bluuebunny/crossref_metadata_2025 huggingface dataset:
Intranasal adjuvant immunotherapy re-activates immunity and enhances survival in murine metastatic Lymphangioleiomyomatosis
Authors: Maisel Katharina
Published: January 2023

Lymphangioleiomyomatosis (LAM) is cystic lung disease affecting primarily women of age. Currently there is no cure for this disease and only modest progress has been made with the introduction of sirolimus as LAM treatment. Recently, we and others have identified that LAM may also induce immunosuppression, similar to cancer. Adjuvant-based therapies like toll-like receptor (TLR) agonists are a promising strategy to activate cancer immunity. We used a murine model of metastatic LAM, to test therapeutic efficacy of TLR agonists PolyI:C, MPLA, and CpG, administered intranasally 2x/week. We found that only CpG enhances survival in murine LAM and that this treatment synergized with anti-PD1 therapy, which we had previously found to enhance survival in murine LAM. We found that the inflammation induced by CpG must be carefully balanced with its beneficial effects – in early disease, 5 μg of CpG was more beneficial than 10 μg. However, in progressive disease, 10 μg did not enhance survival while 20 μg CpG did. Furthermore, we found that a single dose of CpG reduced regulatory T cells and migratory dendritic cells (mDCs) and increased ICOS on T cells in the lungs. CpG also increased mDCs in the lymph nodes, and these were reduced after CCR7 blockade. We also found that repeated administration of CpG reduced immune cell infiltration into the lungs, which suggests a tolerogenic effect that may contribute to the incomplete survival. Overall, our data suggest that CpG may be a new treatment for LAM, that its beneficial effects may be in part mediated by CCR7 dependent DC migration, and that dose and inflammation must be carefully monitored to optimize therapeutic efficacy.

Supported by the ALA Dalsemer Award and LAM Foundation Career Development Award.
Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19
Authors: Zheng Jian , Wong Lok-Yin Roy , Wilhelmsen Kevin , Li Kun , Ortiz Miguel E , Schnicker Nicholas J , Thurman Andrew , Pezzulo Alejandro A , Szachowicz Peter J , Li Pengfei , Pan Ruangang , Klumpp Klaus , Aswad Fred , Rebo Justin , Narumiya Shuh , Murakami Makoto , Zuniga Sonia , Sola Isabel , Enjuanes Luis , Meyerholz David K , Fortney Kristen , McCray Paul B , Perlman Stanley
Published: January 2023

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice. We now demonstrate that mRNA expression of PLA2G2D and PTGDR, a PGD2 receptor, and production of PGD2 increase in human PBMC-derived dendritic cells with aging and after SARS-CoV-2 infection. Using our virulent mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR, or PLA2G2D are protected from severe disease. Further, treatment with a PTGDR antagonist, Asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.

This work is supported in part by grants from the National Institutes of Health USA (NIH) (P01 AI060699 (SP, PBM) and RO1 AI129269 (SP) and BIOAGE Labs (SP). The Pathology Core, is partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH Grant P30 DK-54759), and the Cystic Fibrosis Foundation. PBM is supported by the Roy J. Carver Charitable Trust.
Chemokine CXCL10’s role in the immune response to SARS-CoV-2
Authors: Ivester Hannah , Morrison Holly , Finkielstein Carla , Duggal Nisha , Weger James , Allen Irving C
Published: January 2023

Since the beginning of the SARS-CoV-2 pandemic in early 2020, it was apparent that one of the most striking clinical features of the disease caused by the virus, COVID-19, was the lung damage that occurs. This lung damage is often the result of an immune response gone awry as the host attempts to clear the virus. This overactive immune response, the ‘cytokine storm,’ is responsible for much of the most severe cases of COVID-19 as well as the Multisystem Inflammatory Syndrome in pediatric patients. Since the start of the pandemic, a few key players have been identified in the progression of COVID-19 disease as well as the immune response to it. These include pro-inflammatory cytokines IL-6, TNF-alpha, and CXCL10. Many clinical reports have especially shown the importance of the chemokine CXCL10 as a prognostic biomarker with patients having higher levels of CXCL10 often having more severe outcomes. Recent studies have also shown an increase in CXCL10 and its associated pathways in animal models of COVID-19. Here we continue to provide evidence for the importance of the protein CXCL10 in SARS-CoV-2 immunity through whole genome transcriptomics studies in human and k18-hACE2 mice. Following these, further studies with a mouse-adapted SARS-CoV-2 virus and Cxcl10−/− mice also showed a decreased level of effectiveness in viral clearance in the absence of effective CXCL10. These data continue to show the importance of the chemokine CXCL10 in both disease severity and viral clearance of SARS-CoV-2 infection adding to the growing body of evidence related to SARS-CoV-2 pathobiology.
B cell convergence to distinct broadly reactive epitopes revealed by chimeric hemagglutinin vaccination
Authors: Guthmiller Jenna , Li Lei , Lan Linda Yu-Ling , Henry Carole , Stamper Christopher , Freyn Alec , Utset Henry , Han Julianna , Palese Peter , Coughlan Lynda , Ward Andrew , Krammer Florian , Wilson Patrick C
Published: January 2023

