bluuebunny/crossref_metadata_2025 huggingface dataset:
Barcode Enabled Antigen Mapping (BEAM) enables next-generation systems immunology analysis of the post-COVID-19 immune landscape
Authors: Stubbington Michael J T , Adams Bruce , Reyes Daniel , Royall Ariel , Song Maengseok , Marrache Sean , Shahi Payam , Tsai FuNien , Finnegan Peter , Vollbrecht Thomas , Khadilkar Tanmayee , Jaffe David , Ramenani Ravi , McDonnell Wyatt
Published: January 2023

A more complete understanding of immune responses to disease requires consideration of multiple different cell types across both the innate and adaptive branches of the immune system along with the detection of many different analytes.

We used Barcode Enabled Antigen Mapping (BEAM) and Immune Profiling technology to perform simultaneous multimodal profiling at single cell resolution in hundreds of thousands of peripheral blood mononuclear cells (PBMCs) from a human donor following recovery from COVID-19. In addition to measuring gene and protein expression, we generated full-length, paired sequences of the rearranged T- and B-cell receptors while also screening their specificity for a wide range of antigens from SARS-CoV-2 and other viral pathogens.

These data provide insights into the entirety of the immune landscape after recovery from acute viral disease. The scale and throughput of our experiments gave us high-resolution data from all cell types from the innate and adaptive immune systems.

We identified antigen-specific clones of both B and T lymphocytes, with the high cellular throughput enabling detection of rare clones. Analysis of all PBMC cell types allowed us to place these antigen-specific clones within the overall transcriptional landscape of the post-viral immune system.

Experiments such as these will underpin new systems immunology approaches and will continue to reveal the complex interplay between the components of the immune system. We envisage that these methods will be valuable in the analysis of the immune response to vaccination, infectious disease, cancer, allergy, autoimmune conditions, and ageing that can potentially lead to the development of novel diagnostic and therapeutic approaches.
Development of a point-of-care immunoassay for Ebola Virus Disease through the detection of Ebola virus soluble glycoprotein
Authors: Green Heather R , Hau Derrick , Pandit Sujata , Arias-Umana Jose , DeMers Haley , Chung Chelsea , Foster Abigail , Carrasco Lesly , Gates-Hollingsworth Marcellene A , AuCoin David
Published: January 2023

Ebola Virus Disease (EVD) is a deadly infectious disease that is caused by an infection with Ebola virus (EBOV). EVD is one of the deadliest viral diseases with a human case fatality rate of up to 90%. The largest outbreak to date was the 2013–2016 West African outbreak in which Zaire ebolavirus (ZEBOV) was responsible for over 11,000 deaths. Transmission of the virus can occur through contact with an infected animal or human. Early detection is imperative to prevent the spread of disease and reduce the risk of a potential epidemic by isolating known infected patients. The current gold standard for diagnosing EVD is polymerase chain reaction assays which requires complex laboratory equipment and highly trained personnel that is not easily accessible in resource-limited areas. There is a great need for an EBOV rapid diagnostic test that can be implemented at the point-of-care (POC). Multiple lateral flow immunoassays (LFIs) have been developed for this need and have received emergency use authorization. Many of these tests rely on the detection of Ebola virus VP40 antigen, however, soluble glycoprotein (sGP) has the potential to be a better biomarker for EVD rapid detection as it is abundant in the blood during an infection. The goal of this study was to produce an EBOV sGP LFI prototype. A library of seventeen monoclonal antibodies reactive to EBOV sGP were produced and characterized via enzyme-linked immunosorbent assay, surface plasmon resonance, and Western immunoblotting. From this mAb library, LFI prototypes were developed and assessed for the potential use as a rapid POC diagnostic for EVD.

Supports by the National Institute of Allergy and Infectious Diseases under grant 1R41AI149940
Precision killing of T cells via targeting of T cell receptors using two novel chimeric antigen receptor designs
Authors: Francis Prashanth , Yarnell Michael , Burchill Matthew , Fry Terry
Published: January 2023

Pathologic T cells drive T cell leukemia, solid organ transplant rejection, and many autoimmune diseases. Currently approved treatments have limited ability to target pathogenic vs non-pathogenic T cells. While pan-T cell treatments can be effective, they have significant risks such as infection and malignancy. Chimeric antigen receptors (CAR) are synthetic constructs that retarget T cells to a specific antigen of interest; we hypothesized that we could use this technology for targeted killing of T cells.

We developed two novel CAR designs which target T cells via their T cell receptor (TCR). We opted to target OT1 T cells, a model system with a known antigenic epitope. The anti-Vβ5 CAR is a second-generation CAR with a traditional extracellular scFv domain that targets Vβ5 of the OT1 TCR. The H2Kb bait CAR instead has an extracellular H2Kb complex loaded with the SIINFEKL cognate epitope. Both anti-Vβ5 and H2Kb bait CAR T cells demonstrate degranulation, activation, and proliferation upon binding OT1 T cells. In vitro co-culture with OT1 T cells results in internalization of the OT1 TCR and specific killing of OT1 T cells. Interestingly, the changes induced by the H2Kb bait CAR were delayed relative to the anti-Vβ5 CAR. In vivo experiments show both CAR designs kill adoptively transferred OT1 T cells without significant effect on the other T cell populations.

In conclusion, we report the development of two novel CAR designs which allow for the targeted killing of T cells via their T cell receptor. While the anti-Vβ5 CAR T cells generated a more rapid response, the H2Kb bait CAR offers the promise of more precisely targeting T cells. This work has the potential to transform the treatment of conditions where removal of pathogenic T cells is critical for cure.

