bluuebunny/crossref_metadata_2025 huggingface dataset:
Pneumoviruses can impair the central nervous system by different mechanisms
Authors: Kalergis Alexis M , Bohmwald Karen , Andrade Catalina A
Published: August 2023

Pneumoviruses such as the respiratory syncytial virus (hRSV) and the human metapneumovirus (hMPV) are the leading cause of acute lower tract respiratory infection, mainly in infants, elderly and immunocompromised individuals, causing high morbidity and mortality rates. However, these viruses can cause neurological alterations, such as encephalitis and encephalopathy. Viral RNA and pro-inflammatory molecules have been found in patients with neurological signs, supporting the notion of neuroinvasion and/or neuroinflammation caused by hRSV and hMPV. Therefore, this work seeks to evaluate the effects of these viruses on the brain. Accordingly, mice were infected with either hRSV or hMPV or treated with non-infectious controls (mock). Despite detecting viral load in the lungs of hMPV-infected mice, no viral load was detected in their brains, as it was shown for hRSV. However, pro-inflammatory cytokines such as IL-6 and IFN-g were increased only in the sera of hMPV-infected mice. Also, we observed differential patterns in the increase of cytokines in the brain of hRSV or hMPV-infected mice, including IL-6, TNF-a, and IL-4. Moreover, increased blood-brain barrier permeability was observed in mice infected with both viruses. Additionally, after several weeks post-infection, a Marble Burying (MB) test was performed, and we observed an impaired cognitive performance in mice infected with both viruses. All these results suggest that infection with these pneumoviruses can cause long-term behavioral impairment in mice. Our work provides new insight into the effect of hRSV and hMPV on the central nervous system and underscores the need to further understand how respiratory virus can damage brain function in humans.

This work was supported by ANID/FONDECYT grants #11221280; #1190830, ANID scholarship # 21210662, the Millennium Institute on Immunology and Immunotherapy ACE 210015, ICN09_016 / ICN 2021_045; former P09/016-F.
Redox extracellular vesicles induce neurotoxic cytokine production from innate immune cells
Authors: Bondada Subbarao , Zhao Yanming , Alhakeem Sara S , Ho Jenni , Collard James C , Chaiswing Luksana , Butterfield David Alan , St Clair Daret K
Published: August 2023

Therapy induced cognition impairment (TICI) or chemobrain is a well-recognized side effect of cancer therapy, which reduces quality of life for cancer survivors. Most cancer therapies induce reactive oxygen species, which can lead to cancer cell death, but they also promote neuronal cell death both directly and indirectly leading to TICI. High grade gliomas are one of the cancers that respond poorly to therapy and are associated with development of chemobrain. Currently, the mechanisms underlying chemobrain are not well understood. The systemic side effects such as cachexia, fatigue, and cognitive impairment are associated with sustained elevation of inflammatory cytokines and microglial cell activation. We tested the hypothesis that chemotherapy and radiation treatment of glioblastoma (GBM) leads to production of extracellular vesicles (EVs) with oxidized proteins that uniquely stimulate specific immune cells to produce cytokines like TNF-α which was shown to induce therapy induced cognition impairment (TICI) by causing neurotoxicity. Previously we showed that TNF-α has a major role in TICI, as it can cross the blood brain barrier and induce neurotoxicity. We found that chemotherapy or radiation induces production of EVs from cells and tissues that are targets of the drugs. These EVs contain more proteins adducted to 4-hydorxy nonenal (HNE) compared to controls. These EVs contain proteins released from tissues such as brain that are damaged by therapy. The HNE adducted EVs were better than control EVs in inducing macrophages and microglial cells to produce the pro-inflammatory cytokines, TNF-α, and IL-6. Serum IL-6 levels and IL-6 producing cells were significantly increased in vivo when C57BL/6 mice were injected with Redox EVs.

NIH R01 CA21793
Activation of PHACTR2 with oral NIM-1001 treatment enhances regulatory CD4+ T cell function and provides protection from systemic lupus erythematosus
Authors: Leber Andrew J , Hontecillas Raquel , Juni Nuria Tubau , Fitch Sarah N , Bassaganya-Riera Josep
Published: August 2023

Phosphatase and actin regulator 2 (PHACTR2) is an RPEL domain containing protein recently shown to be differentially expressed in regulatory CD4+ T cells (Tregs). Within rheumatic conditions, specifically systemic lupus erythematous, Tregs possess lesser suppressive capacity, are more likely to lose CD25 and FOXP3 expression and to experience low IL-2 environments. Using a combined loss-of-function and therapeutic approach, we sought to characterize the role of PHACTR2 in Treg biology and rheumatic disease. In a study of in vitro differentiation of naïve CD4+ T cells, Phactr2−/− cells differentiated into CD25hi FOXP3+ Tregs at a lesser proportion than WT cells. Using a bm12 adoptive transfer model of alloactivation, Phactr2−/− mice exhibit greater inflammation, demonstrated through increased spleen weight, greater incidence of ascites, and increased Th1 cells in the spleen post-transfer. Activation of PHACTR2 in an MRL/lpr model of lupus, by daily oral treatment with NIM-1001, protected against proteinuria and provided a three-fold increase in splenic Tregs. Similarly, in the NZB/W F1 model, oral treatment with NIM-1001 significantly reduced histological severity of disease in the kidney, increased splenic Tregs and decreased bone marrow plasma cells. NIM-1001 down-regulated multiple plasma chemokines and cytokines including CXCL1 and CCL4 in a dose-dependent manner. This data suggests that the PHACTR2 pathway merits development as a promising therapeutic mechanism for lupus and other diseases exhibiting impairments in Treg regulatory function.
Liver cells transduced with AAV.MOG increase the frequency of MOG-specific T regulatory cells capable of reducing EAE by adoptive transfer
Authors: Senior Kevin G , Min Daniel , Keeler Geoffrey D , Gaddie Cristina D , Chan Christopher , Wilson Olivia , Baker Maya , Hoffman Brad E
Published: August 2023