Seasonal influenza virus vaccines provide modest protection against endemic viruses but lack effectiveness against pandemic-threat viruses. Therefore, new immunogens and vaccine platforms that induce antibodies against broadly protective epitopes are needed to provide robust protection against seasonal and novel influenza viruses. In a phase 1 clinical trial, the chimeric hemagglutinin (cHA) immunogen induced antibody responses against the conserved HA stalk domain. However, the landscape of the B cell specificities and subsets induced by this vaccine remain undetermined. Using single cell RNA-sequencing and repertoire sequencing, we identified that formulation of the cHA vaccine differentially induced B cell responses against distinct epitopes. Notably, those participants that received an inactivated vaccine with a squalene-based adjuvant had a greater proportion of B cells targeting two conserved neutralizing epitopes of the stalk domain: the central stalk and anchor epitopes. Moreover, participants that received an inactivated vaccine with adjuvant had distinct transcriptional B cell subsets, including an increase in atypical memory B cells. B cell clonal overlap between acute and memory timepoints was substantial, with several multi-donor clones that targeted distinct broadly protective epitopes. In addition, we identified stalk targeting germline antibodies that could neutralize recent pandemic H1N1 viruses. Altogether, our study reveals that the inactivated cHA vaccine with an oil-in-water adjuvant promotes B cell activation and maturation against broadly protective epitopes.

Supported by National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health grant numbers K99AI159136 (JJG), U19AI082724 (PCW), U19AI109946 (PCW), U19AI057266 (PCW), P01AI097092 (PP), R01AI145870-01 (PP), R21AI146529 (LC), and T32AI007244-36 (JH), the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) grant number HHSN272201400005C (PCW), HHSN272201400008C (LC, FK, PP), and the NIAD Centers of Excellence for Influenza Research and Response (CEIRR) grant number 75N93019R00028 (PCW, FK, PP). This work was also partially supported by the NIAID Collaborative Influenza Vaccine Innovation Centers (CIVIC; 75N93019C00051, FK, PP, ABW, PCW).
Identifying maternal humoral immune responses associated with control of viremia after primary maternal CMV infection in a rhesus macaque model
Authors: Otero Claire E , Nelson Cody S , Scheef Elizabeth , Sprehe Lesli , Mostrom Matilda , Malouli Daniel , Fruh Klaus , Chan Cliburn , Kaur Amitinder , Permar Sallie R
Published: January 2023

Congenital cytomegalovirus (cCMV) is the most common in utero infection and causes major neurodevelopmental deficits, but there remains no licensed vaccine to prevent cCMV. Little is known about maternal immune responses that can prevent placental CMV transmission, which could guide rational design of an effective vaccine. Using the rhesus macaque (RM) model of primary RM CMV (RhCMV) infection during pregnancy, we established that pre-existing RhCMV-neutralizing IgG protected against cCMV, even in the setting of CD4+ T cell depletion, where vertical transmission occurs consistently in RhCMV-seronegative RMs. However, the antibody (Ab) functions mediating this protection have not been fully defined. To identify humoral immune correlates of containment of viremia and protection against cCMV we used samples from previous studies in this model to measure Ab binding to whole RhCMV virus and key glycoproteins, neutralization, Ab dependent cellular phagocytosis (ADCP), and cytotoxicity (ADCC). Immunocompetent RMs developed both RhCMV-specific neutralizing and Fc-mediated effector functions (ADCP, ADCC) 2–4 weeks post infection. However, Ab responses were not statistically distinct between transmitters and non-transmitters during this period. Maternal viremia outperformed maternal Ab responses as a predictor of placental transmission. Therefore, we correlated each Ab response with maternal plasma viral load at day 21 post infection. Interestingly, IgG binding to gB and the pentamer, immunodominant viral glycoproteins involved in viral entry, displayed significant inverse relationships with maternal viremia (n=15, Spearman r=−0.71, p=0.003 for both), highlighting these as critical targets for vaccine design.

Supported by grants from NIH (NIAID P01-AI129859, NCI T32-CA009111)
Impact of pre-existing immunity on the development of de novo virus-specific TRM following live attenuated influenza vaccination
Authors: Lobby Jenna L , Danzy Shamika , Lowen Anice , Kohlmeier Jacob E
Published: January 2023

Live attenuated influenza vaccine (LAIV) elicits both humoral and cellular immune memory in children, but its efficacy is limited in adults. We hypothesize that pre-existing immunity from past infections and/or immunizations prevents the attenuated vaccine from establishing an immune response. To determine if we can overcome this limitation by increasing the antigenic distance of the vaccine strain from previous circulating seasonal strains, we generated a series of drifted LAIVs with successive mutations in the HA protein, allowing for increasing levels of escape from pre-existing antibody. We also inserted a CD8+ T cell epitope from the Sendai virus nucleoprotein (SeV-NP) as a readout for generation of a de novo TRM response following immunization. Surprisingly, we were unable to identify SeV-NP+ CD8+ TRM following LAIV immunization in PR8-immune mice, even with LAIV strains that can fully escape pre-existing antibody. As these data suggested a role for cell-mediated immunity in limiting LAIV efficacy, we investigated several scenarios to assess the impact of pre-existing LAIV-specific TRM in the upper and lower respiratory tract. Ultimately, we found that deletion of the immunodominant influenza NP366–374 epitope was sufficient to escape pre-existing CD8+ TRM and to establish de novo CD8+ TRM. Combined, these studies demonstrate that both pre-existing humoral and cellular immunity can limit the effectiveness of LAIV, thereby informing future design of vaccine vectors against respiratory pathogens.