Supported by NIH HL152756
The role of autophagy in thymic stromal cell maintenance with age
Authors: Emtage Simon Nicholas Bruce , Semwal Manpreet , Wedemeyer Sarah , Xiao Yangming , Griffith Ann
Published: January 2023

T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells (TSCs) directs the progenitors along a well-characterized program of differentiation. However, the biology of the TSCs comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. TSCs exhibit high basal levels of autophagy at the steady state, which is critical for self-antigen presentation and T cell selection. The mechanisms governing high basal autophagy in TSCs are unknown, however autophagy is induced by many stressors, including high H202 levels. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the H202 quenching enzyme catalase (CAT) in TSCs, and found that CAT deficiency results in high H202 levels in this population, eventually leading to thymic atrophy. Our current studies address the possibility that, in addition to a role in self-antigen presentation, autophagy may also mitigate thymic atrophy through its role in removing organelle damaged by H202. Our preliminary studies suggest that increased basal autophagy in Beclin 1 knock-in (Becn1F121A/F121A) mice promotes thymus function during aging. Supported by N.I.H. grant R01AI121367
Augmenting oxidation of branched-chain amino acids (BCAAs) treats murine chronic Graft-Versus-Host Disease (cGVHD)
Authors: Jin Sujeong , Wagers Sara Bolivar , Walker Kirsti L , Mirek Emily T , Sage Peter T , Arany Zoltan , Anthony Tracy G , Blazar Bruce R
Published: January 2023

Aberrant immune responses, such as high Tfollicular helper (Tfh) and germinal center B cells (GCB) in cGVHD inducing antibody (Ab) secretion with tissue deposition and fibrosis, require distinct metabolic reprogramming to sustain pathogenesis. Leucine, isoleucine, and valine are essential BCAAs that signal via mTORC1 to support glycolysis and protein synthesis. Alternatively, BCAAs can be oxidized to metabolites that directly enter the tricarboxylic acid cycle or become substrates for gluconeogenesis and ketogenesis. Branched chain ketoacid dehydrogenase kinase (BDK) is a negative regulator of BCAA metabolism, decreasing downstream oxidation and increasing mTORC1 and glycolysis. We sought to determine whether increasing BCAA oxidation could be exploited to inhibit cGVHD. BDK is inhibitable by 3,6-Dichlorobenzo[β] thiophene-2-carboxylic acid (BT2), increasing BCAA oxidation and decreasing T effector (Teff) activation. In a multiorgan cGVHD model with bronchiolitis obliterans, mice that received BDK knockout vs wildtype donor T cells had improved survival and pulmonary function with decreased GCB frequencies (p-value 0.005) and a trend toward decreased Tfh/Tfollicular regulatory ratios. An in vitro assay measuring Tfh-mediated B cell Ab production showed that BT2 downregulates IgG class switching, which is key for cGVHD pathogenesis. BT2-treated T effectors but not T regulatory cells (Treg) exhibited increased oxidative phosphorylation (OXPHOS). Therefore, limiting BCAAs inhibits Teff switch from a highly glycolytic state to OXPHOS. In contrast, BCAAs are not required for Treg energy production or survival. These data suggest that augmenting BCAA oxidation could be a new therapeutic approach for treating cGVHD.

Supported by NIH P01 AI056299
Leveraging CAR T cells to Achieve Desensitization and Enable Transplantation
Authors: Markmann Caroline Anna , Zheng Zheng , Yu Ming , Rostami Susan , Wang Wei , Ochoa Trini , Parvathaneni Kalpana , Xu Xiaoming , Scholler John , Zhang Qian , Posey Avery , Allman David , Milone Michael , Arruda Valder , Jones Ben Samelson , Naji Ali , Bhoj Vijay
Published: January 2023

Pre-existing allo-antibodies (allo-Abs), that preclude transplant due to the risk of hyperacute rejection, lead to prolonged wait times and high mortality rates. Current desensitization approaches are ineffective as they do not adequately deplete allo-specific B cells and plasma cells (PCs). We hypothesize that stringent depletion of these cells is required to eliminate pre-existing allo-Abs. We leverage the exquisite ability of CAR T cells to eliminate target cells to desensitize transplant candidates. We constructed CARs targeting murine CD19 or BCMA, which cover the entire B cell-PC continuum. We first evaluated the function of CAR T cells against B cells and PCs in vitro. C57BL/6 mice were sensitized with BALB/c skin grafts. After skin rejection, sensitized mice received total body irradiation followed by treatment with either control T cells, CART-19 T cells, or a combination of CART-19 and CART-BCMA T cells (combo-CART). Allo-Abs, total Ig, and B cells were measured over 13 weeks. Functional desensitization was then assessed by induction of diabetes followed by BALB/c-derived islet cell transplant and glucose were measured to assess graft survival. CD19- and BCMA-targeted CARs effectively depleted primary B cells and PCs in vitro and in vivo. Control and CART-19 T cells were ineffective at desensitizing mice, but combo-CART treatment resulted in significant decrease of allo-Abs. Islet cell grafts succumbed to hyperacute rejection in 80% of control and CART-19 treated mice. However, combo-CART treatment resulted in prolonged graft survival in all mice (mean 35 days, range 16–60). Thus, CAR T cells targeting B cell and PC antigens represent a promising approach to desensitization and could enable lifesaving transplantation.
Class Switching is Required for Antigen-Dependent B cell Anti-Tumor Activity
Authors: Guo Zihan , Claudio Natalie , Ziglari Tahereh , Pucci Ferdinando
Published: January 2023