Multiple Sclerosis is a demyelinating autoimmune disease brought on by self-reactive, myelin-specific T effector cells. Immunosuppressants are the most utilized tool to combat disease progression; however, they bring unwanted side effects. We have previously published on a novel gene immunotherapy that uses an adeno-associated virus (AAV) containing the full sequence of the myelin oligodendrocyte glycoprotein (MOG) that restores tolerance through liver-directed gene therapy, resulting in the generation of antigen-specific T regulatory cells (Tregs) that can ameliorate Experimental Autoimmune Encephalomyelitis (EAE). While the data is promising, the mechanism of this therapy remains enigmatic. To begin defining the mechanism initially, we cultured MOG-specific CD4+ T cells from 2D2 mice with liver cells from B6 mice that received either AAV.MOG or a saline control. We found a significant increase in the frequency of CD4+CD25+FoxP3+ Tregs when 2D2 T cells were cultured with liver cells from AAV.MOG-vectored mice in a 1:2 ratio compared to the control. Next, we injected 2D2 mice with AAV.MOG and isolated CD4+CD25+ T cells 2 weeks later. To evaluate their suppressive ability they, or control cells, were adoptively transferred to recipient naïve B6 mice. After 24 hours, active EAE was induced. By day 15, disease severity in mice that received AAV.MOG donor 2D2 T cells was significantly reduced compared to the controls (CD4+CD25+T cells from wildtype B6 mice given either AAV.MOG or AAV.AQP4) Overall, these results demonstrated that livers transduced with AAV.MOG can induce 2D2 Tregs in vitro and that CD4+CD25+ T cells from vectored 2D2 mice reduce EAE severity.

Supported by a grant from the NIH (R01AI128074) and the Children’s Miracle Network
First-in-class PDE4D bifunctional degraders for inflammatory skin diseases
Authors: Shakya Arvind , Erdman Paul , Lai Chon , Baker Sherry , Orlandi Chiara , Greene Steven , Chaurasia Aparajita , Fung Leah
Published: August 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by erythema and pruritis, affecting over 15 million people in the US. Th2/Th17 cytokines play a key role in AD pathogenesis, resulting in an increased inflammatory response, disrupted skin barrier, increased susceptibility to infections and allergen sensitization. Phosphodiesterase 4 (PDE4) is a cyclic 3’,5’-adenosine monophosphate (cAMP)-specific phosphodiesterase and its expression is elevated in AD patients sustaining a highly inflammatory environment. PDE4 inhibition has been shown to effectively suppress proinflammatory cytokines in clinically validated approaches for several chronic inflammatory conditions. The current standard of care therapies including PDE4 inhibitors are linked to broad adverse effects due to their off-target activities. Utilizing our proprietary PRODEGY discovery platform, we have designed potent and selective PDE4D bifunctional degraders. PDE4D degradation dramatically decreases the pro-inflammatory cytokines released by activated T cells in vitro. Additionally, PDE4D is degraded efficiently in mice spleens treated with the lead PDE4D degrader in a dose-dependent manner, with no adverse effects. Importantly, our PDE4D degraders do not induce significant degradation of known neo-substrates, suggesting high specificity. Taken together, we propose the introduction of a more potent, specific, and safe PDE4D degrader as novel therapy for inflammatory skin diseases, such as AD.
Cytokine-driven integrin expression controls CD8 T cell homeostasis
Authors: Li Can , Park Jung-Hyun
Published: August 2023

The peripheral CD8 T cell pool comprises a mixture of naïve and memory-phenotype (MP) cells. While naïve CD8 T cells are continuously produced and supplied through thymic export, MP CD8 T cells are mostly generated by antigen activation or cytokine-mediated differentiation and maintained by homeostatic cytokines, such as IL-15. Both naïve and MP CD8 T cells are necessary to mount vigorous immune responses against new and reoccurring pathogens. But, the mechanisms through which MP CD8 T cells would avoid competing with naïve CD8 T cells for limited space and survival cues remain largely unsolved. Here, we report that integrin CD103 plays a critical role in this process. CD103 is highly expressed on naïve CD8 T cells to mediate adhesion to cells and tissues that are abundant for its ligand, E-cadherin. Interestingly, homeostatic cytokine signaling that drives MP CD8 T cell differentiation potently downregulated the expression of CD103, dissociating them from potential competition with CD103-positive naïve CD8 T cells. Conversely, the forced expression of CD103 on MP CD8 T cells resulted in the accumulation of MP CD8 T cells and selective loss of naïve CD8 T cells. Mechanistically, we identified a subset of dendritic cells that expresses E-cadherin (E-cad+ DCs), that would interact with CD103-positive CD8 T cells to provide survival signals and control their maintenance. Collectively, our results suggest that IL-15-mediated downregulation of CD103 contributes to balancing the size and composition of the peripheral CD8 T cell pool, revealing a previously unappreciated role of cytokine-driven integrin expression in controlling CD8 T cell homeostasis.

