal that different cytokine profiles were induced in subjects depending on the vaccine modality and whether it was given as the prime or boost dose. LAV appeared to be most immunogenic, particularly when received as a booster dose after PIV priming. Production of cytokines, including sCD30, APRIL, sTNF-R2, and IL-1RA, occurred in the majority of subjects tested approximately 10–14days post-vaccination. This study supports other data from this trial demonstrating that the type of vaccine and the order in which it is received impacts immunogenicity. Correlations of soluble factor responses with traditional adaptive immune responses, such as neutralizing antibody and IFN-γ ELISpot assays, will be presented.
Paracrine rather than systemic glucocorticoids are biologically active in the thymus
Authors: Taves Matthew Donald , Mittelstadt Paul R , Korol Anastasia M , Hamden Jordan E , Abraham Ninan , Soma Kiran K , Ashwell Jonathan D
Published: January 2023

Thymocyte positive and negative selection are critical for generation of a competent and self-tolerant T cell repertoire. Glucocorticoids (GCs) protect thymocytes from T cell receptor (TCR)-induced death, and thymocyte-specific GC receptor (GR) deletion amplifies negative selection, weakening the TCR repertoire. Circulating GCs are secreted by the adrenals, but levels fluctuate widely with time of day and in response to stressors. To avoid such variation, thymus GCs might be regulated independently of the adrenals, as thymic epithelial cells (TECs) and possibly thymocytes express GC-synthetic enzymes. Whether local GC production is sufficient to affect thymocyte development in the presence of adrenal GCs, however, is unknown. Here, we have found that corticosterone, the major mouse GC, was locally elevated in the thymus compared to the blood, and that cultured thymus produced corticosterone from endogenous substrates via GC-synthetic enzyme activity. To test the source and importance of local GC synthesis in vivo, we generated mice with targeted deletion of the GC-synthetic enzyme Cyp11b1 in TECs (Cyp11b1foxn1-Cre) or thymocytes (Cyp11b1lck-Cre). As a measure of GC signaling we quantified thymocyte expression of the GC-responsive gene Gilz. Gilz mRNA was normal in Cyp11b1lck-Cre but reduced in Cyp11b1foxn1-Cre thymocytes, with a reduction equivalent to that in GR-deficient thymocytes. Basal GR signaling is thus driven overwhelmingly by TEC-rather than adrenal-derived corticosterone. These findings demonstrate the importance of paracrine GC function in vivo, and are consistent with a role for paracrine GCs in thymocyte selection.
Analysis of expression of genes encoding neutrophil and eosinophil granule proteins during in vitro eosinophilopoiesis
Authors: Chung Il Yup , Kim Sung Min , Hwang Sae Mi , Kim Karam
Published: January 2023

Neutrophils and eosinophils, the two prominent granulocytes in circulation, are characteristically distinguished by neutrophil primary granule proteins (NPGPs) and eosinophil specific granule proteins (ESGPs). They differentiate from myeloid progenitors in the bone marrow commonly through a successive stage encompassing promyelocyte, myelocyte, metamyelocyte, and band form. The purpose of this study is to analyze bifurcation of the two granulocytes by examining expression of the NPGP- and ESGP-encoding genes during eosinophilic differentiation of cord blood (CB) CD34+ cells for 24 days. All transcripts of the NPGP-encoding genes, including PRTN3, MPO, CTSG, AZU1, and ELANE, reach peaks at day 12 and steeply decrease thereafter, while the transcripts of ESGP-encoding genes, such as MBP and ECP, reached peaks at day 18. Similarly, upon differentiation of immature eosinophilic EoL-1 cells, the NPGP transcripts are markedly downregulated accompanying by upregulated ESGP transcripts. Thus, the NPGP-encoding genes appear to be transcriptionally active even at eosinophilic promyelocyte and myelocyte stages. As these cells undergo maturation through metamyelocyte and band form stages, a transcription network come into a play that favorably shifts expression the ESGP-encoding genes.
The human CD34(−)CD117(+)CD56(−)NKp44(−) population in secondary lymphoid tissue serves as a common progenitor to NK cells and ILC2s
Authors: Chen Luxi , Carson Mary , Nalin Ansel , Young Karen A , Scoville Steven D , Mundy-Bosse Bethany L , Caligiuri Michael A , Freud Aharon G
Published: January 2023

