The tumor microenvironment affects the outcome of radiotherapy against head and neck squamous cell carcinoma (HNSCC). We recently found that tolerogenic myeloid cells accumulate in circulation of HNSCC patients undergoing radiotherapy. Here, we analyzed tumor-containing lymph nodes biopsies collected from these patients. After two-weeks of radiotherapy, we found an increase in tumor-associated macrophages (TAMs) with activated STAT3, while CD8 T-cells were reduced as detected using multiplex IHC. Gene expression profiling indicated upregulation of M2 macrophage-related genes (CD163, CD206), immunosuppressive mediators (ARG1, LIF, TGFB1) and Th2 cytokines (IL4, IL5) in irradiated tumors. We next validated STAT3 as a potential target in human HNSCC-associated TAMs, using UM-SCC1 xenotransplants in humanized mice. Local injections of myeloid cell-targeted STAT3 antisense oligonucleotide (CpG-STAT3ASO) activated human DCs/macrophages, promoted CD8 T-cell recruitment and thereby arrested UM-SCC1 tumor growth. Furthermore, CpG-STAT3ASO synergized with tumor irradiation against syngeneic HPV+ mEERL and HPV– MOC2 HNSCC tumors in mice, triggering tumor regression and/or extending animal survival. The antitumor immune responses were CD8+ and CD4+ T-cell-dependent and associated with the activation of antigen-presenting cells (DCs/M1 macrophages) and increased CD8+ to regulatory T-cell ratio. Our observations suggest that targeted inhibition of STAT3 in tumor-associated myeloid cells augments the efficacy of radiotherapy against HNSCC.
Dayson Moreira, Sagus Sampath, Haejung Won, Seok Voon White, Yu-Lin Su, Marice Alcantara, Chongkai Wang, Peter P. Lee, Ellie Maghami, Erminia Massarelli, Marcin Kortylewski
TH17 cell subpopulations have been defined that contribute to inflammation and homeostasis, yet the characteristics of TH17 cells that contribute to host defense against infection are not clear. To elucidate the antimicrobial machinery of the TH17 subset, we studied the response to Cutibacterium acnes, a skin commensal that is resistant to IL-26, the only known TH17 secreted protein with direct antimicrobial activity. We generated C. acnes-specific antimicrobial TH17 clones (AMTH17) with varying antimicrobial activity against C. acnes, which we correlated by RNA-seq to the expression of transcripts encoding proteins that contribute to antimicrobial activity. Additionally, we validated that AMTH17-mediated killing of C. acnes as well as bacterial pathogens, was dependent on the secretion of granulysin, granzyme B, perforin and histone H2B. We found that AMTH17s can release fibrous structures composed of DNA decorated with the histone H2B that entangle C. acnes that we call T cell extracellular traps (TETs). Within acne lesions, H2B and IL-17 colocalized in CD4+ T cells, in proximity to TETs in the extracellular space composed of DNA decorated with H2B. This study identifies a functionally distinct subpopulation of TH17 cells with an ability to form TETs containing secreted antimicrobial proteins that capture and kill bacteria.
George W. Agak, Alice Mouton, Rosane Teles, Thomas A. Weston, Marco Morselli, Priscila R. Andrade, Matteo Pellegrini, Robert L. Modlin
Human herpes simplex virus-1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway resulting in impairment of central nervous system (CNS) cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-a/b induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-b protein, thereby also constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-b secretion and ISGs mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro, and by which the human central nervous system prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-b immunity, rather than viral recognition triggering an amplification of IFN-a/b production.
Daxing Gao, Michael J. Ciancanelli, Peng Zhang, Oliver Harschnitz, Vincent Bondet, Mary Hasek, Jie Chen, Xin Mu, Yuval Itan, Aurélie Cobat, Vanessa Sancho-Shimizu, Benedetta Bigio, Lazaro Lorenzo, Gabriele Ciceri, Jessica L. McAlpine, Esperanza Anguiano, Emmanuelle Jouanguy, Damien Chaussabel, isabelle Meyts, Michael S. Diamond, Laurent Abel, Sun Hur, Gregory A. Smith, Luigi D. Notarangelo, Darragh Duffy, Lorenz Studer, Jean-Laurent Casanova, Shen-Ying Zhang
FOXP3+ regulatory T cells (Tregs) rely on fatty acid -oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for differentiation and function. Recent data demonstrate a role for Tregs in the maintenance of tissue homeostasis with tissue-resident Tregs possessing tissue-specific transcriptomes. However, specific signals that establish tissue-resident Treg programs remain largely unknown. Tregs metabolically rely on FAO, and considering the lipid-rich environments of tissues, we hypothesized that environmental lipids drive Treg homeostasis. First, using human adipose tissue to model tissue residency, we identified oleic acid as the most prevalent free fatty acid. Mechanistically, oleic acid amplified Treg FAO-driven OXPHOS metabolism, creating a positive feedback mechanism that increased the expression of FOXP3 and phosphorylation of STAT5, which enhanced Treg suppressive function. Comparing the transcriptomic program induced by oleic acid to the pro-inflammatory arachidonic acid, we found that Tregs sorted from peripheral blood and adipose of healthy donors transcriptomically resembled the oleic acid in vitro treated Tregs, whereas Tregs from the adipose of MS patients more closely resembled an arachidonic acid transcriptomic profile. Finally, we found oleic acid concentrations were reduced in the adipose of MS patients, and exposure of MS Tregs to oleic acid restored defects in their suppressive function. These data demonstrate the importance of fatty acids in regulating tissue inflammatory signals.