Supported by grants from NIH (F31 HL156639-01, R35 HL150803)
Requirement for robust T and B cell responses for immune protection against Powassan virus (POWV) in virus-like particle (VLP) vaccination.
Authors: Stone E Taylor , Hassert Mariah , Geerling Elizabeth , Wagner Colleen , Brien James , Ebel Gregory , Hirsch Alec , German Cody , Smith Jessica , Pinto Amelia K
Published: January 2023

Powassan virus (POWV) is a tick-borne pathogen for which humans are an incidental host. POWV infection can be fatal or result in long-term neurological sequelae; however, there are no approved vaccinations for POWV. Integral to efficacious vaccine development is the identification of correlates of immune protection, which we accomplished in this study by utilizing a murine model of POWV infection. Using POWV lethal and sub-lethal challenge models, we show that 1) robust B and T cell responses are necessary for immune protection, 2) POWV lethality can be attributed to both viral- and host-mediated drivers of disease and 3) knowledge of the immune correlates of protection against POWV can be applied in a virus-like particle (VLP)-based vaccination approach that provides protection from lethal POWV challenge. We also provide evidence that POWV-experienced CD8+ T cells may potentiate immune-mediated pathogenesis during infection. Identification of beneficial and detrimental aspects of POWV-immune protection is significant as it will aid in the rational design of POWV vaccines.

This research was supported in part by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID): 5R01AI152192-02, 5R01AI137424-03 and F31AI152460-01. This research was also supported in part by the Department of Defense (Award#: PR192269). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Dysregulation of CD8+ T cell function in the setting of altered cytokine signaling due to inborn errors of immunity
Authors: Campos Duran Jose S , Conrey Peyton E , Sayed Samir , Di Chao , Gonzalez Elena N , Romberg Neil D , Bergerson Jenna R E , Holland Steve M , Freeman Alexandra F , Su Helen C , Leiding Jennifer W , Forbes Lisa R , Vogel Tiphanie P , Henrickson Sarah E
Published: January 2023

A key outcome of chronic antigenic stimulation in the setting of sustained inflammation is the development of CD8+ T cell exhaustion. However, the relative roles of inflammation and antigen stimulation on T cell dysfunction remain unclear. Inborn errors of immunity (IEIs) provide an opportunity to study the effect of genetic alterations in signaling pathways on immune function. Here, we focus on an IEI resulting from genetically amplified cytokine signaling: gain-of-function mutations in STAT3 (STAT3 GOF). STAT3 signals downstream of multiple inflammatory cytokines (e.g., IL-6 and IL-27) and we used STAT3 GOF as a genetic mimic of chronic inflammation. To assess how amplified STAT3 signaling impacts immune cell subset distribution and activation status, PBMCs from STAT3 GOF patients and age-matched healthy controls were profiled via mass cytometry (CyTOF). This revealed altered states of CD8+ T cell activation including increased expression of immunoregulatory receptors (e.g., PD-1 and CD39). STAT3 GOF CD8+ T cells also showed impaired cytokine production and reduced proliferative capacity. Bulk RNA sequencing of naïve STAT3 GOF CD8+ T cells identified an exhausted-like transcriptional profile. Quantitation of metabolic function shows alterations in glucose use and oxidative phosphorylation in STAT3 GOF. Our studies suggest that inflammation alone may directly impair CD8+ T cell function. Understanding the mechanisms by which inflammation impacts T cell function may identify components of T cell dysregulation that can be targeted therapeutically in these rare patients, as well as those with more common inflammatory diseases (e.g., obesity) with shared aspects of T cell dysfunction.

Supported by CHOP Research Institute, The Primary Immune Deficiency Treatment Consortium (PIDTC) and Immune Deficiency Foundation (IDF).
Association of elevated CD151+ T cell frequencies with impaired immune function
Authors: Perez Mildred , Lowman Kelsey , Moylan David C , Tidwell Christopher M , Schroeder Andrew , Duverger Alexandra , Wagner Frederic H , Erdmann Nathan , Goepfert Paul , Hu Hui , Sabbaj Steffanie , Kutsch Olaf
Published: January 2023

We recently reported that in humans the tetraspanin CD151 marks phenotypically distinct T cell subsets. The frequency of CD151+ T cells differs between T cell lineages and in between memory T cell populations. CD151+ T cells are hyper-proliferative and hyper-activated, as shown by phospho-proteomic analysis. We found that CD151 is an activation marker that is upregulated following TCR/CD3 activation, but at baseline, the presence of CD151 was associated with the loss of CD28 expression, a sign of T cell senescence. We hypothesized that the presence of CD151+ T cells is associated with immune hyperactivation and in extension, impaired immune surveillance. Unfortunately, we found that not even basic human CD151 T cell biology is reproduced in mouse models and thus had to resort to the study of pathological human conditions to elucidate the role(s) of CD151+ T cells. As cancer development requires immune escape, cancer diagnosis could be used as an indicator of impaired immune surveillance. Benefitting from the availability of longitudinal patient sample and data collections from people living with HIV (PLWH) we found that PLWH that developed cancer all had CD8+CD151+ T cell frequencies outside a normal reference range. CD8+CD151+ T cell frequency increase was not cancer-specific and could be detected years before the cancer diagnosis (0.5–12 years), indicating that the increase was not part of an anti-tumor response. Other established immune markers showed no correlation with the development of cancer. The findings associate elevated CD8+CD151+ T cell frequencies with impaired immune surveillance and suggest that elevated CD8+CD151+ T cell frequencies, at least in PLWH can be used as prognostic marker for cancer development.