This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Two developmental pathways generate functionally distinct populations of natural killer cells
Authors: Ding Yi , Lavaert Marieke , Das Arundhoti , Harly Christelle , Malin Justin , Zhao Yongge , Bhandoola Avinash
Published: August 2023

Natural killer (NK) cells function by eliminating virus-infected cells or tumor cells during early defenses. However, the early development of NK cells and lineage relationships between NK cells and helper innate lymphoid cells (ILCs) remain elusive. Common precursors for ILCs (ILCPs) can differentiate into both helper ILCs and NK cells. Here, we identified a NK lineage-restricted progenitor population, early NK progenitor (ENKP), which does not develop from ILCPs, thus ENKP may represent the ILCP-independent pathway of NK cell development. Competitive chimera experiment shows ENKPs generate NK cells more efficiently than ILCPs, suggesting that ENKP-dependent pathway is the major pathway for NK cell development. scRNA-seq shows ENKP-derived NK cells express Ly49 receptors and higher levels of cytotoxic genes whereas ILCP-derived NK cells have very low expression of Ly49 receptors and express higher levels of genes implicated in tissue residency such as CD69 and CD200R. Furthermore, Ly49H+ NK cells which response to MCMV infection mostly develop from ENKPs but not ILCPs. Consistently, ENKP-derived NK cells but not ILCP-derived NK cells expanded dramatically after MCMV infection. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets.
Normalizing pathogen exposure via pet shop mouse cohousing leads to preferential maintenance of long-lived effector memory CD8 T cells
Authors: Lucas Erin D , Huggins Matthew A , Pierson Mark , Hamilton Sara
Published: August 2023

Studies of the CD8 T cell memory compartment in mice have primarily been done in young mice maintained in specific pathogen free (SPF) environment. The murine compartment is skewed towards central memory CD8 T cells, whereas adult human memory T cells are dominated by effector memory T cells. The murine memory compartment is akin to that of neonate humans, suggesting there are factors missing in SPF mice that are required to mature the T cell compartment. We wanted to ask how pathogen exposure changes the memory compartment of mice, so we utilized a model of pet shop mouse co-housing (COH), which exposes mice to a wide variety of normal mouse pathogens. COH mice display a substantial increase in long-lived effector CD8 T cells (LLEC). LLEC are a CD8 T cell memory population described by the Hamilton Lab that is phenotypically more effector-like and are highly effective at clearing systemic infections when compared to other memory T cells. However, LLEC display reduced homeostatic and antigen-driven proliferation in SPF mice leading to a lower representation in the memory pool. The factors in COH that support LLEC expansion and persistence are currently unknown, and it is also unclear if COH impacts LLEC function. Thus, the goal of this study is to understand what intrinsic and environmental factors contribute to LLEC formation and maintenance, as well as determine how LLEC function contributes to memory responses in COH mice. I have found that COH LLEC are more polyfunctional compared to SPF LLEC, and my preliminary work has found that LLEC turnover is increased in COH mice. Future studies will determine what specific inflammatory cues lead to LLEC expansion, and if this process can be altered to improve T cell responses to infection and vaccination.

Supported by grants from the NIH (R01 AI155468)
The role of GPR132 in regulating T cell function
Authors: Tovar Giuseppina Marchesini , Bergsbaken Tessa
Published: August 2023

CD8+ T cells are vital in controlling pathogens and undergo robust expansion upon priming. Tissue-resident memory T cells (Trm) that reside in non-lymphoid tissues are less responsive in terms of expansion; however, the signals within the tissue that direct the quantity and quality of CD8+ Trm pools are still unclear. G protein coupled receptors (GPCRs) sense and integrate environmental signals and are essential for regulating T cell function. GPR132 or G2A modulates homeostasis in mature peripheral T cells, and Gpr132−/− mice develop an autoimmune syndrome accompanied by expansion of the T cell compartment. GPR132 ligands are synthesized by intestinal epithelial cells and commensal bacteria, making GPR132 a potential target for regulating T cell expansion in the intestinal tissue. We investigated the function of GPR132 during intestinal infection by Y. pseudotuberculosis.GPR132 expression was elevated on T cells early after infection . Gpr132−/− cells displayed enhanced expansion, outnumbering wild-type (WT) cells by 3–4 fold in all tissues. Early differentiation was not impacted by GPR132-deficiency, as there were no differences in circulating and Trm memory precursor subpopulations between WT and Gpr132−/− cells. To dissect the mechanism of GPR132 action, we examined the transcriptome of WT and Gpr132−/− naïve T cells. Minimal differences in gene expression were observed, and both showed comparable proliferation upon TCR-engagement in vitro. These data suggest enhanced responsiveness of Gpr132−/−T cells was not programmed at the naïve stage but develops during T cell activation. Our results expose a role for GPR132 signaling axis in regulating T cell immunity and demonstrate GPCRs can be targeted to modulate T cell responses.

