Cytomegalovirus (CMV) is a globally ubiquitous pathogen with a seroprevalence of approximately 50% in the UK. CMV infection induces expansion of immunosenescent T cell and NK cell populations with these cells demonstrating lower responsiveness to activation and reduced functionality upon infection and vaccination. In this study, we found that CMV+ participants had normal T cell responses after single dose or homologous vaccination with the viral vector ChAdOx1. In contrast, CMV seropositivity was associated with a loss of T cell IFN-γ secretion following heterologous ChAd-MVA viral vector vaccination. Analysis of participants receiving a single dose of ChAdOx1 demonstrates that T cells from CMV+ donors have a more terminally differentiated profile of CD57+PD1+ CD4+ T cells and CD8+ T cells expressing less IL-2Rα (CD25), and fewer polyfunctional CD4+ T cells 14 days post-vaccination. NK cells from CMV-seropositive individuals also have a reduced activation profile. Overall, our data suggest that although CMV infection enhances immunosenescence of T and NK populations, it does not affect antigen-specific T cell IFN-γ secretion or antibody IgG production after vaccination with the current ChAdOx1 nCoV-19 vaccination regimen in the UK.
Hannah R. Sharpe, Nicholas M. Provine, Georgina S. Bowyer, Pedro Moreira Folegatti, Sandra Belij-Rammerstorfer, Amy Flaxman, Rebecca Makinson, Adrian V.S. Hill, Katie J. Ewer, Andrew J. Pollard, Paul Klenerman, Sarah Gilbert, Teresa Lambe
NADPH deficiency exacerbates lupus in murine models and in patients, but the mechanisms remain unknown. One hypothesis is that NADPH oxidase suppresses autoimmunity by facilitating dead cell clearance via LC3-associated phagocytosis (LAP). The absence of LAP reportedly causes an autoinflammatory syndrome in aged, non-autoimmune mice. Prior work implicated cytochrome b-245, beta polypeptide (CYBB), a component of the NADPH oxidase complex, and RUN and cysteine-rich domain containing Beclin 1 interacting protein (RUBICON), as requisite for LAP. To test the hypothesis that NADPH oxidase deficiency exacerbates lupus via a defect in LAP, we deleted Rubicon in the B6.Sle1.Yaa and MRL.Faslpr lupus mouse models. Under this hypothesis, RUBICON deficiency should phenocopy NADPH oxidase deficiency, as both work in the same pathway. However, we observed the opposite—RUBICON deficiency resulted in reduced mortality, renal disease, and autoantibody titers to RNA-associated autoantigens. Given that our data contradicts the published role for LAP in autoimmunity, we assessed whether CYBB and RUBICON are requisite for LAP. We found instead that LAP is not dependent on either of these two pathways. Our data thus reveal RUBICON as a novel regulator of SLE, possibly by a B cell-intrinsic mechanism, but do not support a role for LAP in lupus.
Rachael A. Gordon, Christina C. Giannouli, Chirag Raparia, Sheldon I. Bastacky, Anthony Marinov, William Hawse, Richard T. Cattley, Jeremy S. Tilstra, Allison M. Campbell, Kevin M. Nickerson, Anne Davidson, Mark J. Shlomchik
Duration of protection from SARS-CoV-2 infection in people with HIV (PWH) following vaccination is unclear. In a sub-study of the phase 2/3 the COV002 trial (NCT04400838), 54 HIV positive male participants on antiretroviral therapy (undetectable viral loads, CD4+ T cells >350 cells/ul) received two doses of ChAdOx1 nCoV-19 (AZD1222) 4-6 weeks apart and were followed for 6 months. Responses to vaccination were determined by serology (IgG ELISA and MesoScale Discovery (MSD)), neutralisation, ACE-2 inhibition, gamma interferon ELISpot, activation-induced marker (AIM) assay and T cell proliferation. We show that 6 months after vaccination the majority of measurable immune responses were greater than pre-vaccination baseline, but with evidence of a decline in both humoral and cell mediated immunity. There was, however, no significant difference compared to a cohort of HIV-uninfected individuals vaccinated with the same regimen. Responses to the variants of concern were detectable, although were lower than wild type. Pre-existing cross-reactive T cell responses to SARS-CoV-2 spike were associated with greater post-vaccine immunity and correlated with prior exposure to beta coronaviruses. These data support the on-going policy to vaccinate PWH against SARS-CoV-2, and underpin the need for long-term monitoring of responses after vaccination.