Recent clinical studies demonstrated a positive correlation between B cell parameters and response to immune checkpoint blockade therapy. However, the potential mechanisms underlying B cell anti-tumor activities are still unclear. A better understanding of B cell-mediated immune responses in cancer will highlight novel vantage points to exploit therapeutically. To this end, we engineered tumor cells to express the B cell model antigen hen egg lysozyme (HEL). Adoptive transfer of CD45.1+ HEL-specific B cells (SWHEL B cells) into wild-type mice bearing HEL+ tumors significantly delayed tumor growth. SWHEL B cells from tumor-draining lymph nodes showed higher levels of MHCII expression, germinal center (GC) formation and class switch rate than non-tumor specific B cells. Of note, MD4 B cells, which recognize HEL but cannot class switch, were unable to control tumor growth, suggesting that immunoglobulin class M are not required for B cell anti-tumor activities. Accordingly, depletion of CD4 T cells (which are required for class switch) in mice bearing HEL+ tumors reverted the anti-tumor activity of SWHEL B cells. GC SWHEL B cells class switched to immunoglobulin G1 with a frequency between 20 and 80%, and resulted in a 10-fold increase of plasmablast (CD19+CD138+) and memory B cell (CD19+IgD-CD38+CCR6+) generation, as compared to non-tumor specific B cells. Collectively, these data point to a role for class-switched B cells and CD4 T cells in tumor immunosurveillance. Future studies will assess the ability of tumor antigen-specific B cells in priming naïve CD4 T cells and the role of other immunoglobulin classes in tumor control and escape.

Supported by V Foundation for Cancer Research V2019-012
A tumor-restricted glycoepitope of podocalyxin correlates with immune evasion in high-grade serous ovarian carcinoma.
Authors: Brassard Julyanne , Hernaez Diana Canals , Hughes Michael R , Milne Katy , Dean Pamela , Warren Mary , Zhang Kevin , Banville Allyson C , Smazynski Julian , Bond David , Nelson Brad H , Roskelley Calvin D , McNagny Kelly M
Published: January 2023

High-grade serous ovarian carcinoma (HGSOC) is an aggressive tumor with a 5-year disease-free survival of roughly 15%, partly because it is usually diagnosed at an advanced stage. Podocalyxin (Podxl) is a highly glycosylated sialomucin normally expressed by vascular endothelia and kidney podocytes. Strikingly, Podxl expression is frequently upregulated by a variety of tumors (including HGSOC) and is consistently associated with poor prognosis. We capitalized on the fact that glycosylation pathways are frequently dysregulated in cancer to develop an antibody, PODO447, that recognizes a tumor-restricted glycoform of Podxl not expressed on normal tissue. While the exact epitope remains to be identified, our results suggest that PODO447 binds an epitope comprising a peptide domain of Podxl in combination with the core 1 O-GalNAc glycan (T-antigen). When coupled to a cytotoxin, a PODO447-antibody-drug conjugate (ADC) effectively kills human tumor cells in vitro and in xenografted mice. While the vast majority of ovarian tumors highly express the Podxl core protein, only a subset of these express the PODO447 epitope. Strikingly, tumors that express a high level of PODO447 epitope tend to be those that lack infiltrating CD8+ T cells and CD20+ B cells: a phenotype that has previously been linked to immune evasion and poorest disease-free survival. Furthermore, we find that PODO447 is a more consistent marker of these immunologically “cold” tumors than a number of other markers, including CA125, mesothelin and folate receptor α. These results highlight the PODO447-epitope as a highly selective diagnostic marker of poor outcome tumors and the PODO447-ADC as a novel strategy for therapeutic intervention.

This research was supported by the Canadian Institutes of Health Research (Grant Number: PJT-166180), the School of Biomedical Engineering (The University of British Columbia) postdoctoral fellowship and the Michael Smith Foundation for Health Research (MSFHR) research trainee award.
Role of the extracellular ATP/adenosine pathway in neutrophil-mediated T cell suppression in ovarian cancer microenvironment
Authors: Giridharan Thejaswini , Suzuki Sora , Emmons Tiffany R , Khan A N M Nazmul , Yaffe Michael B , Zsiros Emese , Odunsi Kunle , Bhalla Manmeet , Ghanem Elsa Bou , Segal Brahm H
Published: January 2023

The extracellular ATP (eATP)/adenosine (ADO) pathway is a key modulator of innate and adaptive immunity and is a target for immunotherapy for cancer. In the tumor microenvironment (TME) and conditions of chronic injury, ATP is released by dying cells and is hydrolyzed by the ecto-nucleotidase CD39 to AMP, and further to ADO by CD73. We previously observed that normal neutrophils (PMN) acquire a T cell suppressor phenotype mimicking PMN-MDSCs when exposed to the TME. Using a coculture system consisting of ascites supernatants (ASC) from patients with ovarian cancer (OC) as an authentic component of the TME and concentrations of healthy donor PMN and T cells that mimic those in OC ascites, we found that ASC-primed PMNs inhibit stimulated T cell proliferation, activation and metabolic responses. This suppressor activity was dependent on several PMN pathways, including complement signaling and NADPH oxidase. Since PMNs regulate and respond to signaling from the eATP/ADO pathway, we asked whether eATP/ADO mediates PMN suppressor function. PMNs had high constitutive expression of CD39 that was unaffected by ASC exposure, and inhibition of CD39 by POM-1 abrogated PMN suppressor function. CD73 inhibitor Adenosine 5′-(α,β-methylene) diphosphate also abrogated PMN suppressor function while using a different inhibitor (AB-680) had no effect. Neither ADO receptor inhibitors nor the addition of adenosine deaminase to deplete ADO affected PMN suppressor function. Together, these studies point to eATP as a modulator of PMN suppressor function in the TME and the potential to abrogate suppression by targeting CD39. Future studies will address the specific signaling functions of eATP on PMN in the TME that drive suppressor function.