New Jersey Commission on Cancer Research Predoctoral Fellowship Grant COCR23PRF011.
Sublethal whole-body irradiation induces permanent loss and dysfunction in pre-existing pathogen-specific memory CD8 T cell populations
Authors: Heidarian Mohammad , Jensen Isaac J , Hassert Mariah , Kannan Shravan Kumar , Xue Hai-Hui , Harty John T , Badovinac Vladimir P
Published: August 2023

The increasing use of nuclear energy sources inevitably increases the risk of accidental or deliberate radiation-exposure. However, the extent to which radiation-exposure impacts memory CD8 T cells, potent mediators of immunity to recurring intracellular infections and malignancies, remains understudied. Using P14 LCMV immune chimeric mice (P14 chimeras), we observed that sublethal (5Gy) whole body irradiation (WBI) induced a rapid decline in the number of naive (TN) and P14 circulatory memory CD8 T cells (TCIRCM) with the former being more susceptible to radiation-induced death. While TN cell numbers rapidly recovered, the number of P14 TCIRCM cells remained low at least nine months after radiation-exposure. Additionally, the remaining P14 TCIRCM in irradiated hosts exhibited an inefficient transition to a central memory (CD62Lhi) phenotype compared to non-irradiated P14 chimeras. WBI also resulted in long-lasting T-cell intrinsic deficits including diminished cytokine and chemokine production in response to antigen-stimulation along with impaired secondary expansion upon cognate Ag re-encounter. Irradiated P14 chimeras displayed significantly higher bacterial burden after challenge with L. monocytogenes expressing GP33 compared with the non-irradiated controls, likely due to radiation-induced numerical and functional impairments. Taken together, our findings suggest that sublethal radiation-exposure caused long-term numerical decline, impaired differentiation, and functional dysregulation in pre-existing TCIRCM, which rendered previously-protected hosts susceptible to re-infections.

Supported by NIH Grants GM134880, AI114543, The Holden Comprehensive Cancer Center at The University of Iowa and its National Cancer Institute Award P30CA086862
Deciphering molecular and functional codes of human memory lymphocytes
Authors: Singh Amit K , Roy Roshni , Kaileh Mary , Sarantopoulou Dimitra , Hernandez Dena , Arepalli Sampath , Bektas Arsun , Kim Jaekwan , McKelevy Julia , Zukley Linda , Dunn Christopher , Nguyen Cuong , Wallace Tonya , Wood William , Piao Yulan , De Supriyo K , Sen Jyoti Misra , Weng Nan-ping , Ferrucci Luigi , Sen Ranjan
Published: August 2023

Long-term immunity against infections and effective response to vaccination are dependent on unique feature of lymphocytes referred as “memory generation”. Majority of our understanding is established on murine data and our understanding of human naive to memory differentiation is limited. In our ongoing study we explored epigenetics (DNA methylation and accessible chromatin), transcriptomics (basal, activation and proliferation induced gene expression), intracellular cytokine assay (high-parameter flow cytometry) of naive and memory B and T lymphocytes subsets from the healthy donors. We explored the epigenetic changes associated with memory differentiation. However, relationship between epigenetics and transcriptomic outcomes didn’t explain entirely the properties of being memory or naïve lymphocyte. To understand mechanistic differences, naive and memory cells of B and CD4 T lymphocytes were activated in vitro using either antigen receptor (BCR or TCR crosslinking with antibodies) or PMA+Ionomycin (pan-activation) and assayed for early or late gene expression and for intracellular cytokine profiles. Surprisingly, majority of transcriptomic patterns were similar between naive and memory lymphocytes while their cytokine expression profiles were significantly different. These findings indicate the significant role of translational machinery influencing the memory and naïve lymphocyte functions. Our results are in direction of segregating transcription and translation mediated dependencies of adaptive memory. These findings will provide the foundation for future studies related to dysregulation of memory cell differentiation and function in aging and infections.

This project was entirely supported by intramural funding of NIA-NIH.
Transcription factor Nfil3 is a regulator of CD8 T cell differentiation in mouse Influenza A infection
Authors: Sportiello Michael John , Topham David J
Published: August 2023

After an acute respiratory infection is cleared, most T cells that controlled the infection die off. Tissue resident memory CD8 T cells (TRM) remain in the lung, poised to fight off a recurrent infection. These TRM have large impacts on the survival and morbidity of subsequent infection. While many have studied this subset to understand what causes differentiation into this essential T cell subset, a master regulator of this phenotype remains elusive. To these ends, RNA and ATAC sequencing experiments of TRM were conducted after viral clearance to investigate the existence of a master regulator. Transcription factor Nfil3 became a likely candidate after integrated omics analyses. Flow cytometry to fully characterize the levels of Nfil3 in all T cell subsets of the lung, spleen, and draining lymph node was conducted 7, 14, and 43 days post infection (dpi). At days 7 and 14 dpi, Nfil3 correlated with pre-TRM markers. At 43 dpi, Nfil3 was highest in TRM and lowest in central memory T cells. Finally, experiments were performed to investigate if Nfil3 played a causal role in TRM development or maintenance. T cells were transduced with Nfil3 overexpression constructs and adoptively transferred into mice, which were then infected. Notable changes in differentiation were observed between treatment and control. T cell proliferation was also affected ex vivo in a proliferation dye assay, implying that Nfil3 plays a role in the acute response. Finally, using a CD8-specific inducible floxout system, Nfil3 was floxed out during acute infection, recently post viral-clearance, and 6 weeks after during the memory phase to appreciate the effect on T cell differentiation. Overall, we argue Nfil3 plays a role in CD8 T cell differentiation in respiratory infections.