Supported by grants from NIH R01-AI122842, NIH R33-AI116188, NIH R33-AI133679
Comprehensive analysis of the durability and the quality of humoral and T cell responses in SARS-CoV-2 vaccinated and infected subjects.
Authors: Woloszczuk Kyra , Connors Jennifer R , Cusimano Gina , Bell Matthew , Furukawa Sara , Melnyk Nataliya , Lin Edward , Venkateswaran Kodumudi , Bernui Mariana , Kutzler Michele , Cairns Charles , Haddad Elias K
Published: January 2023

Understanding adaptive immune memory to SARS-CoV-2 is relevant for the development of vaccines and new immunotherapies. Specifically, understanding the interactions between memory B cells and T-cell subsets can shed light on persistent immune memory and its effects on antibody durability and antibody affinity. Studying the frequencies of SARS-CoV-2 memory B cells and antigen-specific T-cells 3–6 months following natural infection or vaccination would aid us in vaccine development and vaccination in infected subjects. In this study, we analyzed these responses in individuals who were hospitalized with COVID-19 (n=20), individuals who were vaccinated after COVID hospitalization (n=20), and uninfected individuals (n=40) with two doses of the Pfizer or Moderna SARS-CoV-2 vaccine. We hypothesize individuals who were vaccinated after hospitalization will have the highest frequency of memory T follicular helper cells, memory B cells and antibody durability. To accomplish this, we will analyze anti-SARS-CoV-2 antibodies, including 7 RBD variants as well as 65-cytokines in all patients via Luminex and examine frequencies of memory T follicular helper cells, persistent memory B-cells specific to SARS-CoV-2 using flow cytometry. All immune responses will be analyzed 3–6 months after hospitalization or after a second vaccination. These results show the differences in durability and quality of humoral and T cell responses between vaccinated and infected subjects.

Supported by grants from NIH supplement #U19AI128910-04S1
Neuroinflammatory Signatures of Complement Component 4 in the Subventricular Zone of Autism Spectrum Disorder and Schizophrenia
Authors: Mou Ta-Chung , Lane Malcolm , Ireland Derek D C , Clark Sarah M , Tonelli Leonardo H
Published: January 2023

Early inflammatory insults are the most recognized environmental risk factor associated with neurodevelopmental psychiatric disorders. The subventricular zone (SVZ) is a region heavily involved in neurodevelopment and neuroimmune interactions through the lifespan. Complement component 4 (C4), a classical molecule involved in inflammatory responses, has been associated with schizophrenia (SZ) by genetic studies. However, whether C4 is involved in other neurodevelopmental disorders, such as autism (ASD), is a matter of current studies. Moreover, while C4 in SZ has been studied in the context of synaptic pruning, little is known about its neuroinflammatory role. Using in situ hybridization with radioactive riboprobes and RNAscope, we identified robust astrocytic expression of C4 in the SVZ and in the septum pellucidum. C4 was also expressed in ependyma, neurons, and Ki67+ cells, but not microglia, with higher expression in SZ as compared to controls and ASD. Moreover, the number of Ki67+ cells was significantly higher in ASD compared to SZ and controls. Targeted transcriptomic analysis of inflammatory pathways revealed a strong association of complement system genes with SZ, and to a lesser extent ASD, as well as generalized inflammation without an association with known infectious pathways. Additional analysis of differentially expressed genes (DEGs) showed that ASD DEGs were enriched in adaptive immune system functions such as Th cell differentiation, while SZ DEGs were enriched in innate immune system functions, including NF-κB and toll like receptor signaling. Taken together, these results support a role for C4 into inflammatory-neuroimmune dysregulation observed in SZ and ASD pathology.

Supported by National Institutes of Mental Health grant P50 MH103222 (Silvio O. Conte Center for Translational Mental Health Research).
PD-1 restricts the development and effector function of tissue regulatory T cells in experimental autoimmune encephalomyelitis
Authors: Liang Dan , Judge Jennifer , Markson Samuel , Guinn Samantha , Collier Jenna Lynn , Shahid Osmaan , Manne Sasikanth , Kuchroo Juhi , Fung Megan , Pauken Kristen E , Vignali Dario A A , Wherry John E , Sharpe Arlene H
Published: January 2023

PD-1 restricts T cell effector functions, regulates T cell tolerance, and maintains immune homeostasis, thereby playing pivotal roles in cancer, autoimmune and infectious diseases. Our studies and others show that PD-1 can restrain Treg suppressive function in autoimmunity and cancer. Here, we investigate the role of Treg intrinsic PD-1 in regulating pathogenic and protective CD4+ T cell responses in experimental autoimmune encephalomyelitis (EAE) by inducing PD-1 deletion on FoxP3+ cells prior to disease development. This deletion of PD-1 only in Treg protects mice from severe disease, and both flow cytometry and transcriptional analysis of central nervous system (CNS) CD4+ T cells show enrichment of tissue effector Treg (eTreg) subsets at peak of disease. We find that PD-1 restrains the generation and suppressive function of these effector Treg. This tissue eTreg signature is also observed in human and mouse Treg subsets from the tumor microenvironment, and are potent suppressor populations. Further studies are underway to determine how PD-1 impacts TCR signaling and FoxP3 transcriptional co-activators in Treg cells. Our study provides deeper insights into how PD-1 regulates T cell tolerance, underscoring the important role of PD-1 in regulating Treg differentiation and suppressive function during autoimmune diseases, and provide mechanistic insights for PD-1 modulation in cancer and autoimmunity.