Saige L. Pompura, Allon Wagner, Alexandra Kitz, Jacob Laperche, Nir Yosef, Margarita Dominguez-Villar, David Hafler
Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed death (PD)-1 blockade is poorly understood. Here, we performed dynamic single-cell RNA sequencing of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete sub-population of immunotherapy persister cells (IPCs) that resisted CD8 T-cell mediated killing. These cells expressed Snai1 and stem cell antigen-1 (Sca-1), and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by interleukin-6 (IL-6) but were vulnerable to tumor necrosis factor-alpha (TNF-α)-induced cytotoxicity, relying on Birc2 and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.
Kartik Sehgal, Andrew J. Portell, Elena V. Ivanova, Patrick H. Lizotte, Navin R. Mahadevan, Jonathan R. Greene, Amir Vajdi, Carino Gurjao, Tyler Teceno, Luke J. Taus, Tran C. Thai, Shunsuke Kitajima, Derek Liu, Tetsuo Tani, Moataz Noureddine, Christie J. Lau, Paul T. Kirschmeier, David Liu, Marios Giannakis, Russell W. Jenkins, Prafulla C. Gokhale, Silvia Goldoni, Maria Pinzon-Ortiz, William D. Hastings, Peter Hammerman, Juan J. Miret, Cloud P. Paweletz, David A. Barbie
SARS-CoV-2 causes a wide spectrum of clinical manifestations and significant mortality. Studies investigating underlying immune characteristics are needed to understand disease pathogenesis and inform vaccine design. In this study, we examined immune cell subsets in hospitalized and non-hospitalized individuals. In hospitalized patients, many adaptive and innate immune cells were decreased in frequency compared to healthy and convalescent individuals, with the exception of B lymphocytes which increased. Our findings show increased frequencies of T-cell activation markers (CD69, Ox40, HLA-DR and CD154) in hospitalized patients, with other T-cell activation/exhaustion markers (CD25, PD-L1 and TIGIT) remaining elevated in hospitalized and non-hospitalized individuals. B cells had a similar pattern of activation/exhaustion, with increased frequency of CD69 and CD95 during hospitalization, followed by an increase in PD1 frequencies in non-hospitalized individuals. Interestingly, many of these changes were found to increase over time in non-hospitalized longitudinal samples, suggesting a prolonged period of immune dysregulation following SARS-CoV-2 infection. Changes in T-cell activation/exhaustion in non-hospitalized patients were found to positively correlate with age. Severely infected individuals had increased expression of activation and exhaustion markers. These data suggest a prolonged period of immune dysregulation following SARS-CoV-2 infection highlighting the need for additional studies investigating immune dysregulation in convalescent individuals.
Jacob K. Files, Sushma Boppana, Mildred D. Perez, Sanghita Sarkar, Kelsey E. Lowman, Kai Qin, Sarah Sterrett, Eric Carlin, Anju Bansal, Steffanie Sabbaj, Dustin M. Long, Olaf Kutsch, James Kobie, Paul Goepfert, Nathaniel Erdmann
The mechanism by which inflammasome activation is modulated remains unclear. In this study, we identified an AIM2-interacting protein, the E3 ubiquitin ligase HUWE1, which was also found to interact with NLRP3 and NLRC4 through the HIN domain of AIM2 and the NACHT domains of NLRP3 and NLRC4. The BH3 domain of HUWE1 was important for its interaction with NLRP3, AIM2, and NLRC4. Caspase-1 maturation, IL-1β release, and pyroptosis were reduced in Huwe1-deficient bone marrow–derived macrophages (BMDMs) compared with WT BMDMs in response to stimuli to induce NLRP3, NLRC4, and AIM2 inflammasome activation. Furthermore, the activation of NLRP3, NLRC4, and AIM2 inflammasomes in both mouse and human cells was remarkably reduced by treatment with the HUWE1 inhibitor BI8622. HUWE1 mediated the K27-linked polyubiquitination of AIM2, NLRP3, and NLRC4, which led to inflammasome assembly, ASC speck formation, and sustained caspase-1 activation. Huwe1-deficient mice had an increased bacterial burden and decreased caspase-1 activation and IL-1β production upon Salmonella, Francisella, or Acinetobacter baumannii infection. Our study provides insights into the mechanisms of inflammasome activation as well as a potential therapeutic target against bacterial infection.
