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
ARID1A, a component of the chromatin-remodeling complex SWI/SNF, is one of the most frequently mutated genes in human cancer. We sought to develop rational combination therapy to potentiate the efficacy of immune checkpoint blockade in ARID1A-deficient tumors. In a proteomic analysis of a data set from The Cancer Genomic Atlas, we found enhanced expression of Chk2, a DNA damage checkpoint kinase, in ARID1A-mutated/deficient tumors. Surprisingly, we found that ARID1A targets the nonchromatin substrate Chk2 for ubiquitination. Loss of ARID1A increased the Chk2 level through modulating autoubiquitination of the E3-ligase RNF8 and thereby reducing RNF8-mediated Chk2 degradation. Inhibition of the ATM/Chk2 DNA damage checkpoint axis led to replication stress and accumulation of cytosolic DNA, which subsequently activated the DNA sensor STING-mediated innate immune response in ARID1A-deficient tumors. As expected, tumors with mutation or low expression of both ARID1A and ATM/Chk2 exhibited increased tumor-infiltrating lymphocytes and were associated with longer patient survival. Notably, an ATM inhibitor selectively potentiated the efficacy of immune checkpoint blockade in ARID1A-depleted tumors but not in WT tumors. Together, these results suggest that ARID1A’s targeting of the nonchromatin substrate Chk2 for ubiquitination makes it possible to selectively modulate cancer cell–intrinsic innate immunity to enhance the antitumor activity of immune checkpoint blockade.
Lulu Wang, Lin Yang, Chen Wang, Wei Zhao, Zhenlin Ju, Wei Zhang, Jianfeng Shen, Yang Peng, Clemens An, Yen T. Luu, Shumei Song, Timothy A. Yap, Jaffer A. Ajani, Gordon B. Mills, Xuetong Shen, Guang Peng
BACKGROUND. Serological assays are of critical importance to investigate correlates of response and protection in COVID-19, to define previous exposure to SARS-CoV-2 in populations and to verify the development of an adaptive immune response in infected individuals. METHODS. We studied 509 confirmed COVID-19 patients from the San Raffaele Hospital of Milan and 480 pre-pandemic organ donor sera collected in 2010-2012. Using fluid-phase luciferase immune precipitation (LIPS) assays, we characterized IgG, IgM, IgA antibodies to the spike Receptor Binding Domain (RBD), S1+S2, nucleocapsid, and ORF6 to 10 of SARS-CoV-2, to the HCoV-OC43 and HCoV-HKU1 betacoronaviruses spike S2, and the H1N1Ca2009 flu virus hemagglutinin. Sequential samples at 1 and 3 months post-hospital discharge were also tested in 95 patients for SARS-CoV-2 RBD antibodies. RESULTS. Antibodies developed rapidly against multiple SARS-CoV-2 antigens in 95% of patients by 4 weeks post-symptoms onset and IgG to the RBD increased until the 3rd month of follow-up. We observed a major synchronous expansion of antibodies to the HCoV-OC43 and HCoV-HKU1 spike S2. A likely co-infection with influenza was neither linked to a more severe presentation of the disease nor to a worse outcome. Of the measured antibody responses positivity for IgG against the SARS-CoV-2 spike RBD was predictive of survival. CONCLUSIONS. The measurement of antibodies to selected epitopes of SARS-CoV-2 antigens can offer a more accurate assessment of the humoral response in patients and its impact on survival. The presence of partially cross-reactive antibodies with other betacoronoviruses is likely to impact on serological assay specificity and interpretation.
Massimiliano Secchi, Elena Bazzigaluppi, Cristina Brigatti, Ilaria Marzinotto, Cristina Tresoldi, Patrizia Rovere-Querini, Andrea Poli, Antonella Castagna, Gabriella Scarlatti, Alberto Zangrillo, Fabio Ciceri, Lorenzo Piemonti, Vito Lampasona
Protection of the brain from viral infections involves the type I interferon (IFN-I) system, defects in which renders humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels leads to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we here show that microglia and other immune cells undergo apoptosis in the HSV-1-infected brain through a mechanism dependent on the cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, while lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1-infected organotypic brain slices, or mice treated with caspase inhibitor, exhibited lower viral load and improved outcome of infection. Collectively, we identify an activation-induced apoptosis program in brain immune cells which down-modulates local immune responses.