Supported by NIH P01AI108545
Regulation of myeloid-derived suppressor cell expansion by TRAF3 during chronic inflammation
Authors: Jung Jaeyong , Zhu Sining , Lalani Almin , Sant’Angelo Derek B , Covey Lori R , Ostrand-Rosenberg Suzanne , Xie Ping
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) expand under pathological conditions and orchestrate the immunosuppressive and inflammatory network, and thus are recognized as a prime therapeutic target of human cancers and inflammatory diseases. In this study, we identified the tumor suppressor TRAF3 as a critical regulator of MDSC expansion. We found that myeloid cell-specific Traf3-deficient (M-Traf3−/−) mice exhibited MDSC hyperexpansion during chronic inflammation. Using mixed bone marrow chimeras, we demonstrated that TRAF3-mediated regulation of MDSC expansion involves cell-extrinsic mechanisms. To delineate the environmental factors that induce the aberrant expansion of MDSCs in M-Traf3−/− mice, we performed cytokine protein array analyses and in vivo neutralization experiments. Our results revealed that G-CSF is a major driver of MDSC hyperexpansion in M-Traf3−/− mice during chronic inflammation. Furthermore, we found that G-CSF was mainly produced by CCR4+ inflammatory monocytes and macrophages in mice during chronic inflammation. Taken together, our findings provide novel insights into the cellular and molecular mechanisms underlying TRAF3-mediated regulation of MDSC expansion, and open up new avenues for the development of MDSC-targeting therapeutics in disease conditions.

This study was supported by the National Institutes of Health grant R21 AI128264 (P. Xie), a New Jersey Commission on Cancer Research (NJCCR) grant DCHS19CRF005 (P. Xie), a Pilot Award of Cancer Institute of New Jersey through Grant Number P30CA072720 from the National Cancer Institute (P. Xie), and a Busch Biomedical Grant (P. Xie).
Setting the pace: CD4 T cell-intrinsic Arginase 1 orchestrates Th1 induction and contraction
Authors: West Erin E , Freeley Simon , Kaminski Marcin M , Merle Nicolas S , Veenbergen Sharon , Lee Duck-Yeon , St. John-Williams Lisa , Thompson J Will , Green Douglas R , Scholl-Buergi Sabine , Karall Daniela , Huemer Martina , Kemper Claudia
Published: January 2023

CD4 T cell-intrinsic engagement of the complement receptor CD46 controls nutrient influx and the metabolic reprogramming events that are essential for both the initiation and contraction of human Th1 responses, characterized by IFN-g and IL-10 production respectively. Here, we demonstrate that CD46 also orchestrates T cell arginine metabolism by upregulating the arginine transporter CAT-1 and, unexpectedly, Arginase 1 (Arg1). Arg1 has been well characterized in macrophages where it is associated with the IL-10 secreting M2 type, but its expression or function in T cells has not been described. Surprisingly, in CD4 T cell, Arg1 seems to restrain IL-10 production and contraction: T cells isolated from four patients with rare Arg1 deficiency mount strong Th1 responses but display significantly increased IL-10 switching and early contraction when compared to healthy control cells. Similarly, Arg1fl/fl CD4-cre+ mice infected with influenza virus are characterized by an enhanced Th1 response that contracts more rapidly, resulting in viral control and significantly reduced lung pathology. Unexpectedly, both Arg1-deficient mouse and human T cells produce normal levels of nitric oxide (NO) and polyamines. Metabolic profiling rather revealed that T cells lacking Arg1 have enhanced glycolysis, reduced TCA-cycle intermediates, and engage an “alternative” glutamine pathway often utilized by cancer cells. Normalization of these metabolic perturbations, through the targeting of specific metabolic enzymes, restored typical Th1 induction/contraction. Overall, these data unveil an unexpected intrinsic role for Arginase 1 as a pacemaker of the Th1 lifecycle, which could be harnessed for the amelioration Th1-driven pathologies.
Characterization of urine-derived immune cells from bladder cancer patients and comparison to tumor and peripheral blood
Authors: Tran Michelle A , Farkas Adam , Beaumont Kristin , O’Donnell Timothy , Mehrazin Reza , Wiklund Peter , Horowitz Amir , Galsky Matthew , Sfakianos John , Bhardwaj Nina
Published: January 2023

Background

Although PD-1/PD-L1 immune checkpoint blockade (ICB) therapy has achieved durable clinical responses in a subset of bladder cancer patients (15–25%), the majority do not respond. This has led to a need to identify clinically predictive biomarkers. Urine is an accessible material that may reflect cellular and/or genetic signatures related to ICB response. It has been shown that bladder cancer patient urine contains immune cells in addition to tumor cells. To study the concordance between urinary immune cells and the tumor immune microenvironment (TIME), we un-biasedly characterized bladder cancer patient urine and compared it to tumor and peripheral blood mononuclear cells (PBMC).

Methods

Matched tumor, urine, and PBMC from 8 bladder cancer patients were dissociated for single cell RNA sequencing (scRNAseq) and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) using 10X Genomics.