This work was funded by a Program Project grant through the National Institutes of Health, National Institute of Allergy and Infectious Diseases P01-AI102851 and through the National Institutes of Health training grant T32GM007356-46 and the National Institutes of Health training grant T32HL066988-20.
The majority of circulating human B cells belong to a naïve anergic B cell subset distinguished by expression of CD73
Authors: Dizon Brian , Pierce Susan
Published: August 2023

In humans, it is estimated that ~40% of peripheral blood (PB) naïve (CD19+ CD27− CD45RBMEM− CD38−) B cells are in a state of anergy defined as IgD+ IgMlow. Presumably these anergic B cells express only weakly autoreactive B cell receptors (BCR) that allowed them to escape elimination by central tolerance mechanisms in the bone marrow, but were sufficient to induce anergy in the periphery. Although the ultimate fate of these B cells is not known, it is hypothesized that anergic B cells undergo a process of redemption in which they are activated and somatically hypermutate away from self, allowing them to be recruited into foreign antigen-specific responses. To better characterize human anergic B cells, we utilized a panel of 18 antibodies specific for B cell surface markers and BCR isotypes, which allowed identification of the known human B cell subsets by flow cytometry. We determined that ~50% of B cells were contained in the naïve compartment, of which ~60% were anergic (IgD+ IgMlow) cells distinguished by the expression of CD73. In contrast, CD11c+ CD27− B cells (termed ABCs) and CD45RB+ CD27− MBCs comprised only 0.6% and 7.0% of circulating anergic B cells, respectively. Using 9G4 to identify inherently autoreactive B cells expressing germline VH4–34, we found that the majority of 9G4+ cells were naïve anergic B cells that express CD73. Thus, in humans the CD73+ naïve compartment appears to be the major reservoir of anergic B cells, possibly for redemption and participation in immune responses to pathogens. Studies are underway to define the responses of these B cells in health and disease.

This work was supported by the Intramural Research Program of the US National Institutes of Health, National Institute of Allergy and Infectious Diseases, and National Institute of Arthritis and Musculoskeletal and Skin Diseases.
Neutrophils show distinct patterns of activation in the context of infection with different pathogens
Authors: Chen Fei , Espinosa Vanessa , Wu Wenhui , Peignier Adeline , Wang Keyi , Lothstein Katherine , Rivera Amariliz , Parker Dane , Gause William C
Published: August 2023

Alternatively activated neutrophils (N2) enhance lung anti-helminth trained macrophage effector function in response to infection with the helminth Nippostrongylus. brasiliensis (Nb) (Fei Chen, et al., NI, 2014, CR, 2022). However, potential differences in activation patterns of neutrophils with different pathogens is not yet well studied. We report here lung neutrophils bear unique gene expression profiles at 2 days after infection with Nb, fungi (Aspergillosis fumigatus (Af)), and bacteria (Staphylococcus aureus (SA). AF and SA triggered genes associated with cell death and autophagy but differed greatly in metabolic activity. In contrast, Nb induced gene expression patterns characteristic of type 2 immunity, xenobiotic pathways, and cell proliferation. Further analysis revealed increased edu incorporation, KI-67 and IL-13 expression in neutrophils in the lung but not in the blood and bone marrow. Furthermore, the proliferating neutrophil subset predominantly expressed IL-13 and was IL-4R dependent. To directly examine neutrophils proliferating in the lung after Nb infection, Nb-primed neutrophils (CD45.2) were transferred to CD45.1 Nb inoculated recipients. At day 10 after inoculation, donor neutrophils were readily detected and were proliferating as measured by ki-67 staining and edu incorporation. These studies suggest that neutrophils exhibit distinct activation states following infection with different pathogens. Unlike neutrophils typically associated with microbial infection, where rapid turnover from cell death typically occurs, neutrophils activated following Nb infection persist and undergo cell cycling within the remodeled lung tissue microenvironment.
Plasmodium falciparum mRNA liver stage antigens induce antibody and liver CD8 TRM and CD8 TE cell responses in mice
Authors: Tan Steven , Alameh Mohamad-Gabriel , Renn Jonathan P , Allen Joselyn Natasha , Pichugin Alexander V , Duffy Patrick E , Weissman Drew , Krzych Urszula
Published: August 2023

Plasmodium spp. cause more than half a million malaria deaths annually and the only approved vaccine, RTS,S, based on P.falciparum (Pf) circumsporozoite protein (CSP), confers incomplete and non-durable protection. A strategy to improve malaria vaccines involves combining Ags of different Plasmodium stages such as sporozoite (spz) and liver stage (LS). This strategy promises a broader repertoire of specific antibodies (Ab), CD4 and CD8 T cells. We have shown that mice immunized with a combination of P. berghei (Pb) CSP and LS Ags expressed as DNA-Adenovirus generate higher protection than CSP alone. Protection is associated with Ag-specific Abs and liver CD8 TRM cells. We hypothesized that spz that escaped CSP-specific Abs were targeted by LS-Ag-specific responses. Our current efforts are directed towards utilizing mRNA template as it can accommodate many Ags. We designed mRNA-LNP expressing single Pf-LS Ags, 305w and SHMT, based on protective rodent parasite LS Ag orthologues, for immunization of mice. Analyses of mRNA-Pf-305w- and Pf-SHMT-induced immune responses show a divergence of reactivities. Whereas Pf-305w induced high Ab titers, Pf-SHMT induced liver CD8 TRM and CD8 TE cells that exceeded Pf-305w-induced CD8 T cells. Peptides corresponding to Pf-SHMT recalled high IFN-g responses, a signature of protective immunity. We continue to characterize and evaluate contributions of T cell and B cell responses induced with combinations of Pf-LS Ags including Pf-CSP to protective immunity against transgenic parasites. mRNA vaccines provide a rapid, flexible platform to explore malaria vaccine improvements where liver CD8 TRM and CD8 TE cells, B cells and Abs may be crucial for highly efficacious and protracted protection.
Natural antibodies mediate resistance to Acinetobacter baumannii respiratory infections
Authors: Wilson Timothy James , Islam Aminul A , Actis Luis A
Published: August 2023