Line S. Reinert, Ahmad S. Rashidi, Diana N. Tran, Georgios Katzilieris-Petras, Astrid K. Hvidt, Mette Gohr, Stefanie Fruhwürth, Chiranjeevi Bodda, Martin K. Thomsen, Mikkel H. Vendelbo, Ahmad Raza Khan, Brian Hansen, Petra Bergström, Lotta Agholme, Trine H. Mogensen, Maria H. Christensen, Jens R. Nyengaard, Ganes C. Sen, Henrik Zetterberg, Georges M.G.M. Verjans, Soren R. Paluden
Immune evasion is a pivotal event in tumor progression. To eliminate human cancer cells, current immune checkpoint therapy is set to boost the CD8+ T cell-mediated cytotoxicity. However, this action is eventually dependent on the efficient recognition of tumor-specific antigens via T cell receptors. One primary mechanism by which tumor cells evade immune surveillance is to downregulate their antigen presentation. Little progress has been made towards harnessing potential therapeutic targets for enhancing antigen presentation on the tumor cell. Here, we identified MAL2 as a key player that determines the turnover of the antigen-loaded MHC-I complex and reduces the antigen presentation on tumor cells. MAL2 promotes the endocytosis of tumor antigens via direct interaction with the MHC-I complex and endosome-associated RAB proteins. In preclinical models, depletion of MAL2 in breast tumor cells profoundly enhanced the cytotoxicity of tumor-infiltrating CD8+ T cells and suppressed breast tumor growth, suggesting that MAL2 is a potential therapeutic target for breast cancer immunotherapy.
Yuanzhang Fang, Lifei Wang, Changlin Wan, Yifan Sun, Kevin Van der Jeught, Zhuolong Zhou, Tianhan Dong, Ka Man So, Tao Yu, Yujing Li, Haniyeh Eyvani, Austyn Colter, Edward Dong, Sha Cao, Jin Wang, Bryan P. Schneider, George Sandusky, Yunlong Liu, Chi Zhang, Xiongbin Lu, Xinna Zhang
BACKGROUND. The T cell responses to the common cold coronaviruses have not been well characterized. Pre-existing T cell immunity to SARS-CoV-2 has been reported, and a recent study suggested that this was due to cross-recognition of the novel coronavirus by T cells specific for the common cold coronaviruses. METHODS. We used the ELISpot assay to characterize the T cell responses against peptide pools derived from the spike protein of three common cold coronaviruses (HCoV-229E, HCoV-NL63, and HCoV-OC43) and SARS-CoV-2 in 21 healthy donors who were seronegative for SARS-CoV-2 and had no known exposure to the virus. An in vitro expansion culture assay was also used to analyze memory T cell responses. RESULTS. We found responses to the spike protein of the three common cold coronaviruses in many donors. We then focused on HCoV-NL63 and demonstrated broad T cell responses to the spike protein and identified 22 targeted peptides. Interestingly, only one subject had a significant response to SARS-CoV-2 spike or nucleocapsid protein in the ELISpot assay. In vitro expansion studies suggested that T cells specific for the HCoV-NL63 spike protein in this subject could also recognize SARS-CoV-2 spike protein peptide pools. CONCLUSIONS. Healthy donors have circulating T cells specific for the spike proteins of HCoV-NL63, HCoV-229E, and HCoV-OC43. T cell responses to SARS-CoV-2 spike and nucleocapsid proteins were present in only one subject and were potentially the result of cross-recognition by T cells specific for the common cold coronaviruses. Further studies are needed to determine whether this influences COVID-19 outcomes.
Bezawit A. Woldemeskel, Abena K. Kwaa, Caroline C. Garliss, Oliver Laeyendecker, Stuart C. Ray, Joel N. Blankson