Supported by grant from NIH (5R01CA188900)
“X”ploring the multifaceted functions of NLRX1 in triple-negative breast cancer
Authors: Nagai Margaret A , Woolls Mackenzie , Imran Khan M , Tupik Juselyn , Allen Irving C
Published: January 2023

Breast cancer is a devastatingly common and deadly malignancy, and despite improved survival resulting from improved detection and treatment, triple-negative breast cancer (TNBC) remains difficult to treat. While historically studied in the context of infectious diseases, a multifaceted anti-inflammatory pattern recognition receptor called NLRX1 has recently been investigated in several types of cancers due to its involvement in several pathways significant to tumorigenesis and disease progression. This project seeks to define the role of NLRX1 in TNBC. To achieve this, we generated novel Nlrx1−/−(KO) mice compatible with the widely-used 4T1 murine model of TNBC. We also generated 4T1 cells that either overexpress or knockdown NLRX1, as well as their respective controls (4T1OE, 4T1OE-CTL, 4T1KD, 4T1KD-CTL). KO and wild-type (WT) mice were then injected with unaltered 4T1 cells or one of the four altered 4T1 cell lines. We then quantified tumor growth, final tumor volume, and lung metastasis as a measure of disease burden. This in vivo work demonstrated that NLRX1 is protective against disease burden when expressed in healthy host tissues but is problematic when overexpressed by 4T1 cells. Ex vivo and in vitro experiments then revealed a pro-migratory phenotype in 4T1OE cells, likely as the result of altered TGF-β signaling and/or the PI3K-Akt-mTOR pathway as a means to impact epithelial-mesenchymal transition. Combined, we believe the function of NLRX1 in TNBC depends on the cell in which it is expressed (i.e. healthy vs cancerous), and that this function may ultimately be a result of altered epithelial-mesenchymal transition pathways.
Leveraging the Treg-intrinsic CTLA4-PKCη signaling pathway for cancer immunotherapy
Authors: Liu Hsin Yu , Pedros Christophe , Kong Kok-Fai , Canonigo-Balancio Ann J , Xue Wen , Altman Amnon
Published: January 2023

Our previous studies revealed a critical role of a novel CTLA4-PKCη signaling in mediating the suppressive activity of Tregs in antitumor immunity. Here, we extended these findings into a biologically and clinically more relevant context. We have analyzed the role of PKCη in antitumor immunity in intact tumor-bearing mice with Treg− or CD8+ T cell-specific Prkch deletion, including in a therapeutic model of combinatorial treatment. Using two models of mouse transplantable cancer and a genetically engineered autochthonous hepatocellular carcinoma (HCC) model, we found that mice with Treg-specific Prkch deletion displayed a significantly reduced growth of B16-F10 melanoma and TRAMP-C1 adenocarcinoma tumors. Tumor growth reduction was associated with a less immunosuppressive tumor microenvironment, indicated by increased numbers and function of tumor-infiltrating CD8+ T cells and elevated expression of the costimulatory ligand CD86 on CD103+ DCs. In contrast, CD8+ T cell-specific Prkch deletion had no effect on tumor growth and the abundance or functionality of CD8+ T cells. Similar beneficial antitumor effects were found in mice with germline or Treg-specific Prkch deletion that were induced to develop an autochthonous HCC. Lastly, using a therapeutic model, we found that monotherapies consisting of Treg-specific Prkch deletion or vaccination with irradiated Flt3L-expressing B16-F10 tumor cells post-tumor implantation significantly delayed tumor growth. This effect was more pronounced in mice receiving a combination of the two immunotherapies. These findings demonstrate the potential utility of PKCη inhibition as a viable clinical approach to treat cancer patients, especially when combined with adjuvant therapies.

Funded by the NIH grant 5R01CA233862
Identification of CPAF as the immunoprevalent antigen of Chlamydia trachomatis
Authors: Li Yanli , Warren Joanna , Poston Taylor , Shaw Fiona , Conrad Shayla , Xu Yinyan , Zheng Xiaojing , O’Connell Catherine M , Hillier Sharon L , Wiesenfeld Harold C , Darville Toni , Goonetilleke Nilu
Published: January 2023

Chlamydia trachomatis (CT) is a common sexually transmitted bacterial infection, that in women can cause pelvic inflammatory disease and infertility. No preventative vaccine has been developed against CT. Immunity to CT is primarily mediated by Th1 CD4+ T cells. We are defining immunoprevalent CT proteins in a well-defined cohort of CT seropositive women with the goal of defining novel vaccine immunogens. We screened 30 women one month after a CT-positive test by cultured IFN-γ ELISpot. Ten-day short-term cell lines (STCL) were generated against overlapping peptides spanning 21 CT antigens. The threshold for a positive CT-specific T cell response (≥ 300 spot-forming cells, SFU, per 106 cells) was defined following a screening of 12 CT seronegative donors. CT− specific T cell responses were detected in 27/30 CT-seropositive women. On average, women harbored T cell responses to two CT proteins (range 0–6). Strikingly, CT858 (CPAF) elicited a T cell response in 16/30 women with an average of 966 IFN-g SFU/106 cells (range 300–3,633 SFU/106 cells). Data to date suggest CPAF-specific T cell responses are predominantly CD4-restricted. In preliminary studies, we have also detected CT-specific T cell responses in men with documented CT infection, including a T cell response to CPAF. We are currently mapping CPAF T cell epitopes and expanding our screen to other CT secreted proteins. In summary, CT858 (CPAF) is an immunoprevalent antigen in women and a promising vaccine immunogen.