Results

scRNAseq of bladder cancer patient urine revealed CD4+ and CD8+ T, T regulatory (Treg), natural killer (NK), and B cells as well as neutrophils, dendritic cells, monocytes, and macrophages. The composition and transcriptional profiles of these cells were more similar to the tumor immune cells than to PBMC. Urine immune cells expressed hypoxia, anergy, and pro-inflammatory gene signatures that were more similar to tumor immune cells than PBMC.

Conclusions

Our work represents the first scRNAseq and CITE-seq of cancer patient urine. Our study shows several immune cells shed in bladder cancer patient urine and suggests they look phenotypically similar to the TIME. This has implications for future clinical applications as urine can be sampled non-invasively in scenarios when tumor resection may not be feasible.

Supported by the NIH grant: R01 CA249175-01.
Mechanisms by which Factor H protects Trypanosoma cruzi from the alternative pathway of complement.
Authors: Menon Smrithi Sugumaran , Ramirez-Toloza Galia , Wycoff Keith L , Shaughnessy Jutamas , Ram Sanjay , Ferreira Viviana P
Published: January 2023

Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi (T. cruzi). Complement (C) is a critical arm of the immune system. Various pathogens, including T. cruzi, hijack host regulators such as Factor H (FH) to inactivate C. FH regulates the alternative pathway (AP) of C in blood and on cell surfaces. FH contains 20 short consensus repeat (SCR) domains and binds to C activation products (i.e., C3b/C3d) or polyanionic host cell markers (e.g., sialic acid, glycosaminoglycans) on cell surfaces, and uses its 4 N-terminal SCRs to inactivate the AP. T. cruzi potentially binds FH by transferring host sialic acids onto its surface using its trans-sialidase enzyme. This study asked whether FH binds directly to T. cruzi in the absence of C3b and defines the FH domains involved. Only C-resistant infective trypomastigotes, but not C-sensitive noninfective epimastigotes, bound FH directly in a dose-dependent manner. Although SCR 19–20 was hypothesized as important for this interaction given it is the only known sialic acid-binding region, domain mapping indicated SCR 5–8 competitively inhibited FH binding by >35% with lower contributions of other regions. FH related protein-5 (FHR-5), whose SCRs bear sequence homology to all known polyanion-binding regions (6–7, 10–14, 19–20), fully competitively inhibited FH binding to trypomastigotes. In addition, competitive survival experiments with serum and FHR-5 led to >80% killing when the AP or all C pathways were active. This is the first study to show consequences of completely inhibiting FH protection on T. cruzi and to suggest multiple FH domains are involved in the interaction. These data may be harnessed for developing therapeutics and vaccine candidates against T. cruzi.

This work was supported by The University of Toledo Biomedical Research Innovation Program (VF), and The American Association of Immunologists Careers in Immunology Fellowship Program (VF and SSM).
T cell immunity to a classical strain of Klebsiella pneumoniae
Authors: Mackel Joseph J , Wasbotten Rachel , Dahler Aubree , Smith Catherine Morffy , Rosen David A
Published: January 2023

Klebsiella pneumoniae (Kp) is a versatile pathogen capable of causing numerous infections that are becoming increasingly difficult to treat due to increasing antibiotic resistance. Kp strains are categorized into two distinct pathotypes: classical and hypervirulent, which exhibit critical differences in pathogenesis. Despite causing the majority of clinical cases, adaptive immunity resulting from classical Kp infections is poorly understood. Our laboratory has recently developed a model of adaptive immunity to live infection with classical Kp in which wild-type mice surviving primary infection are protected from morbidity and mortality upon rechallenge. Passive transfer of convalescent serum from surviving mice to naïve recipients does not confer protection against cKp infection, suggesting antibody responses alone are inadequate to protect against cKp. CD69+CD4+ T cells and γδ T cells expand dramatically in the lung following primary cKp infection and mice deficient in T cells are not protected from reinfection, indicating a requirement for T cells in the protective response. Further analysis of mice deficient in either classical αβ T cells or γδ T cells indicates that either T cell subset is capable of mediating protective immunity against Kp reinfection. Production of IL-17A by both CD4 and γδ T cells is enhanced during secondary infection. Together these data indicate that a natural course of infection with classical Kp elicits populations of classical T cells and γδ T cells that mediate protection against reinfection, likely through the rapid production of IL-17A. These results will ultimately inform rational design of vaccines and immunotherapies to prevent and treat Kp infection.

Supported by AAI Careers in Immunology Fellowship
Longitudinal interactions between levels of serum cytokine and the microbiome from four body sites
Authors: Zhou Xin , Shen Xiaotao , Johnson Jethro , Spakowicz Daniel J , Zhu Chenchen , Zhou Wenyu , Zhou Yanjiao , Weinstock George , Snyder Michael
Published: January 2023

The human body is co-habituated with trillions of microbes, which are actively interacting with the human immune system. Our previous study demonstrated that a subset of individuals, characterized by a chronic absence of serum Interleukin (IL)-17 and IL-22, is more likely to be resistant to insulin compared with individuals with detectable serum IL-17/IL-22. Additional analysis pointed out that such an absence of IL-17 and IL-22 is associated with the low abundance of the gut microbiome that belongs to the class of Clostridia.