Ane Ogbe, Matthew Pace, Mustapha Bittaye, Timothy Tipoe, Sandra Adele, Jasmini Alagaratnam, Parvinder K. Aley, M. Azim Ansari, Anna Bara, Samantha Broadhead, Anthony Brown, Helen Brown, Federica Cappuccini, Paola Cinardo, Wanwisa Dejnirattisai, Katie Ewer, Henry Fok, Pedro M. Folegatti, Jamie Fowler, Leila Godfrey, Anna L. Goodman, Bethany Jackson, Daniel Jenkin, Mathew Jones, Stephanie Longet, Rebecca A. Makinson, Natalie G. Marchevsky, Moncy Mathew, Andrea Mazzella, Yama F. Mujadidi, Lucia Parolini, Claire Petersen, Emma Plested, Katrina Pollock, Thurkka Rajeswaran, Maheshi N. Ramasamy, Sarah Rhead, Hannah Robinson, Nicola Robinson, Helen Sanders, Sonia Serrano Fandos, Tom Tipton, Anele Waters, Panagiota Zacharopoulou, Eleanor Barnes, Susanna Dunachie, Philip Goulder, Paul Klenerman, Gavin R. Screaton, Alan Winston, Adrian V.S. Hill, Sarah C. Gilbert, Miles Carroll, Andrew J. Pollard, Sarah Fidler, Julie Fox, Teresa Lambe, John Frater
Clinical outcomes in colorectal cancer (CRC) have been correlated with T cell infiltrates, but the specific populations of T cells, their functions, and how they influence clinical outcomes remains unclear. To comprehensively investigate the diverse phenotype and function of T cells in CRC, we profiled 37,931 single T cells from tumors and adjacent normal colon of 16 treatment-naïve CRC patients with respect to transcriptome, TCR sequence, and 23 cell surface markers. Our single-cell analysis identified phenotypically and functionally distinguishable effector CD4+ and CD8+ T cell-types within human tumors. We employed single-cell gene signatures from these T cell subsets to query the TCGA database to assess the prognostic significance of these subsets. Among CD8+ T-cell infiltrates, we found two distinct cytotoxic T cell types differentiated into clonally-expanded exhausted T cells. GZMK+ KLRG1+ cytotoxic T cells with a less dysfunctional phenotype were enriched in CRC patients with good outcomes. Strikingly, GNLY+ CD103+ cytotoxic T cells, including intraepithelial lymphocytes (IELs) with a more dysfunctional phenotype, were not associated with good clinical outcomes, despite high co-expression of CD39 and CD103, markers which denote tumor-reactivity. Together, this suggests that tumor-reactive cytotoxic T cells are effectively targeted to the tumor, yet their presence alone does not contribute to anti-tumor activity due to their impaired function, as reflected in clinical outcomes. Among CD4+ T cell-infiltrates, we found two distinct regulatory T cells (Treg) subtypes associated with opposite clinical outcomes. While total Tregs, predominantly Helios+ cells, were associated with good outcomes, Helios- CD38+ peripherally-induced Treg cells (pTregs) were strongly associated with bad outcomes independent of stage. CD38+ pTregs, which shared gene signatures with Th17 cells, possessed a highly suppressive phenotype, suggesting they are the elusive Treg population that inhibits anti-tumor immunity in CRC. These findings highlight the potential utility of these subpopulations in predicting clinical outcomes independent of stage. Furthermore, these observations support the potential for novel CRC therapies directed at CD38+ pTregs or CD8+ CD103+ T cells to augment existing T cell-targeted immunotherapies.
