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Background Rhinoviruses are the predominant cause of respiratory viral infections and are strongly associated with asthma exacerbations. While humoral immunity plays an important role during virus infections, cellular aspects of this response are less well understood. Here, we investigated the antiviral response of circulating B cells upon experimental rhinovirus infection in healthy individuals and asthma patients. Methods We purified B cells from experimentally infected healthy individuals and patients with asthma and subjected them to total RNA-sequencing. Rhinovirus-derived RNA was measured in isolated B cells using a highly sensitive PCR. B cells were stimulated with rhinovirus in vitro to further study gene expression, expression of antiviral proteins and B-cell differentiation in response rhinovirus stimulation. Protein expression of pro-inflammatory cytokines in response to rhinovirus was assessed using a proximity extension assay. Results B cells isolated from experimentally infected subjects exhibited an antiviral gene profile linked to IFN-alpha, carried viral RNA in vivo and were transiently infected by rhinovirus in vitro. B cells rapidly differentiated into plasmablasts upon rhinovirus stimulation. While B cells lacked expression of interferons in response to rhinovirus exposure, co-stimulation with rhinovirus and IFN-alpha upregulated pro-inflammatory cytokine expression suggesting a potential new function of B cells during virus infections. Asthma patients showed extensive upregulation and dysregulation of antiviral gene expression. Conclusion These findings add to the understanding of systemic effects of rhinovirus infections on B-cell responses in the periphery, show potential dysregulation in patients with asthma and might also have implications during infection with other respiratory viruses.

Context.— The ongoing COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has elicited a surge in demand for serologic testing to identify previously infected individuals. In particular, antibody testing is crucial in identifying COVID-19 convalescent plasma, which has been approved by the Food and Drug Administration under the Emergency Use Authorization for use as passive immunotherapy for hospitalized patients infected with COVID-19. Currently, high-titer COVID-19 convalescent plasma can be qualified by Ortho's Vitros COVID-19 IgG antibody test. Objective.— To explore the use of an efficient testing method to identify high-titer COVID-19 convalescent plasma for use in treating COVID-19–infected patients and track COVID-19 positivity over time. Design.— We evaluated an enzyme-linked immunosorbent assay (ELISA)–based method that detects antibodies specific to the SARS-CoV-2 receptor binding domain (RBD) with individual and pooled plasma samples and compared its performance against the Vitros COVID-19 IgG antibody test. Using the pooled RBD-ELISA (P-RE) method, we also screened more than 10 000 longitudinal healthy blood donor samples to assess seroprevalence. Results.— P-RE demonstrates 100% sensitivity in detecting Food and Drug Administration–defined high-titer samples when compared with the Vitros COVID-19 IgG antibody test. Overall sensitivity of P-RE when compared with the Vitros COVID-19 IgG antibody test and our individual sample RBD-ELISA (I-RE) were 83% and 56%, respectively. When screening 10 218 healthy blood donor samples by P-RE, we found the seroprevalence correlated with the local infection rates with a correlation coefficient of 0.21 (P < .001). Conclusions.— Pooling plasma samples can be used to efficiently screen large populations for individuals with high-titer anti–RBD antibodies, important for COVID-19 convalescent plasma identification.

Kids armed with anti-coronavirus B cells It remains unclear whether B cell repertoires against coronaviruses and other pathogens differ between adults and children and how important these distinctions are. Yang et al. analyzed blood samples from young children and adults, as well as tissues from deceased organ donors, characterizing the B cell receptor (BCR) repertoires specific to six common pathogens and two viruses that they had not seen before: Ebola virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Children had higher frequencies of B cells with convergent BCR heavy chains against previously encountered pathogens and higher frequencies of class-switched convergent B cell clones against SARS-CoV-2 and related coronaviruses. These findings suggest that encounters with coronaviruses in early life may produce cross-reactive memory B cell populations that contribute to divergent COVID-19 susceptibilities. Science, this issue p. 738, Blood taken from children before the COVID-19 pandemic contains memory B cells that bind SARS-CoV-2., Vaccination and infection promote the formation, tissue distribution, and clonal evolution of B cells, which encode humoral immune memory. We evaluated pediatric and adult blood and deceased adult organ donor tissues to determine convergent antigen-specific antibody genes of similar sequences shared between individuals. B cell memory varied for different pathogens. Polysaccharide antigenspecific clones were not exclusive to the spleen. Adults had higher clone frequencies and greater class switching in lymphoid tissues than blood, while pediatric blood had abundant class-switched convergent clones. Consistent with reported serology, prepandemic children had class-switched convergent clones to severe acute respiratory syndrome coronavirus 2 with weak cross-reactivity to other coronaviruses, while adult blood or tissues showed few such clones. These results highlight the prominence of early childhood B cell clonal expansions and cross-reactivity for future responses to novel pathogens.