To better understand the interactions between serum cytokines and microbiome, we curated a dataset from the iHMP that contains 86 individuals with a study duration from 5 months to 6 years, whose microbiome has been characterized from four body sites include: stool (n=927), skin (n=1128), nasal (922), oral (n=1001). Their serum cytokines had been characterized using 62-plex Luminex antibody-conjugated bead capture assay. The longitudinal interaction between microbiome abundance and serum cytokine are modeled by Bayesian Mixed-Effects Model, and the interaction between microbiome richness and serum cytokine are modeled by Linear Mixed Models.

We found the interactions are strongest in the gut compared to the other three body sites, but it is highly territory specific. For example, Moraxella from the skin is negatively correlated with multiple cytokines, but not Moraxella from the nasal. Among all cytokines, IL-1 signaling is the strongest modulator of microbiome abundance in all four body sites, but LEPTIN and GMCFS are more potent when focused on richness. Our results indicate that the immune regulation of the microbiome is active on multiple body sites, and such interaction is genera and cytokine specific.

This work was supported by the National Institutes of Health Common Fund Human Microbiome Project (HMP) (1U54DE02378901). Dr. Xin Zhou received support from the Stanford Aging and Ethnogeriatrics (SAGE) Research Center under NIH/NIA grant P30AG059307. The SAGE Center is part of the Resource Centers for Minority Aging Research (RCMAR) Program led by the National Institute on Aging (NIA) at the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and does not necessarily represent the official views of the NIA or the NIH.
Aberrant survival of uterine natural killer subsets in uterus transplant recipients
Authors: Porrett Paige , Gonzalez Michael V , Garifallou James , Wright Emma D , Lucander Aaron C K , Bell Mar’Kayla Joy , Tyson Kennedy , Smiler Jacqueline , Mafra Fernanda , Da Silva Renata Pellegrino , Johnston Sarah , Naziruddin Bashoo , Testa Giuliano , Johannesson Liza , George James , Freud Aharon , O’Neill Kathleen
Published: January 2023

Uterine natural killer cells (uNKs) are critical mediators of pregnancy success, but how aberrancies in uNK survival or differentiation underpin pregnancy complications is unknown. To improve our understanding of normal and abnormal uNK biology, we studied 1) uNKs in uterus transplant (UTx) recipients at high risk for pregnancy complications and 2) uNK survival after exposure to pharmacologic immunosuppression in vitro. To address the first question, we analyzed uNKs isolated from endometrial biopsies of healthy controls (n=3) or UTx recipients (n=5) using scRNA-seq. In healthy controls, CD103-expressing uNK3 cells were the dominant uNK subset (30% of all uNK cells). In contrast, uNK1 cells were the most frequent (30%) in the majority of UTx recipients. This dominance of uNK1 cells in UTx recipients did not appear to arise only from loss of uNK3 cells, as the uNK1 transcriptional signature was abnormally upregulated in immature proliferating uNKs. Next, we used multiparameter flow cytometry to study single-cell suspensions of whole endometrium from deceased organ donors (n=4) that were cultured for one week with or without the calcineurin inhibitor FK506. Although FK506 significantly impaired the survival of CD103+ uNK3 cells in vitro, there was no evidence of uNK1 enhancement. Instead, FK506 appeared to selectively deplete CD39+ cells – a marker currently used to identify human decidual NK1 cells. Altogether, these results demonstrate that the distribution of uNK subsets is often altered in UTx recipients, and that FK506 can impact the survival of specific uNK subsets. Identification of the additional factors which impact uNK differentiation and survival will be necessary to understand the genesis of pregnancy complications.

Supported by NIH/NIAID (R01 AI 145905) University of Pennsylvania Institute for Immunology
ILC3 Expansion in Acute Myeloid Leukemia
Authors: Dinh Thanh Thanh Trinh , Lordo Matthew R , Shilo Nikolas , Altynova Ekaterina , Kronen Parker , Broughton Megan , Sellers Victoria , Collins Patrick , Freud Aharon G , Mundy-Bosse Bethany L
Published: January 2023

At homeostasis, type 3 innate lymphoid cells (ILC3s) are tissue-resident and have key roles in maintaining mucosal immunity. Recently, the expansion of ILC3s has been described in solid tumors; however, the mechanism driving this finding is not completely understood. In the setting of mouse and human acute myeloid leukemia (AML), we have observed an expansion of ILC3s. Using an AML murine model, we observed a 7-fold increase in ILC3s in the intestine (the normal ILC3 niche) of leukemic mice relative to controls and a 3-fold increase of ILC3s in the bone marrow (intestine: 0.208% +/− 0.04 AML vs 0.032% +/− 0.04 WT, n=5, p<0.001; bone marrow: 1.312% +/− 0.20 AML vs 0.067% +/− 0.20 WT, n=5, p<0.0001). We hypothesized that the ILC3 increase was due at least in part to increased ILC3 differentiation. To address this hypothesis, we isolated the innate lymphoid cell precursor (ILCP) from normal human donors and co-cultured with AML cells which resulted in increased ILC3 frequency (59% +/− 5% with AML vs 28% +/− 5% without AML, n=15, p <0.001). This observation was also recapitulated in a human PDX model in NSG mice (3.22% +/− 7.8% without AML vs 23.91% +/− 7.8% with AML, n=10; p<0.0001). Our published data have shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. By manipulating AHR activity ex vivo, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (60% +/− 5.5% with AML vs 27% +/− 5% AML+AHR inhibitor, n=15, p<0.001). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion by skewing ILCP differentiation towards ILC3. Future studies will define the role of ILC3s in AML progression.