The developmental pathways of human innate lymphoid cells (ILCs) have not been fully elucidated. We reported that human natural killer (NK) cells can derive from progenitor cells naturally residing in tonsils. We hypothesized that Group 2 ILCs (ILC2s) also develop in tonsils through a shared pathway. In support of this, ex vivo flow cytometry analyses of enriched human tonsil ILCs revealed a putative pathway of human CD294(+) ILC2 development stemming from a lineage (Lin)(−)CD34(−)CD117(+) population from which non-overlapping CD94(+) NK cells also emerged. Furthermore, when purified tonsil Lin(−)CD34(−)CD117(+) cells were cultured for 28 days with murine OP9 stroma expressing the human Notch ligand, Delta-like 1, in the presence of human interleukin-7 and Flt3 ligand, they produced mutually distinct and functionally mature populations of CD94(+) NK cells and CD294(+) ILC2s. The tonsil Lin(−)CD34(−)CD117(+) population is heterogeneous for CD56 and NKp44. Evaluation of the in vitro lineage differentiation potentials of subsets within the Lin(−)CD34(−)CD117(+) population according to NKp44 and CD56 revealed that NKp44(−)CD56(−) cells gave rise to both NK cells and ILC2s, whereas fractions expressing NKp44 and/or CD56 gave rise to NK cells but only to negligible ILC2s. In fact, the NKp44(−)CD56(−) population gave rise to a significantly higher percentage of ILC2s (7.50 ± 0.6) compared to the NKp44(+)CD56(+/−) populations (1.22 ± 0.4) (n = 5, p < 0.001). These data support a model whereby human NK cells and ILC2s can develop from a common Lin(−)CD34(−)CD117(+)CD56(−)NKp44(−) progenitor in tonsils and that the acquisition of NKp44 and/or CD56 restricts lineage differentiation potential towards the NK cell lineage.
3D visualization of sex differences in the immune response to influenza infection in the mouse lung
Authors: Condiff Emily K , Milanez-Almeida Pedro , Li Weizhe , Gerner Michael Y. , Germain Ronald N
Published: January 2023

Recent developments of imaging techniques, including histo-cytometry and a new tissue clearing and staining protocol called Clearing-enhanced 3D (Ce3D), allow for high-resolution, 3D multiplex quantitative imaging and analysis of cell populations in intact organ material. We have established a panel of 10–14 markers for use in Ce3D-cleared 250μm-thick sections of lung tissue to visualize both innate and adaptive aspects of the immune response as comprehensively as possible. This panel, combined with quantitative analysis provided by histo-cytometry, allows for the collection of larger, more informative imaging datasets than typically generated by conventional histochemistry methods. In our study, we applied these techniques to the mouse model of influenza A virus (IAV) infection to investigate the patterns of immune cell distribution and viral spread in the lungs of male and female mice. In C57BL/6J mice, we observed increased mortality in female mice compared to males when infected with the same dose of the lethal PR8 strain of IAV. Literature research as well as data from the infection of different transgenic mouse lines suggest that higher levels of innate inflammation in female mice may lead to the observed difference in mortality. Ce3D multiplex images show multiple differences in the distribution of both innate immune cells and infected areas in lungs from male and female mice. This study will contribute to a better understanding of both the immune response to IAV infection and sex differences in immunity in general, potentially leading to better design of tailored vaccines and treatments in the future.
Quantifying the relative impact of influenza virus mutations on viral fitness and escape from cellular immunity
Authors: Li Zheng-Rong Tiger , Zarnitsyna Veronika I , Lowen Anice C , Kohlmeier Jacob E , Antia Rustom
Published: January 2023