Supported by UNC Chlamydia Vaccine Initiative (U19AI144181)
Establishment of influenza-specific lung tissue-resident helper memory CD4+ T cells relies on expression the eATP sensor P2RX7
Authors: da Silva Henrique Borges , Santiago-Carvalho Igor , de Gois Macedo Bruna , Vardam-Kaur Trupti , van Dijk Sarah
Published: January 2023

Influenza remains an infection of high medical relevance. Development of universal anti-influenza vaccines will require a better knowledge of lung immune responses. That includes influenza-specific CD4+ T cells, which establish long-term lung residency and induce a diversified local protective response to subsequent infection. Lung-resident memory CD4+ T cells are heterogeneous, including Th1-like cells, and a subset that shares phenotypic and functional features with follicular helper (Tfh) cells – called tissue-resident helper (Trh). Notably, both Tfh and lung Trh cells express the extracellular ATP (eATP) sensor P2RX7 at high levels. Given its known deleterious role in Tfh cells, we investigated if P2RX7 could also limit the Trh pool in influenza-infected lungs. Contrary to our hypothesis, T cell-specific P2RX7 ablation led to a significant decrease in the Trh cell subset. Consequently, lungs of T cell-P2RX7 knockout mice harbor reduced B cell and CD8+ T cell responses. Initial Trh cell establishment is not affected by P2RX7 knockout; rather, P2RX7-deficient Trh cells gradually decay over time. This is associated with increased apoptosis and decreased in situ proliferation of P2RX7-knockout Trh cells. Our bulk RNA-seq analysis of lung CD4+ T cells revealed many cell cycle-associated genes poorly expressed in P2RX7-knockout cells. Some (but not all) Trh-fate genes are also decreased in P2RX7 deficiency. We are currently performing single-cell RNA-seq to define whether P2RX7 promotes the Trh fate in a particular subset of lung precursor cells. Our data shows that, in response to influenza, P2RX7 promotes lung-resident Trh cells, suggesting this eATP sensor has opposite roles in Tfh-like CD4+ T cells in a tissue-specific manner.

Supported by grant from NIH (R00 AI139381)
Induction of Autophagy reduces IFN-I mediated Inflammation and restores anti-HIV-1 T Cell response in vivo
Authors: Zhen Anjie , Mu Wenli , Rezek Valerie , Martin Heather , Carrillo Mayra , Hamid Philip , Lizarraga Miguel , Yang Otto , Jamieson Beth , Kitchen Scott
Published: January 2023

A hallmark of HIV-1 infection is chronic inflammation. Chronic immune activation drives the pathogenesis of HIV-1 infection, leading to loss of CD4+ T cells and immune exhaustion. Previously, we demonstrated that persistent type I interferon (IFN-I) signaling drives T cell exhaustion during chronic HIV infection and combined anti-retroviral therapy (ART) and IFN-I receptor blockade could lead to reduced inflammation, accelerated viral suppression and HIV reservoir. However, as IFN-Is are key regulators for antiviral immunity, more specific interventions to fine-tune IFN-I signaling and chronic inflammation are needed. Autophagy is a homeostatic mechanism for disposal of damaged cellular organelles and elimination of intracellular pathogens, which is pivotal for cellular homeostasis, T cell development and function. Autophagy is also impaired during HIV-1 infection. Here we demonstrate that autophagy is directly linked to IFN-I signaling. Impairment of autophagy leads to accumulation of damaged mitochondria and spontaneous IFN-I signaling. Autophagy inducers reduce IFN-I signaling in activated macrophages and restore functions of exhausted anti-HIV-1 T cells in vitro. Importantly, autophagy inducer treatment in HIV-1 infected humanized bone marrow/liver/thymus (BLT) mice significantly reduced persistent IFN-I signaling and immune activation, restored exhausted antiviral T cell responses, and accelerated viral suppression by ART. Autophagy inducer treatment also led to reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy is a promising approach for treating persistent immune activation and improve immune control of HIV replication.

This work was funded by NIAID 1R21AI140866 (to Zhen), NIDA R01DA-52841 (to Zhen), NIAID R2120200174 (PIs: Xie&Zhen), NCI 1R01CA239261-01 (to Kitchen), NIH Grants NIH Grants P30AI28697 (the UCLA CFAR Virology Core, Gene and Cell Therapy Core, and Humanized Mouse Core), U19AI149504(PIs: Kitchen & Chen), CIRM DISC2-10748, UPLIFT: UCLA Postdocs’ Longitudinal Investment in Faculty (Award # K12 GM106996) (to Carrillo), R01 AG052340 (to Jamieson). This work was also supported by the UCLA AIDS Institute, the James B. Pendleton Charitable Trust, and the McCarthy Family Foundation. We would also like to thank Drs. Romas Geleziunas and Jeff Murry and the people at Gilead for providing the antiretroviral drugs used in this study.
Characterization of cytokine measurement in human serum, plasma, urine, tears, milk, CSF, and saliva with a MILLIPLEX® multiplex immunoassay
Authors: Steiner Harold L , Luo Hong , Ahmed Tanzir , Gilliam Brooke , Saporita Anthony J , Qiang Xiao
Published: January 2023

Multiplex immunoassays are valuable research tools for studying circulating cytokines. Here we evaluate the ability of the MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A (Cat. No. HCYTA-60K) 48-plex immunoassay to reproducibly measure cytokines in matched human serum and plasma samples from healthy donors. For comparison, three other bead-based cytokine multiplex kits, featuring many of the same analytes, were analyzed in parallel. Two different lots from each kit were tested with the same matched sample cohort and data were analyzed with Belysa® software. The HCYTA-60K kit was unique in its use of a fixed standard curve, resulting in superior consistency in curve performance between lots. Sample value ratios and correlation demonstrated remarkable consistency of serum and plasma measurement between lots of the HCYTA-60K kit.