Supported by grant from NIH (1R01CA255860-01A1)
Combining Immunohistochemistry and In Situ Hybridization to Characterize Immunosuppression in the Tumor Microenvironment
Authors: Cartwright Emily , Oliver Ashley , Kalyuzhny Alex
Published: January 2023

While immune-based cancer treatments have been successfully deployed in the clinic for hematological malignancies, treatment of solid tumors has proven more challenging. Comprehensive understanding of the dynamic landscape of the tumor microenvironment (TME) will lead to new therapies and improved efficacy of current therapies. Immunohistochemistry (IHC) and in situ hybridization (ISH) are the two techniques at the forefront of TME characterization. Combining RNA and protein detection with ISH and IHC reveals cell-type specific gene expression and the cellular sources of secreted proteins. Traditional ISH-IHC workflows require harsh pre-treatment of tissues prior to detection with primary antibodies, destroying the target epitope for some antibody clones. Bio-Techne’s RNAscopeTM RNA protein Codetection Assay improves the likelihood of successful antibody staining by incubating with primary antibody first, before initiating the RNAScope pretreatment step. With minimal optimization time, we were able to visualize protein expression using IHC validated antibodies for important immunosuppression biomarkers such as HIF-1 alpha, FoxP3, PD-L1, and CD11b. Using a cancer tissue microarray (TMA), we show this workflow is compatible with many tissue types, including pancreas, uterus, liver, breast, lung, prostate, and colon tissue. In combination with RNAScope probes CD8A, CD4, PD-1, TGFB1, and VEGF we were able to identify immunosuppressive cells in the TME, including Regulatory T cells (Tregs) and tumor associated macrophages (TAMs). Adoption of this workflow will streamline profiling of the TME, facilitating a more comprehensive evaluation of solid tumors for biomarker discovery and immunotherapy development.
An automated approach to high-plex cytometric immunophenotyping with CyTOF XT
Authors: Li Stephen K H , Zabinyakov Nick , Bouzekri Alexandre , Straus Rita , Jong Raymond , Sullivan Michael , Loboda Alexander , Majonis Daniel , Loh Christina
Published: January 2023

CyTOF® mass cytometry is a single-cell analysis platform that uses isotope-tagged antibodies to resolve 50-plus markers in a single tube without signal compensation, making CyTOF ideal for routine immunophenotyping. CyTOF XT™, the latest CyTOF system, features automated sample acquisition. Stained samples were acquired in parallel using the automated CyTOF XT system and manually, using the Helios™ system, to assess performance of the automated system.

Multiple suspension mass cytometry staining workflows were evaluated. Population frequencies and resolution indices for markers were assessed by manual gating. There was no significant difference between population frequencies analyzed between the two CyTOF systems. On average, samples acquired on CyTOF XT resulted in greater resolution between positive and negative populations compared to Helios.

The Maxpar® Direct™ Immune Profiling System, which comprises the Maxpar® Direct™ Immune Profiling Assay™ and Maxpar Pathsetter™ software, was also compared on the CyTOF XT and Helios systems. The Maxpar Direct Immune Profiling Assay includes a 30-marker panel in a dry, single-tube format for staining human whole blood or PBMC. Maxpar Pathsetter automates reporting of population statistics and stain assessments for the panel. Maxpar Pathsetter showed comparable population frequencies between the two CyTOF systems and improved staining assessment on CyTOF XT.

Overall, these studies find that the CyTOF XT system generates better signal resolution than the Helios system. Automated acquisition by CyTOF XT enables researchers to accurately and reproducibly streamline human immunophenotyping.

For Research Use Only. Not for use in diagnostic procedures.
An integrated single-cell multiomics approach to characterize mRNA, intracellular, and surface proteins using intracellular AbSeq and BD® AbSeq Immune Discovery Panel
Authors: Song Hye-Won , Hatami Elham , Vadrevu Samatha , Martin Jody , Snowden Eileen , Zhu Timothy Zheng , Wright Adam , Nakamoto Margaret , Dillmore Shannon , Ayer Aruna
Published: January 2023

DNA-barcoded antibodies enable single-cell multiomics approaches that examine protein alongside mRNA in a single cell. Although it is a powerful approach, leading methods are currently limited to surface proteins, which limits understanding of signal-transduction and transcriptional pathways that are often governed by expression changes and posttranslational modifications of intracellular proteins. The ability to analyze intracellular proteins alongside the transcriptome is complicated by the fix and perm processes necessary to access intracellular epitopes, which can negatively impact RNA assay sensitivity. Furthermore, nonspecific binding of antibody-oligos to nucleic acids can generate high background signals.

To address these issues, we generated a protocol that can recover high-quality RNA alongside specific intracellular and surface protein information. Using this protocol in conjunction with the BD Rhapsody™ Single-Cell Analysis System, we simultaneously profiled over 50 protein markers including 14 intracellular proteins with the whole transcriptome. We identified predicted patterns, including increased phosphorylation of AKT protein correlated with the proliferation marker Ki67 upon immune stimulation, and transcription factor (T-bet) expression in a specific cell subset. Together these preliminary results suggest that analysis of intracellular proteins together with the transcriptome may be possible using single-cell sequencing technologies and can enable a deeper understanding of cellular states.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.