The relative conservation of T cell epitopes in human influenza viruses compared to antibody epitopes may be explained by two mechanisms: (1) mutations of the T cell epitopes are constrained by their potential to decrease viral fitness by altering protein function, or (2) the selection benefits gained by ‘escaping’ mutations are small due to the breadth of MHC polymorphisms among human population. The level of influenza-specific cellular immunity may further complicate the relative contribution of these two mechanisms. We developed a mathematical model incorporating the mutation cost, selection benefit, and allele frequency of ‘escapable’ MHC that indicated certain combinations of these three parameters determined the fate of influenza viruses harboring mutations in T cell epitopes. To measure the mutation cost and selection benefit, we developed a novel digital droplet PCR method for the simultaneous detection of wild-type and mutant influenza viruses harboring mutations in the immunodominant NP366–374 epitope. Primary infections with WT, NP-mutated, and 1:1 mixture of WT and mutated x31 influenza viruses showed no significant difference between the two viruses, while the secondary infection of PR8-primed B6 mice with the 1:1 mixture resulted in a 10-fold decrease in the peak of viral load of WT compared to NP-mutated viruses. Together, these data show that the mutation of a single immunodominant T cell epitope provides significant selection benefit with minimal fitness cost to the virus. Future studies in mice with diverse MHC alleles are underway to quantify the impact of varying levels of cellular immune pressure on the in vivo ability of mutant influenza viruses to escape T cell immunity.
Metabolic subsets of plasma cells and their turnover in the marrow
Authors: Wols Heather A Minges , Aguilera Alexya N , Witte Pamela L
Published: January 2023

The ability of long-lived plasma cells (LLPC) to maintain longevity and avoid metabolic burnout has been largely unexplored until recently. Our lab has shown that LLPC reside in hypoxic niches and are divided into two distinct subsets based on fluorescence following MitoTracker Red CM H2Xros staining: a predominant MitoHI subset, suggestive of aerobic respiration, and a rarer MitoLO subset, suggestive of glycolysis. These MitoHI and LO PC are comparable in function as detected by ELISPOT and ELISA following 24h ex vivo culture at oxygen levels as low as 0.5% (severe hypoxia). Taken together, these data suggest LLPC have the flexibility to use glycolysis when needed, although respiration may be the preferable metabolic strategy. To determine whether the MitoHI and LO PC subsets differed in turnover, we looked at 5-bromo-2′-deoxyuridine (BrdU) incorporation after ten days of labeling post antigen-boost to label PC forming from memory cells. Mice were sacrificed 13, 20, and 43 days following boost, and the frequency of BrdU+ PCs in the MitoHI and LO marrow compartments determined. The proportions of BrdU+ MitoHI PCs remained relatively stable from days 13 to 20 and slightly declined by day 43. Frequency of BrdU+ MitoLO PCs was initially greater than that in the MitoHI PC compartment, but rapidly diminished at subsequent time points. These data suggest that the turnover in the MitoLO compartment is much higher compared to the MitoHI compartment, which is more stable. Tracking antigen-specific PCs supported this conclusion. Thus, although marrow PC reside in hypoxic niches, they may prefer to use aerobic respiration and employ glycolysis only as an ‘as needed’ basis.
Development of an experimental model of Campylobacter jejuni intestinal colonization in commercial turkeys
Authors: Sylte Matthew Jon , Looft Torey , Johnson Timothy A. , Wu Zuowei , Zhang Qijing
Published: January 2023

The cecum is the main site where poultry are asymptomatically colonized by thermophillic Campylobacter spp. Consumption of contaminated poultry products is one of the main sources of human campylobacteriosis, for which Campylobacter jejuni is responsible for approximately 90% of the cases. In chickens, experimental models of C. jejuni colonization to examine the host-response have been established, but a model remains to be developed for commercial turkeys. We sought to develop a model to characterize intestinal colonization and host response in commercial turkeys using mutants of C. jejuni (strain NCTC 11168) constructed to express different antibiotic resistance markers (chloramphenicol (CjCm) or kanamycin (CjK)). Turkey poults were colonized by oral gavage at 21 days of age with up to 5×108 cfu of either CjCm, CjK or sterile PBS. Up to 8 poults/treatment were euthanized at days 2, 7 and 14 post-exposure and intestinal samples were assessed for colonization and host-response. Both mutants were consistently recovered from cecum, but not ileum, throughout the study, with ~107 cfu/g detected from cecal contents at day 14 post-exposure. Mild inflammatory changes were histologically noted in the cecum and cecal tonsil of CjCm or CjK colonized poults at days 7 and 14 post-exposure. Using IHC, Campylobacter antigen was detected between cecal villi by day 7, indicating persistent colonization. Host-response for cytokines (IL-6, -8, -10, -17A, -22 and others) and β-defensins was analyzed by qRT-PCR from intestinal tissues. This study demonstrates that CjCm and CjK persistently colonize the cecum of commercial turkeys and will be useful to evaluate strategies to reduce intestinal colonization by C. jejuni in turkeys and promote food safety.