Further, detectability of additional sample types (urine, tears, milk, CSF, and saliva) was determined with the HCYTA-60K kit. Samples from healthy donors for each biofluid were tested to determine detectability. All analytes could be measured in at least one of the additional sample types. To characterize neuroinflammation during progressive stages of neurological disease, CSF cytokines were measured in samples from mild cognitive impairment, Alzheimer’s Disease, and healthy controls.

Our study illustrates the reliability of the MILLIPLEX® HCYTA-60K kit for reproducible measurement of cytokines in serum and plasma between different kit lots. Additionally, we characterize its ability to measure cytokines in urine, tears, milk, CSF, and saliva. These results highlight the utility and robustness of the HCYTA-60K multiplex panel for the quantification of cytokines in diverse biofluids.
Ablation of cDC2 lineage specification by mutations within the −165 kb Zeb2 enhancer
Authors: Liu Tiantian , Kim Sunkyung , Desai Pritesh , Kim Do-Hyun , Huang Xiao , Ferris Stephen T , Wu Renee , Ou Feiya , Egawa Takeshi , Van Dyken Steven J , Diamond Michael S , Kubo Masato , Murphy Theresa L , Murphy Kenneth M
Published: January 2023

Efforts to determine the unique functions of the two types of classical dendritic cells, cDC1 and cDC2, are hindered by limited understanding of the divergence of the common dendritic cell progenitor (CDP). Some transcription factors act in commitment of already specified progenitors, such as Batf3 which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer, but how other factors control CDP divergence remains unknown. Here, we report the transcriptional mechanism of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis suggested that Nfil3 acts upstream of Id2, Batf3, and Zeb2 in cDC1 development but has not revealed its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and ChIP-seq analysis identified NFIL3 binding in the −165 kb Zeb2 enhancer at sites that also bind CCAAT-enhancer-binding proteins (C/EBPs). Mutation of these NFIL3/C/EBP sites by in vivo CRISPR/Cas9 targeting revealed functional redundancy, with C/EBPs and NFIL3 competing in binding these sites to support or repress Zeb2 expression, respectively. Mutation of these three NFIL3/C/EBP sites eliminated Zeb2 expression in myeloid, but not lymphoid progenitors, producing mice that completely lacked pre-cDC2 specification and mature cDC2 in vivo. These mice failed to make an appropriate TH2 response against H. polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths. Notably, Zeb2 expression is not required to maintain cDC2 identity, but acts only to block cDC1 specification. Thus, the competition between C/EBPs and NFIL3 binding at the −165 kb Zeb2 enhancer determines CDP divergence.
Pathological metabolic adaptation of neutrophils recruited to cystic fibrosis airway microenvironment can not be reversed by modulator therapy
Authors: Cammarata-Mouchtouris Alexandre , Collins Genoah L , Kim Susan O , Moncada Diego , Tirouvanziam Rabindra , Chandler Joshua D
Published: January 2023

Background

Neutrophilic inflammation is a hallmark of cystic fibrosis (CF) lung disease. Prior studies from our group show that blood neutrophils undergo metabolic and functional adaptations upon entry into the CF lung lumen that causes them to become anabolic, while repressing bacterial killing function and enhancing degranulation. The recent advent of highly effective modulator therapy (HEMT) for the CFTR channel (mutated in CF) has improved patient outcomes, but it is unclear how HEMT impact CF lung neutrophilic inflammation.

Method

We used an organotypic model in which blood neutrophils are transmigrated through differentiated airway epithelial cells towards a chemoattractant control (leukotriene B4, LTB4), or airway supernatant from CF patients not on HEMT (CFASN) or on HEMT (CFMOD). We conducted phenotypic (FACS, CFU-assay) and metabolomic (metabolomics,13C6-glucose tracing) analysis of the transmigrated neutrophils.

Results

CFMOD-recruited neutrophils contained ivacaftor (a HEMT drug), demonstrating exposure to the therapy. Compared to LTB4-recruited neutrophils, CFASN/CFMOD-recruited neutrophils showed increased AMP, GMP and intracellular and secreted metabolites related to citric acid cycle activity. 13C6-glucose flux indicated similar rates of extracellular glucose utilization in all transmigrated neutrophils, but higher total glycolysis to lactate in CFASN/CFMOD-transmigrated neutrophils.

Conclusion

Pathological metabolic adaptations of neutrophils following transmigration into CF airway fluid are not substantially altered by HEMT. Further studies are needed to assess the potential impact on functional adaptations by these cells.

