T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates T cells’ exit from quiescence. The increased nutrient requirements of activated lymphocytes are met in part by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine (Asn) is a non-essential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular Asn and ASNS activity in CD8+ T cell activation. At early timepoints of activation in vitro, CD8+ T cells expressed little or no ASNS and, as a consequence, viability and TCR-stimulated growth, activation and metabolic reprogramming were substantially impaired under conditions of Asn deprivation. At later timepoints (>24h of activation), TCR-induced mTOR-dependent signals resulted in upregulation of ASNS, that endowed CD8+ T cells with the capacity to function independently of extracellular Asn. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular Asn is involved in optimal T cell effector responses.
Helen Carrasco Hope, Rebecca J. Brownlie, Christopher M. Fife, Lynette Steele, Mihaela Lorger, Robert J. Salmond
Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is lack of biomarkers, specific medications, and a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NFκB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed new light on the role of IgG glycosylation in the development of podocyte injury and propose the development of ‘liquid kidney biopsy” approaches to diagnose LN.
Rhea Bhargava, Sylvain Lehoux, Kayaho Maeda, Maria G. Tsokos, Suzanne Krishfield, Lena Y. Ellezian, Martin Pollak, Isaac E. Stillman, Richard D. Cummings, George C. Tsokos
Antibodies that neutralize SARS-CoV-2, are thought to provide the most immediate and effective treatment for those severely afflicted by this virus. Because coronavirus potentially diversifies by mutation, broadly neutralizing antibodies are especially sought. Here we report a novel approach to rapid generation of potent broadly neutralizing human anti-SARS-CoV-2 antibodies. We isolated SARS-CoV-2 Spike protein-specific memory B cells by panning from the blood of convalescent human subjects after infection with SARS-CoV-2, sequenced and expressed Ig genes from individual B cells as human monoclonal antibodies (mAbs). All of 43 human mAbs generated in this way neutralized SARS-CoV-2. Eighteen of the 43 human mAbs exhibited half-maximal inhibitory concentration (IC50s) of 6.7 x10-12 M to 6.7x10-15 M for spike pseudotyped virus. Seven of the human mAbs also neutralized with IC50<6.7 x10-12 M viruses pseudotyped with mutant spike proteins (including receptor binding domain mutants and the S1 C-terminal D614G mutant). Neutralization of the Wuhan Hu-1 founder strain and of some variants decreased when coding sequences were reverted to germline, suggesting that potency of neutralization was acquired by somatic hypermutation and selection of B cells. The results indicate that infection with SARS-CoV-2 evokes high affinity B cell responses, some products of which are broadly neutralizing and others highly strain-specific. We also identify variants that would potentially resist immunity evoked by infection with the Wuhan Hu-1 founder strain or by vaccines developed with products of that strain, suggesting evolutionary courses SARS-CoV-2 could take.
Mayara Garcia de Mattos Barbosa, Hui Liu, Daniel Huynh, Greg Shelley, Evan T. Keller, Brian T. Emmer, Emily J. Sherman, David Ginsburg, Andrew A. Kennedy, Andrew W. Tai, Christiane E. Wobus, Carmen Mirabelli, Thomas M. Lanigan, Milagros Samaniego, Wenzhao Meng, Aaron M. Rosenfeld, Eline T. Luning Prak, Jeffrey L. Platt, Marilia Cascalho
The programmed death-1 (PD-1) and the PD ligand 1 (PD-L1) interaction represents a key immune checkpoint within the tumor microenvironment (TME), and PD-1 blockade has led to exciting therapeutic advances in clinical oncology. Although IFN-γ–dependent PD-L1 induction on tumor cells was initially thought to mediate the suppression on effector cells, recent studies have shown that PD-L1 is also expressed at high level on tumor-associated macrophages (TAMs) in certain types of tumors. However, the precise role of PD-L1 expression on TAMs in suppressing antitumor immunity within the TME remains to be defined. Using a myeloid-specific Pdl1-knockout mouse model, here we showed definitive evidence that PD-L1 expression on TAMs is critical for suppression of intratumor CD8+ T cell function. We further demonstrated that tumor-derived Sonic hedgehog (Shh) drives PD-L1 expression in TAMs to suppress tumor-infiltrating CD8+ T cell function, leading to tumor progression. Mechanistically, Shh-dependent upregulation of PD-L1 in TAMs is mediated by signal transducer and activator of transcription 3, a cascade that has not been previously reported to our knowledge. Last, single-cell RNA sequencing analysis of human hepatocellular carcinoma revealed that PD-L1 is mainly expressed on M2 TAMs, supporting the clinical relevance of our findings. Collectively, our data revealed an essential role for Shh-dependent PD-L1 upregulation in TAMs in suppressing antitumor immunity within the TME, which could lead to the development of new immunotherapeutic strategies for treating cancer.