Acinetobacter baumannii is a growing public health threat due to increasing incidence of carbapenem-resistant strains in U.S. hospitals. With widespread antibiotic resistance, there is an urgent need for alternative therapies to treat these infections, which are often acquired in hospital intensive care units. Antibody-based immunotherapies offer one potential treatment avenue, however, adaptive immune responses to A. baumannii have not been well characterized. Pilot experiments, using a mouse pneumonia model to explore the role of adaptive immunity in clearing A. baumannii, uncovered an unexpected and severe defect in resistance to A. baumannii infection in Rag2−/− mice during the first 48 hours after infection. Subsequent experiments showed that intranasally infected Rag2−/− or B cell-deficient μMT mice are impaired compared to wild type mice in clearing bacteria from lung, liver, and spleen at 24 hours post infection. Prophylactic administration of normal mouse serum or purified immunoglobulins from naive mice demonstrates a protective effect of natural antibodies (NAbs) by rescuing Rag2−/− mice from severe infection. Analysis of C3 complement protein binding demonstrated that NAbs increased C3 protein deposition on A. baumannii cells, indicating the activation of the classical complement pathway as a potential mechanism of action. Overall, our study demonstrates a previously unknown role for natural antibodies in the innate resistance to Acinetobacter pneumonia, a finding that may lead to the development of effective therapies against human infections caused by this antibiotic-resistant pathogen.
Improving E. coli-based anti-cancer bacterial therapeutics
Authors: Chen Noah , Danino Tal , Arpaia Nicholas
Published: August 2023

Bacteria-based cancer immunotherapy has existed since Dr. William Coley employed deliberate bacterial infection of tumors over a century ago. Bacteria are inherently immunogenic, self-replicating therapeutics which can be tuned to deliver various payloads, modify specificity, and decrease side effects. Though bacteria are intrinsically capable of selectively colonizing tumor tissue in mouse models, less is understood about the mechanisms behind such colonization. This is especially important as early clinical trials of anti-cancer bacterial therapeutics have been stymied by low colonization rates in human patients. This study aims to identify mechanisms by which E. coli evade clearance by the immune system specifically within tumors. Currently, we have screened E. coli mutants for tumor colonization efficiency by assessing strain prevalence after recovery from tumors in immunocompetent C57BL/6 mice. We have identified pathways which either promote or inhibit E. coli tumor colonization and validated these with competitive infection experiments. This work paves the way for greater understanding of what bacterial mechanisms allow E. coli to home to the tumor niche, which may have implications for both developing anti-cancer bacterial therapeutics as well as for understanding how bacteria colonize solid tumors.

Supported by grants from NIH (R01 CA249160, T32 GM145440)
The CXCR3 chemokine receptor is critical for the protective immune response to Plasmodium parasite infection of the liver
Authors: Reynolds Laura M , Blyn Rebecca , Wegmair Lisa , Lewis Patrick , Sheikh Amina , Okolo Vera , Wilder Brandon , Kappe Stefan , Minkah Nana
Published: August 2023

No highly efficacious anti-malaria vaccine exists, but whole parasite immunization (WPI) where hosts receive live attenuated Plasmodium parasites that arrest in the liver is the only strategy that confers complete protection against future Plasmodium parasite infection. Liver-resident memory CD8 T cells (TRM) that express the chemokine receptor, CXCR3, are essential for WPI-induced protection. Yet, whether CXCR3-dependent signaling plays a functional role in anti-parasite TRM immunity is not fully understood. RNA-seq analyses on hepatocytes isolated from Plasmodium-infected mice showed an upregulation of the CXCR3-dependent chemokines, CXCL9 and CXCL10. We examined protection in WT and CXCR3−/− mice upon WPI and report that in contrast to WT animals, CXCR3−/− mice exhibit impaired control of a parasite challenge given 40 days after immunization. Next, we set out to enumerate T cell recruitment and TRM numbers in the liver after WPI. CXCR3−/− mice exhibited reduced CD8 T cell numbers early after immunization yet by day 40 contained equivalent TRM numbers as compared to immunized WT counterparts. We hypothesize that while CXCR3 signaling is critical for early recruitment of activated CD8 T cells into the Plasmodium-infected liver, other chemokines compensate for the loss of CXCR3 ensuring that equivalent numbers of CD8 T cells are eventually recruited into the liver. However, as CXCR3−/− mice exhibit impaired control of infection despite containing equivalent TRM numbers, the CXCR3 axis must play other unidentified roles. Immune cells are known to be asymmetrically positioned in the liver. Ongoing studies will determine if CXCR3 is critical for the optimal positioning and function of liver TRMs after parasite infection.
Identification and characterization of CXCL13 producers in bone tissue
Authors: Okawa Takuma , Nagai Motoyoshi , Nakata Kazuaki , Dohi Taeko , Kawamura Yuki I , Hase Koji
Published: August 2023