Supported by NIH (R56 HL150658), Cystic Fibrosis Foundation (CAMMAR21F0, TIROUV19G0), I3 Teams Research Award (Emory), and CF@LANTA, a component of Emory University and Children’s Healthcare of Atlanta.
Developing a synbiotic biotherapy to prevent allergic responses to food
Authors: Hesser Lauren A , Maccio-Maretto Lisa , Hubbell Jeffrey , Nagler Cathryn
Published: January 2023

Over the past generation, the prevalence of food allergies has risen dramatically, but the development of treatments has lagged. The commensal gut microbiome is critically involved in regulating allergic responses to food. Our laboratory has described a humanized model of cow’s milk allergy (CMA) in which germ-free mice are colonized with feces from healthy or CMA infants and sensitized to the cow’s milk allergen b-lactoglobulin (BLG). The healthy infant microbiota and specifically a single Clostridial species, Anaerostipes caccae, protect against the development of anaphylaxis, while the CMA microbiota does not induce this protection. We isolated a novel sub-strain of A. caccae (A. caccae_lahuc) from the feces of a healthy infant, sequenced its genome, and characterized its growth on various substrates in vitro. A. caccae_lahuc produces high concentrations of butyrate, a key immunoregulatory molecule in the gut which we hypothesize is critical to its protective impact. A. caccae_lahuc engrafts readily into the dysbiotic CMA microbiota, but co-delivery with lactulose is required for measurable butyrate production in vivo. Lactulose undergoes primary degradation by bacteria in the CMA feces which release small metabolites that A. caccae_lahuc can ferment into butyrate. Preliminary data show that this novel synbiotic biotherapy (A. caccae_lahuc + lactulose) prevents allergic responses to BLG in CMA-colonized mice as measured by serum mMCPT-1 and core body temperature upon challenge, and type 2 cytokine production in BLG-stimulated splenocytes. Further exploration into the mechanisms behind this therapeutic effect will contribute toward the larger goal of developing bacterial biotherapeutics against food allergies.
Novel Association of Lyme disease, Age, and Atopic Dermatitis
Authors: Lee Brandon , Galloway Sarah , Strausz Satu , Shoham Maia , Hansen Paige , Mansfield Paul , Salomon Rachel , Torrez-Dulgeroff Laughing Bear , Saleem Atif , Gars Eric , Sanders Erin , Ollila Hanna , Weissman Irving L , Tal Michal Caspi
Published: January 2023

Borrelia burgdorferi is a bacterial spirochete that can cause Lyme disease (LD) after infecting a susceptible host. Immune responses to the bacteria are highly variable and host specific. The murine substrain, C3H/HeJ, is a frequently utilized model for LD. Interestingly, over a prolonged infection, mice develop dermatitis on tail skin, which shares critical features with human skin. Female C3H/HeJ mice aged 5–8 weeks, 1 year, or 2 years were infected intraperitoneally with 105 B. burgdorferi. Dermatitis was evaluated by gross examination and histology. Dermatitis worsened over the course of untreated infection, with ulceration, hemorrhaging, flaking, hair loss, and dark lesions as well as spongiosis and acanthosis. These features of dermatitis were present in infected mice after 1 year of age. This relationship among LD, atopic dermatitis, and host age seen in the C3H/HeJ mouse model is consistent with a large pool (342,499) of human epidemiological data from Finland. We identified 5,248 individuals with LD and 17,233 with atopic dermatitis in FinnGen. Retrospective analysis shows LD is associated with atopic dermatitis (OR = 1.91 [1.68 −2.37], P < 2e−16). More visits due to LD complications (3 or more visits versus 1 visit) were associated with atopic dermatitis (OR = 2.19 [1.35–3.55], P = 0.0014) and risk of developing atopic dermatitis over time (HR = 2.26 [1.54–3.95], P = 0.0017). Data from mice and humans reveal a novel relationship among LD, age, and atopic dermatitis. Through defined pathological scoring, we demonstrate the onset of murine atopic dermatitis with B. burgdorferi infection, which is further exacerbated by host age at time of infection, and likewise report a similar association in human epidemiological data from FinnGen.

Research was supported by the Fairbairn Family foundation; Bay Area Lyme Foundation; the Younger family foundation; the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation; the Virginia and D. K. Ludwig Fund for Cancer Research; AML grant R01CA086017; the PCBC from NIHLB U01HL099999; as well as grant U19AI109662. M.C.T. was supported by Stanford Immunology training grant 5T32AI007290, and the NIH NRSA 1 F32 AI124558-01 award. L.B.T.D. was supported by a Stanford Diversifying Academia Recruiting Excellence fellowship. S.G. was supported by the California Institute for Regenerative Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Inflammatory responses to Listeria monocytogenes infection in the placenta
Authors: Seveau Stephanie
Published: January 2023

The immunological defense of the developing fetus is crucial for a successful pregnancy. The placenta plays key roles in protecting the fetus against rejection by the maternal immune system, while it ensures its defense against most pathogens. The mechanisms that orchestrate the placental immune functions are still poorly understood. The bacterial pathogen Listeria monocytogenes (Lm) breaches the maternal/fetal barrier, infects the placental chorionic villi, and reaches the fetus resulting in poor pregnancy outcomes. We studied the interplay between Lm and key players of the placental antimicrobial defense: trophoblasts (TCs), which are epithelial cells covering placental chorionic villi at the maternal/fetal interface, and placental macrophages (Hofbauer cells, or HBCs), which are the only leukocytes residing in chorionic villi. Both cell types form a critical immune barrier protecting the fetus from infection. We isolated human primary TCs and HBCs from healthy term placentas to study the Lm intracellular lifecycle as well as the cellular responses to infection (RNAseq and cytokine arrays). We found that both cell types were permissive to Lm infection and mounted a pro-inflammatory response to the pathogen including HBCs repolarization towards a pro-inflammatory phenotype favoring the innate immune responses. However, consistent with their placental homeostatic functions, TCs and repolarized HBCs maintained the expression of tolerogenic factors known to prevent maternal anti-fetal adaptive immunity. We will discuss our published studies (PMID 34399615 and 34367171) and recent data that teased apart the role of the Lm virulence factors in the inflammatory responses of placental cells.