Yu Guo, Longjun Li, Tao Xu, Xiaomin Guo, Chaoming Wang, Yihui Li, Yanan Yang, Dong Yang, Bin Sun, Xudong Zhao, Genze Shao, Xiaopeng Qi
T cell exclusion causes resistance to cancer immunotherapies via immune checkpoint blockade (ICB). Myeloid cells contribute to resistance by expressing signal regulatory protein-α (SIRPα), an inhibitory membrane receptor that interacts with ubiquitous receptor CD47 to control macrophage phagocytosis in the tumor microenvironment. Although CD47/SIRPα-targeting drugs have been assessed in preclinical models, the therapeutic benefit of selectively blocking SIRPα, and not SIRPγ/CD47, in humans remains unknown. We report a potent synergy between selective SIRPα blockade and ICB in increasing memory T cell responses and reverting exclusion in syngeneic and orthotopic tumor models. Selective SIRPα blockade stimulated tumor nest T cell recruitment by restoring murine and human macrophage chemokine secretion and increased anti-tumor T cell responses by promoting tumor-antigen crosspresentation by dendritic cells. However, nonselective SIRPα/SIRPγ blockade targeting CD47 impaired human T cell activation, proliferation, and endothelial transmigration. Selective SIRPα inhibition opens an attractive avenue to overcoming ICB resistance in patients with elevated myeloid cell infiltration in solid tumors.
Vanessa Gauttier, Sabrina Pengam, Justine Durand, Kevin Biteau, Caroline Mary, Aurore Morello, Mélanie Néel, Georgia Porto, Géraldine Teppaz, Virginie Thepenier, Richard Danger, Nicolas Vince, Emmanuelle Wilhelm, Isabelle Girault, Riad Abes, Catherine Ruiz, Charlène Trilleaud, Kerry-Leigh Ralph, E. Sergio Trombetta, Alexandra Garcia, Virginie Vignard, Bernard Martinet, Alexandre Glémain, Sarah Bruneau, Fabienne Haspot, Safa Dehmani, Pierre Duplouye, Masayuki Miyasaka, Nathalie Labarrière, David Laplaud, Stéphanie Le Bas-Bernardet, Christophe Blanquart, Véronique Catros, Pierre-Antoine Gouraud, Isabelle Archambeaud, Hélène Aublé, Sylvie Metairie, Jean-François Mosnier, Dominique Costantini, Gilles Blancho, Sophie Conchon, Bernard Vanhove, Nicolas Poirier
A considerable fraction of B cells recognize SARS-CoV-2 with germline-encoded elements of their B cell receptor resulting in the production of neutralizing and non-neutralizing antibodies. We found that antibody sequences from different discovery cohorts shared biochemical properties and could be retrieved across validation cohorts confirming the stereotyped character of this naive response in COVID-19. While neutralizing antibody sequences were found independently of disease severity in line with serological data, individual non-neutralizing antibody sequences were associated with fatal clinical courses suggesting detrimental effects of these antibodies. We mined 200 immune repertoires of healthy individuals and 500 of patients with blood or solid cancers - all acquired prior to the pandemic - for SARS-CoV-2 antibody sequences. While the largely unmutated B cell rearrangements occurred in a substantial fraction of immune repertoires from young and healthy individuals, these sequences were less likely found in individuals over 60 years of age and in cancer. This reflects B cell repertoire restriction in aging and cancer and may to a certain extent explain the different clinical COVID-19 courses observed in these risk groups. Future studies will have to address if this stereotyped B cell response to SARS-CoV-2 emerging from unmutated antibody rearrangements will create long-lived memory.
Lisa Paschold, Donjete Simnica, Edith Willscher, Maria J.G.T. Vehreschild, Jochen Dutzmann, Daniel G. Sedding, Christoph Schultheiß, Mascha Binder
Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.
Zainab Rahil, Rebecca Leylek, Christian M. Schürch, Han Chen, Zach Bjornson-Hooper, Shannon R. Christensen, Pier Federico Gherardini, Salil S. Bhate, Matthew H. Spitzer, Gabriela K. Fragiadakis, Nilanjan Mukherjee, Nelson Kim, Sizun Jiang, Jennifer Yo, Brice Gaudilliere, Melton Affrime, Bonnie Bock, Scott E. Hensley, Juliana Idoyaga, Nima Aghaeepour, Kenneth Kim, Garry P. Nolan, David R. McIlwain