The bone serves as a reservoir for several immune cell subsets in response to the low-energy status such as fasting. We previously demonstrated that naïve B cells transiently migrate from Peyer’s patches to the bone marrow during fasting in mice. This B-cell dynamics is attributed to upregulation of CXCL13 in the bone during fasting. However, CXCL13 producers in the bone remain to be determined. We observed that lineage-negative population from the bone is responsible for Cxcl13 expression. To further clarify the CXCL13 producers, we performed single-cell RNA-sequencing of bone linage-negative cells from Ad libitum-fed mice and fasting mice. UMAP analysis manifested that the numbers of fibroblasts and multipotent stem cells (MSCs) were increased in the fasting group. Interestingly, Cxcl13-producing cells were identified as osteogenic-MSCs characterized by expressions of Sca-1 (immature cells marker), Sema3c, and Dcn (osteogenic cells maker). This population also expressed an inflammatory cytokine Il6. Meanwhile, mature osteoblasts and chondrocytes expressed neither Cxcl13 nor Il6. Furthermore, co-culture with the plasma from fasting mice upregulated CXCL13 expression in osteogenic-MSCs in vitro, suggesting that a soluble factor(s) in the circulation induces CXCL13. Taken together, osteogenic-MSCs in the bone may sense nutritional status and produce CXCL13 to regulate naïve B-cell dynamics along the gut-bone axis. The pathophysiological role of CXCL13-producing osteogenic-MSCs is currently under investigation.

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The TNF superfamily cytokine receptor Death Receptor 3 is a costimulatory molecule for iNKT cells
Authors: Liman Nurcin , Park Jung-Hyun
Published: August 2023

Invariant NKT (iNKT) cells are a small population of thymus-generated T lymphocytes with innate-like phenotype and function that play a disproportionally important role in immune regulation and surveillance. iNKT cells are generated upon strong agonistic TCR signaling by glycolipid-loaded CD1d molecules, which differs from conventional T cells that are positively selected by weak TCR engagement with peptide-loaded MHC molecules. Whether iNKT cells are also distinct in their costimulatory requirement, however, has remained largely unresolved. Here, we report that the TNF superfamily receptor death receptor 3 (DR3) plays a previously unappreciated costimulatory role in peripheral iNKT cell activation. We found that the co-injection of agonistic anti-DR3 antibodies significantly augmented the activation and proliferation of mature iNKT cells in α-GalCer injected mice. Moreover, iNKT cells of α-GalCer and anti-DR3 co-injected mice dramatically increased their expression of proinflammatory cytokines, resulting in severe thymic atrophy. The DR3 costimulatory effect was strictly dependent on iNKT cells, because iNKT-deficient Traj18−/− mice were refractory to α-GalCer and anti-DR3 costimulation-induced inflammation. Because DR3 ligation alone did not suffice to activate iNKT cells, and DR3 exerted its effects only in the context of TCR costimulation, these results strongly support DR3 being a new bona fide costimulatory molecule for iNKT cells. Considering the interest in utilizing iNKT cells in adoptive T cell therapies and as anti-tumor bioreagents, employing DR3 as a tool to boost iNKT cell immunity will open new venues in translational and clinical research.

This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Interferon regulatory factor 5 involves immune responses through regulation of ly6c expression
Authors: Heo Sun-Hee , Park Suk Young , Kim Na Hyun , Song Eunkyeong , Lee Sei Won
Published: August 2023

Chronic obstructive pulmonary disease (COPD) is characterized by impaired lung function, prolonged airflow obstruction. COPD is mainly induced by long-tern exposure to toxic agents such as particles from cigarette smoke (CS). Interferon regulatory factor 5 (IRF5) is a key transcription factor related to many inflammatory diseases. IRF5 has been involved in polarizing macrophages and recruiting neutrophiles during various conditions. The purpose of this study was to assess immune responsiveness of IRF5 in lung disease. We generated mice deficient in the Irf5 gene (Irf5−/− mice). IRF5 expression was detected in lung and spleen of wild type (WT) mice but not in Irf5−/− mice. After whole-body inhalation exposure of CS, WT and Irf5−/− mice were decreased body wight and showed changed pressure-volume loop. In addition, the number of immune cells in the bronchoalveolar lavage fluid (BALF) was remarkably increased both in WT and in Irf5−/− mice after smoking compared to the non-smoking groups of WT and Irf5−/− mice. The CD11b+F4/80-CCR2+Ly6C+ cells were significantly increased in the BALF of smoking-exposed Irf5−/− mice compared to in that of smoking-exposed WT mice. Also, fecal microbiota analysis showed differences in bacterial abundances between WT smoking and Irf5−/− smoking mice. Taken together, our present findings suggest smoking-exposed Irf5−/− mice showed higher level of Ly6C expression in immune cells of the BALF and differential bacterial abundances in the feces. Further studies are required to elucidate IRF5-regulating Ly6C expression and determine potential therapeutic applications for COPD.
Reciprocity between IL-4 and opioid immune regulation: IL-4 primes human CD4+ T cells for opioid receptor driven MAP kinase signaling
Authors: Golovan Kenneth S , Womelsdorf Michael , Levine Alan D
Published: August 2023