Kazuya Masuda, Adam Kornberg, Jonathan Miller, Sijie Lin, Nathan Suek, Theo Botella, Kerim A. Secener, Alyssa M. Baccarella, Liang Cheng, Matthew Ingham, Vilma Rosario, Ahmed M. Al-Mazrou, Steven A. Lee-Kong, Ravi P. Kiran, Marlon Stoeckius, Peter Smibert, Armando Del Portillo, Paul E. Oberstein, Peter A. Sims, Kelley S. Yan, Arnold Han
Dyslipidemia and autophagy have been implicated in the pathogenesis of blinding neovascular age-related macular degeneration (NV-AMD). Very low-density lipoprotein receptor (VLDLR), expressed in photoreceptors with a high metabolic rate, facilitates the uptake of triglyceride-derived fatty acids (FA). Since FA uptake is reduced in Vldlr-/- tissues, more remain in circulation, and the retina is fuel deficient, driving the formation in mice of neovascular lesions reminiscent of retinal angiomatous proliferation (RAP), a subtype of NV-AMD. Nutrient scarcity and energy failure are classically mitigated by increasing autophagy. We find that excess circulating lipids restrain retinal autophagy, which contributes to pathological angiogenesis in the Vldlr-/- RAP model. Triglyceride-derived FA sensed by free fatty acid receptor 1 (FFAR1) restricted autophagy and oxidative metabolism in photoreceptors. FFAR1 suppressed transcription factor EB (TFEB), a master regulator of autophagy and lipid metabolism. Reduced TFEB, in turn, decreased Sirtuin-3 expression and mitochondrial respiration. Metabolomic signatures of mouse RAP-like retinas were consistent with a role in promoting angiogenesis. This signature was also found in human NV-AMD vitreous. Restoring photoreceptor autophagy in Vldlr-/- retinas, either pharmacologically or by deleting Ffar1, enhanced metabolic efficiency and suppressed pathological angiogenesis. Dysregulated autophagy by circulating lipids might therefore contribute to the energy failure of photoreceptors driving neovascular eye diseases, and FFAR1 may be a target for intervention.
Emilie Heckel, Gael Cagnone, Tapan Agnihotri, Bertan Cakir, Ashim Das, Jin Sung Kim, Nicholas Kim, Geneviève Lavoie, Anu Situ, Sheetal Pundir, Ye Sun, Florian Wünnemann, Kerry A. Pierce, Courtney Dennis, Grant A. Mitchell, Sylvain Chemtob, Flavio A. Rezende, Gregor Andelfinger, Clary B. Clish, Philippe P. Roux, Przemyslaw Sapieha, Lois E.H. Smith, Jean-Sébastien Joyal
Altered islet architecture is associated with β cell dysfunction and Type 2 Diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed “re-activated” β cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, β cell-specific Notch gain-of-function mice. As predicted, we found that Notch activation in post-natal β cells impaired glucose stimulated insulin secretion (GSIS) and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased β cell repulsion, and impaired murine and human pseudo-islet formation. Consistent with our mouse data, Notch and Ephrin signaling are increased in metabolically-inflexible β cells in patients with T2D. These studies suggest than islet architecture can be permanently altered by β cell Notch/Ephrin signaling during a morphogenetic window in early life.
Alberto Bartolomé, Nina Suda, Junjie Yu, Changyu Zhu, Jinsook Son, Hongxu Ding, Andrea Califano, Domenico Accili, Utpal B. Pajvani
Standard-of-care treatment for advanced HER2+ breast cancers (BC) is comprised of two HER2-specific monoclonal antibodies (mAb), Trastuzumab (T) and Pertuzumab (P) with chemotherapy. While this combination (T+P) is highly effective, its synergistic mechanism of action (MOA) is not completely known. Initial studies had demonstrated that Pertuzumab suppressed HER2 hetero-dimerization as the potential therapeutic MOA, thus the improved outcome associated with the T+P combination MOA compared to Trastuzumab alone has been widely reported as being due to Pertuzumab-mediated suppression of HER2 signaling in combination with Trastuzumab-mediated induction of anti-tumor immunity. Unraveling this MOA may be critical to extend this combination strategy to other antigens or other cancers, as well as improving this current treatment modality. Using novel murine and human versions of Pertuzumab, we found it induced both Antibody-Dependent-Cellular-Phagocytosis (ADCP) by tumor-associated macrophages and suppression of HER2 oncogenic signaling. Most significantly, we identified that only T+P combination therapy, but not when either antibody used in isolation, allows for the activation of the classical complement pathway, resulting in both direct complement-dependent cytotoxicity (CDC) as well as complement-dependent cellular phagocytosis (CDCP) of HER2+ BC cells. Notably, we show that tumor expression of C1q was positively associated with survival outcome in HER2+ BC patients, whereas expression of complement regulators CD55 and CD59 were inversely correlated, suggesting the importance of complement activity in clinical outcomes. Accordingly, inhibition of C1 activity in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant anti-tumor MOA for T+P therapy that may be functionally enhanced to augment therapeutic efficacy in the clinic.