Supported by NIH (R01AI157205, R21AI105588, R03AI149371)
Gasdermin D promotes hyperinflammation by triggering necroptosis in the presence of mitochondrial stress
Authors: Weindel Chi Gidley , Zhao Xiao , Martinez Eduardo , Bell Samantha L , Vail Krystal J , Coleman Aja K , VanPortfliet Jordyn J , Mabry Cory J , Li Pingwei , West A Phillip , Karpac Jason , Patrick Kristin L , Watson Robert O
Published: January 2023

Human genetic variants associated with mitochondrial dysfunction have been linked to chronic inflammatory diseases as well as susceptibility to infection. However, the mechanistic impact that these variants have on the immune system is poorly understood. We have discovered that macrophages harboring the common Parkinson’s disease associated variant, Lrrk2G2019S are more prone to cell death in response to both Mycobacterium tuberculosis (Mtb) infection and AIM2 inflammasome activation. Unexpectedly, the enhanced cell death in Lrrk2G2019S macrophages is driven by increased susceptibility to gasdermin D (GSDMD)-mediated mitochondrial pore formation, which releases accumulated mitochondrial ROS and pushes Lrrk2G2019S cells to undergo RIPK3-mediated necroptosis. Consistent with elevated necroptotic cell death, infection of Lrrk2G2019S mice with Mtb elicits dramatic hyperinflammation and exacerbated pathogenesis via enhanced neutrophil infiltration. Remarkably, expression of hLRRK2G2019S in Drosophila melanogaster recapitulates a similar phenotype, suggesting that Lrrk2G2019S plays an evolutionarily conserved role in regulating innate immunity. Our findings demonstrate that altered mitochondrial function can reprogram canonical innate immune and cell death pathways to elicit distinct immune outcomes, providing mechanistic insights into why mutations in LRRK2 are associated with susceptibility to chronic inflammatory and infectious diseases.

Supported by grants from the Parkinson’s Foundation (PF-FBS-1932), NIH (R01 AI125512), Michael J. Fox Foundation (grant 12185), and the Texas A&M Clinical Science and Translational Research (CSTR) Pilot Grant Program.
Aging augments type 2 cytokine responses in ILC2s leading to reparative, M2-like microglia
Authors: Mobley Alexis S , Hamlin Ashley , Garrido Jesus Bautista , Jung Joo Eun , McCullough Louise D , Aronowski Jaroslaw
Published: January 2023

Aging affects immunologic responses by a global suppression of the immune system, including dysregulation of cytokine mediators, leading to increased inflammation throughout all systems, termed inflammaging. However, understanding mechanisms of healthy aging can bypass this effect. Inflammaging also leads to poor outcomes during brain injury, making immune-targeting therapeutics tantamount to overall brain health and longevity. Two candidate immune cells microglia and group 2 innate lymphoid cells (ILC2s) may control immune responses in the aged brain Therefore, we hypothesized that ILC2s are the brains’ gatekeepers of microglia polarization to a reparative, M2-like phenotype under: (1) homeostatic conditions and (2) with aging. To address this, we used flow cytometry to quantify ILC2s between young and aged male and female C57Bl6/J mice, showing an age-related increase in aged males. Moreover, when we stimulated ILC2s, the data indicated an age-related increase in cytokine production in males. We also utilized conditioned media transfer experiments from stimulated young and aged ILC2s to quiescent microglia and assessed their morphologic, genomic, proteomic, and functional changes using microscopy, qRT-PCR, Western immunoblotting, and phagocytosis assays. We showed more complex microglia morphology (indicating microglial priming) and an increase in reparative mRNA, proteins, and phagocytic function in microglia, which was augmented by aged ILC2s. Taken together, we have defined the capacity of ILC2 soluble factors to polarize microglia to a reparative phenotype and maintain this in aging.

Supported by the NIH NINDS F31NS118983
Activation of TLR9 signaling in fibroblastic reticular cells enhances anti-tumor immunity in peritoneal tumor via suppressing peritoneal resident macrophage retention.
Authors: Deng Meihong
Published: January 2023

Immune checkpoint blockade (ICB) has been shown to improve the prognosis of patients with advanced malignancy; however, most patients do not respond to ICB therapy. Peritoneal resident macrophages (PRMs) are critical for peritoneal immunity in health and disease. A recent study has demonstrated that PRMs dampen the therapeutic efficacy of ICB via impairing anti-tumor CD8+ T cell immunity. Here we show that activation of TLR9, an endosomal DNA sensor, in fibroblastic reticular cells (FRCs) negatively regulates PRM retention. Mechanistically, TLR9 in FRCs suppresses retinol metabolism and thus suppresses GATA6 expression in PRMs. Notably, combined treatment of ODN1585, a TLR9 agonist, and anti-PD1 substantially improves the efficacy of anti-PD-1 immune checkpoint blockade therapies in MC-38 and CT26 colorectal carcinoma cell lines-induced peritoneal tumor in mice, evidenced by decreased tumor burden and mortality rate. The improvement is associated with an increase in the percent of CD8+ T cells and a decrease in the percent of CD39+ exhausted CD8+ T cells. Furthermore, global knockout of TLR9 or specific knockout of TLR9 in FRC diminished the beneficial effect of ODN1585 and anti-PD-1 combined treatment on tumor burden and survival. These data demonstrate the potential utility of the FRC-TLR9 signaling in modulating PRM retention to improve the therapeutic efficacy of ICB in peritoneal tumors.

Supported by grants from NIH / startup fund