Amy J. Petty, Rui Dai, Rosa Lapalombella, Robert A. Baiocchi, Don M. Benson, Zihai Li, Xiaopei Huang, Yiping Yang
Intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality rate and few treatment options. Discovery of therapeutic interventions has been slow given the challenges associated with studying acute injury in the human brain. Inflammation induced by exposure of brain tissue to blood appears to be a major part of brain tissue injury. Here, we longitudinally profiled blood and cerebral hematoma effluent from a patient enrolled in the Minimally Invasive Surgery with Thrombolysis in Intracerebral Hemorrhage Evacuation trial, offering a rare window into the local and systemic immune responses to acute brain injury. Using single-cell RNA-Seq (scRNA-Seq), this is the first report to our knowledge that characterized the local cellular response during ICH in the brain of a living patient at single-cell resolution. Our analysis revealed shifts in the activation states of myeloid and T cells in the brain over time, suggesting that leukocyte responses are dynamically reshaped by the hematoma microenvironment. Interestingly, the patient had an asymptomatic rebleed that our transcriptional data indicated occurred prior to detection by CT scan. This case highlights the rapid immune dynamics in the brain after ICH and suggests that sensitive methods such as scRNA-Seq would enable greater understanding of complex intracerebral events.
Brittany A. Goods, Michael H. Askenase, Erica Markarian, Hannah E. Beatty, Riley S. Drake, Ira Fleming, Jonathan H. DeLong, Naomi H. Philip, Charles C. Matouk, Issam A. Awad, Mario Zuccarello, Daniel F. Hanley, J. Christopher Love, Alex K. Shalek, Lauren H. Sansing, the ICHseq Investigators
Autoimmune diseases are characterized by a breakdown of immune tolerance partly due to environmental factors. The short-chain fatty acid acetate, derived mostly from gut microbial fermentation of dietary fiber, promotes anti-inflammatory regulatory T cells and protects mice from type 1 diabetes, colitis and allergies. Here, we show that the effects of acetate extend to another important immune subset involved in tolerance, the IL-10 producing regulatory B cells (B10 cells). Acetate directly promoted B10 cell differentiation from mouse B1a cells both in vivo and in vitro. These effects were linked to metabolic changes through the increased production of acetyl-CoA, which fueled the tricarboxylic acid cycle and promoted post-translational lysine acetylation. Acetate also promoted B10 cells from human blood cell through similar mechanisms. Finally, we identified that dietary fiber supplementation in healthy individuals was associated with increased blood B10 cells. Direct delivery of acetate or indirectly via acetate-producing diets or -bacteria might be a promising approach to restore B10 cells in non-communicable diseases.
Claire I. Daïen, Jian Tan, Rachel Audo, Julie Mielle, Lake-Ee Quek, James R. Krycer, Alexandra S. Angelatos, Martha Duares, Gabriela V. Pinget, Duan Ni, Remy Robert, Md Jahangir Alam, Carmen Balguerías Amián, Frederic Sierro, Arvind Parmar, Gary J. Perkins, Sumaiya Hoque, Alison K. Gosby, Stephen J. Simpson, Rosilene V. Ribeiro, Charles R. Mackay, Laurence Macia
Pre-existing cross-reactivity to SARS-CoV-2 may occur in absence of prior viral exposure. However, this has been difficult to quantify at the population level due to a lack of reliably defined seroreactivity thresholds. Using an orthogonal antibody testing approach, we estimated that ~0.6% of non-triaged adults from the greater Vancouver area, Canada between May 17th and June 19th 2020 showed clear evidence of a prior SARS-CoV-2 infection, after adjusting for false-positive and false-negative test results. Using a highly sensitive multiplex assay and positive/negative thresholds established in infants in whom maternal antibodies have waned, we determine that more than 90% of uninfected adults showed antibody reactivity against the spike, receptor-binding domain (RBD), N-terminal domains (NTD) or the nucleocapsid (N) protein from SARS-CoV-2. This sero-reactivity was evenly distributed across age and sex, correlated with circulating coronaviruses reactivity, and was partially outcompeted by soluble circulating coronaviruses’ spike. Using a custom SARS-CoV-2 peptide mapping array, we found that this antibody reactivity broadly mapped to spike, and to conserved non-structural viral proteins. We conclude that most adults display pre-existing antibody cross-reactivity against SARS-CoV-2, which further supports investigation of how this may impact the clinical severity of COVID-19 or SARS-CoV-2 vaccine responses.