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by the accumulation of myofibroblasts and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-seq on lung fibroblasts isolated from young and aged mice during the early resolution phase post-bleomycin injury. We discovered that relative to injured young fibroblasts, injured aged fibroblasts exhibited a pro-fibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified pro-viral integration site of Moloney murine leukemia virus 1 (PIM1) and its target Nuclear Factor of Activated T Cells-1 (NFATc1) as putative drivers of the sustained pro-fibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts in vitro potentiated their fibrogenic activation in a NFATc1-dependent manner. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Inhibition of PIM1 signaling in IPF lung explants ex vivo inhibited pro-survival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.
Tho X. Pham, Jisu Lee, Jiazhen Guan, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Steven K. Huang, Daniel J. Tschumperlin, Giovanni Ligresti
Kawasaki disease (KD) is the leading cause of non-congenital heart disease in children. Studies in mice and humans propound the NLRP3-IL-1β pathway as the principal driver of KD pathophysiology. Endoplasmic reticulum (ER) stress can activate the NLRP3 inflammasome, but the potential implication of ER stress in KD pathophysiology has not been investigated. We used human patient data and the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to characterize the impact of ER stress on the development of cardiovascular lesions. KD patient transcriptomics and single-cell RNA sequencing of the abdominal aorta from LCWE-injected mice revealed changes in the expression of ER stress genes. Alleviating ER stress genetically, by conditional deletion of Inositol Requiring Enzyme-1 (IRE1) in myeloid cells, or pharmacologically, by inhibition of IRE1 endoribonuclease (RNase) activity, led to significant reduction of LCWE-induced cardiovascular lesion formation as well as reduced caspase-1 activity and IL-1β secretion. These results demonstrate the causal relationship of ER stress to KD pathogenesis, and highlight IRE1 RNase activity as a potential new therapeutic target.
Stefanie Marek-Iannucci, Asli D. Yildirim, Syed M. Hamid, Asli B. Ozdemir, Angela C. Gomez, Begüm Kocatürk, Rebecca A. Porritt, Michael C. Fishbein, Takao Iwawaki, Magali Noval Rivas, Ebru Erbay, Moshe Arditi
Sporozoite-based approaches currently represent the most effective vaccine strategies for induction of sterile protection against Plasmodium falciparum (Pf) malaria. Clinical development of sub-unit vaccines is almost exclusively centered around the Circum-sporozoite Protein (CSP) an abundantly expressed protein on the sporozoite membrane. Anti-CSP antibodies are able to block sporozoite invasion and development in human hepatocytes and subsequently prevent clinical malaria. Here we investigated whether sporozoite-induced human antibodies with specificities different from CSP can reduce Pf-liver stage development. IgG preparations were obtained from 12 volunteers inoculated with a protective immunization regime of whole-sporozoites under chloroquine prophylaxis. These IgGs were depleted for CSP-specificity by affinity chromatography. Recovered non-CSP antibodies were tested for sporozoite membrane binding and for functional inhibition of sporozoite invasion of a human hepatoma cell line and hepatocytes both in vitro and in vivo. Post-immunization IgGs depleted for CSP-specificity of 9 out of 12 donors recognized sporozoite surface antigens. Samples from 5 out of 12 donors functionally reduced parasite-liver cell invasion or development using the hepatoma cell line HC-04 and FRG-huHep mice containing human liver cells. The combined data provide clear evidence that non-CSP proteins as yet undefined do represent antibody targets for functional immunity against Plasmodium falciparum parasites responsible for malaria.
Amanda Fabra-García, Annie S.P. Yang, Marije C. Behet, Xi Zen Yap, Youri van Waardenburg, Swarnendu Kaviraj, Kjerstin Lanke, Geert-Jan van Gemert, Matthijs M. Jore, Teun Bousema, Robert W. Sauerwein
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