Opioid receptor (OR) signaling induces transcriptomic and functional changes in human T cell responses at both a molecular and cellular level, yet the signaling pathways initiated by OR engagement in human T cells are poorly characterized. ORs are G-protein coupled receptors with three subtypes: MuOR, KappaOR, and DeltaOR. Using the human lymphoma Jurkat T cell line and negatively selected human peripheral blood CD4+ T cells, the effects of opioid exposure on the activation kinetics of the MAP kinases ERK 1/2, JNK, and p38, scored as their level of phosphorylation, was followed by immunoblot. Opioid-driven phosphorylation of the MAPKs was dependent on IL-4 pretreatment of Jurkat cells, but not blood-derived CD4+ T cells. After Jurkat cells were primed with a 3-day exposure to IL-4, a series of opioid agonists selective for each OR subtype induced the phosphorylation of all three MAPKs (ERK, JNK, p38) within 2 min. Phosphorylation of the MAPKs was independent of OR subtype and opioid agonist. Notably, the kinetics were different than that observed in neurons. When primary human CD4+ T cells were treated with opioids, only p38 MAPK, not ERK or JNK, was activated with similar kinetics and OR subtype independence as that seen in Jurkat cells. This difference between primary cells and Jurkat cells suggests an additional level of regulation of opioid signaling in primary cells not found in Jurkat cells. The requirement for IL-4 pretreatment suggests that opioids may play a role in Th2 biology. Preliminary data indicate that morphine modestly increases the expression of IL-4 mRNA. Overall, these results reveal that p38 functions as a primary effector of opioid receptor signal transduction in human T cells.

Supported by grants from NIH / NIDA (R01 DA 043253,)
Assessment of The Impact of Oncolytic Virotherapy on The Immunopeptidome Profile of Tumor by Utilizing GEMM Derived Immunocompetent GBM Models
Authors: Piranlioglu Raziye , Ahn Ryuhjin , Nakashima Hiroshi , Islam Anan , Guda Rahul , Charest Al , Goins William F , White Forest M , Chiocca E Antonio A
Published: August 2023

Immunotherapy clinical trials have not shown efficacy in glioblastoma (GBM), arguably the deadliest of all cancers. A microenvironment, characterized by a paucity of T cells recognizing tumor peptides displayed on major histocompatibility complexes of tumor cells is one of the main reasons for GBM’s evasion. Virotherapy provides the opportunity to reshape the GBM TME towards pro-inflammatory phenotype. Data from a recent clinical trial with herpes simplex-1 based oncolytic virus (oHSV) have shown that the administration of these agents leads to a rapid influx of CD8 T cells, and activation of myeloid cells, suggesting that oHSV may offer an in-situ vaccination approach to increase availability of actionable antigens. C57BL/6-derived GBM models do not mimic the outcome observed with patient derived tumors in terms of virus replication. To decipher the impact of oHSV on tumor antigen specific responses, better immunocompetent models are needed.

To assess whether oHSV treatment modulates the immunopeptidome profile (IP) of tumor, we used two transplantable GEMM-derived GBM models, 1620 and 1694. Both have wild type human EGFR, loss of Pten and Cdkn2a in BALB/c background. These models were more receptive to infection than CT2A. In vivo, virus persisted in tumors for up to 6 days, but not for 4 days in CT2A. IP analyses revealed that oHSV promotes epitope presentation on tumor cells and in TME. Moreover, low dose of virus efficiently induced antigen availability in both models though it differentially modulated the MHC ligandome.

Using two new GBM models, we showed that an oncolytic virus used in clinical trial with recurrent GBM (NCT03152318) modulates IP of tumors, and low dose of virus is sufficient to induce the presentation of tumor epitopes.

This project is supported by Bridge Fund given to Drs. Chiocca and White and P01 CA236749 to Dr. Chiocca.
Innate and adaptive mechanisms induced by BCG immunotherapy in melanoma murine model
Authors: de Queiroz Nina Marí Gual Pimenta , Borges Vinícius Martins , Caldeira Christiane Vieira Alves , Marinho Fábio Antônio Vitarelli , Oliveira Sergio Costa
Published: August 2023

BCG (Bacillus Calmette-Guérin) vaccine has been proposed in clinical trials for the treatment of melanoma since 1970, however its mechanism of action still not fully understood. The purpose of this study was to investigate the immune mechanisms involved in the BCG immunotherapy against melanoma. We first characterized the immunological profile of the mouse melanoma B16–F10 cell line compared to BMDMs (bone marrow-derived macrophages) infected with BCG or stimulated with agonists for different innate immune pathways. B16–F10 were only capable to respond after poly I:C and dsDNA90 stimulus inducing CXCL10 and IFN-β, but no response against BCG infection. Moreover, we evaluated BCG intratumoral treatment in a B16–F10 subcutaneous mouse tumor model in C57BL/6 wild type (WT) and several knockout mice (MyD88−/−, TLR3−/−, TLR4−/−, TLR7−/−, TLR3/7/9−/−, cGAS−/−, STING−/−, IFNAR−/−, Caspase 1−/−, Caspase 11−/− and IL-1R−/−). BCG dramatically reduces melanoma in all the mice strains, except for MyD88−/−, suggesting a role played by this adaptor molecule related to the innate immune response. In order to investigate the adaptive immunity, we evaluate BCG treatment in IFNγ−/− and Rag−/−, which demonstrated the importance of lymphocytes for tumor regression. The experiments using B16–F10 infected with BCG and co-cultured with spleen cells or BMDMs from WT or MyD88−/− mice demonstrated that MyD88 is essential for inflammatory cytokines production. MyD88 is also important to regulate the recruitment of immune cells in the tumor microenvironment. Our data suggests that BCG immunotherapy in melanoma depends on MyD88-related innate immune pathway and adaptive response for an efficient tumor control.

This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPQ (152478/2022-1) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG (5.18/2022).