Abdelilah Majdoubi, Christina Michalski, Sarah E. O’Connell, Sarah Dada, Sandeep R. Narpala, Jean P. Gelinas, Disha Mehta, Claire Cheung, Dirk F.H. Winkler, Manjula Basappa, Aaron C. Liu, Matthias Görges, Vilte E. Barakauskas, Michael A. Irvine, Jennifer Mehalko, Dominic Esposito, Inna Sekirov, Agatha N. Jassem, David M. Goldfarb, Steven Pelech, Daniel C. Douek, Adrian B. McDermott, Pascal M Lavoie
With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex ten tumors from subcutaneous MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the two models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.
Soren Charmsaz, Nicole Gross, Elizabeth Jaffee, Won Jin Ho
The increased incidence of whooping cough worldwide suggests that current vaccination against Bordetella pertussis infection has limitations in quality and duration of protection. The resurgence of infection has been linked to the introduction of acellular vaccines (aP) which has an improved safety profile compared to the previously used whole-cell (wP) vaccines. To determine immunological differences between aP vs. wP priming in infancy, we performed a systems approach of the immune response to booster vaccination. Transcriptomic, proteomic, cytometric, and serologic profiling revealed multiple shared immune responses with different kinetics across cohorts, including an increase of blood monocyte frequencies, and strong antigen-specific IgG responses. Additionally, we found a prominent subset of aP-primed individuals (30%) with a strong differential signature, including higher levels of expression for CCL3, NFKBIA, and ICAM1. Contrary to the wP individuals, this subset displayed increased PT-specific IgE responses postboost and higher antigen-specific IgG4 and IgG3 antibodies against FHA and FIM2/3 at baseline and post-boost. Overall, the results show that, while broad immune response patterns to Tdap boost overlap between aP- and wP-primed individuals, a subset of aP-primed individuals present a divergent response. These findings provide candidate targets to study the causes and correlates of waning immunity after aP vaccination.
Ricardo da Silva Antunes, Ferran Soldevila, Mikhail Pomaznoy, Mariana Babor, Jason Bennett, Yuan Tian, Natalie N. Khalil, Yu Qian, Aishwarya Mandava, Richard H. Scheuermann, Mario Cortese, Bali Pulendran, Christopher D. Petro, Adrienne P. Gilkes, Lisa A. Purcell, Alessandro Sette, Bjoern Peters
There are approximately 44,000 cases of human papilloma virus (HPV)‒associated cancer each year in the United States, most commonly caused by HPV16/18. Prophylactic vaccines successfully prevent healthy people from acquiring HPV infections via HPV-specific antibodies. To treat established HPV-associated malignancies, however, new therapies are necessary. Multiple recombinant gorilla adenovirus HPV vaccine constructs were evaluated in NSG β2m-/- peripheral blood mononuclear cell–humanized mice bearing SiHa, a human HPV16+ cervical tumor, and/or in the syngeneic HPV16+ TC-1 model. PRGN-2009 is a new therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T-cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T-cell enhancer agonist epitopes. PRGN-2009 treatment reduced tumor volume and increased CD8 and CD4 T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6-specific T cells, and increased multifunctional CD8 and CD4 T cells in the tumor microenvironment. These studies provide the first evaluation of a therapeutic gorilla adenovirus HPV vaccine, PRGN-2009, showing promising preclinical anti-tumor efficacy and induction of HPV-specific T cells, and the rationale for its evaluation in clinical trials.
Samuel T. Pellom, Claire Smalley Rumfield, Y. Maurice Morillon II, Nicholas Roller, Lisa K. Poppe, Douglas E. Brough, Helen Sabzevari, Jeffrey Schlom, Caroline Jochems
No posts were found with this tag.