Background B cells play a crucial role during rhinovirus (RV) infections by production of virus‐neutralizing antibodies. A main feature of common variable immunodeficiency (CVID) is hypogammaglobulinemia (HG). HG patients have severely reduced levels of antibody‐producing B cells and suffer from prolonged virus infections. Here, we addressed whether antiviral response of peripheral blood lymphocytes differs between HG patients and healthy individuals during natural RV infection. Methods Using fluorescence‐activated cell sorting, B‐cell subsets were analyzed. Simultaneously, CD19 + B cells, CD14 + monocytes, and CD3 + T cells were sorted from frozen peripheral blood mononuclear cells from 11 RV‐infected hypogammaglobulinemia patients, 7 RV‐infected control subjects, and 14 noninfected control subjects. Real‐time PCR was used to study expression of antiviral genes. A pan‐RV PCR was used to detect RV genome in all samples. Results In HG patients, total B‐cell numbers, as well as IgA + and IgG + switched memory B cells, were reduced while naive B cells and T cells were increased. STAT1 expression was increased in HG patients compared to controls in all lymphocyte subsets analyzed. The expression of antiviral genes IFITM1 and MX1 correlated with STAT1 expression in B cells and monocytes. RV RNA was found in 88.9% of monocytes from infected HG patients, 85.7% of monocytes from infected controls, and 7.1% of monocytes from uninfected controls. Conclusions We demonstrate an increased antiviral response in B cells and monocytes in HG patients and their correlation with STAT1 expression. Monocytes of infected HG patients and infected non‐HG controls carry RV RNA.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including 3rd dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is less after infection compared to all vaccines evaluated, but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks post-vaccination in some cases. SARS-CoV-2 antibody specificity, breadth and maturation are affected by imprinting from exposure history, and distinct histological and antigenic contexts in infection compared to vaccination., Human antibody responses to SARS-CoV-2 differ between vaccination and infection, with vaccination (regardless of vaccine type) inducing more productive lymph node germinal center responses and a broader range of IgG neutralizing antibodies capable of recognizing viral variants.

Different vaccines for SARS-CoV-2 are approved in various countries, but few direct comparisons of the antibody responses they stimulate have been reported. We collected plasma specimens in July 2021 from 196 Mongolian participants fully vaccinated with one of four Covid vaccines: Pfizer/BioNTech, AstraZeneca, Sputnik V and Sinopharm. Functional antibody testing with a panel of nine SARS-CoV-2 viral variant RBD proteins reveal marked differences in the vaccine responses, with low antibody levels and RBD-ACE2 blocking activity stimulated by the Sinopharm and Sputnik V vaccines in comparison to the AstraZeneca or Pfizer/BioNTech vaccines. The Alpha variant caused 97% of infections in Mongolia in June and early July 2021. Individuals who recover from SARS-CoV-2 infection after vaccination achieve high antibody titers in most cases. These data suggest that public health interventions such as vaccine boosting, potentially with more potent vaccine types, may be needed to control the COVID-19 pandemic in Mongolia and worldwide.

Different SARS-CoV-2 vaccines are approved in various countries, but few direct comparisons of the antibody responses they stimulate have been reported. We collected plasma specimens in July 2021 from 196 Mongolian participants fully vaccinated with one of four COVID-19 vaccines: Pfizer/BioNTech, AstraZeneca, Sputnik V and Sinopharm. Functional antibody testing with a panel of nine SARS-CoV-2 viral variant receptor binding domain (RBD) proteins reveal marked differences in vaccine responses, with low antibody levels and RBD-ACE2 blocking activity stimulated by the Sinopharm and Sputnik V vaccines in comparison to the AstraZeneca or Pfizer/BioNTech vaccines. The Alpha variant caused 97% of infections in Mongolia in June and early July 2021. Individuals who recover from SARS-CoV-2 infection after vaccination achieve high antibody titers in most cases. These data suggest that public health interventions such as vaccine boosting, potentially with more potent vaccine types, may be needed to control COVID-19 in Mongolia and worldwide., Graphical Abstract, Dashdorj et al. examined antibody responses in COVID-19 vaccinated Mongolian participants. Antibodies blocking ACE2-RBD binding across SARS-CoV-2 variants were highest among Pfizer/BioNTech vaccinees, followed by AstraZeneca, Sputnik V, and then Sinopharm vaccinees. Breakthrough infections in June-July 2021 were predominantly the Alpha variant and induced higher blocking antibodies across vaccination groups.

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, new vaccine strategies including lipid nanoparticle delivery of antigen encoding RNA have been deployed globally. The BioNTech/Pfizer mRNA vaccine BNT162b2 encoding SARS-CoV-2 spike protein shows 95% efficacy in preventing disease, but it is unclear how the antibody responses to vaccination differ from those generated by infection. Here we compare the magnitude and breadth of antibodies targeting SARS-CoV-2, SARS-CoV-2 variants of concern, and endemic coronaviruses, in vaccinees and infected patients. We find that vaccination differs from infection in the dominance of IgG over IgM and IgA responses, with IgG reaching levels similar to those of severely ill COVID-19 patients and shows decreased breadth of the antibody response targeting endemic coronaviruses. Viral variants of concern from B.1.1.7 to P.1 to B.1.351 form a remarkably consistent hierarchy of progressively decreasing antibody recognition by both vaccinees and infected patients exposed to Wuhan-Hu-1 antigens.

SummaryBackgroundThere is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses.MethodsWe describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape.FindingsWe found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape.InterpretationOur results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.FundingThe work was partially funded by The Saban Research Institute at Children’s Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.

We present the case of an inpatient with pneumonia and repeatedly negative nasopharyngeal SARS-CoV-2 testing. In such challenging cases, alternative diagnostic options include lower respiratory tract and plasma SARS-CoV-2 RNA testing, of which the latter may be particularly useful where bronchoscopy is deferred due to clinical factors or transmission risk.

Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients., SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.

Background Respiratory infections with rhinoviruses (RV) are strongly associated with development and exacerbations of asthma, and they pose an additional health risk for subjects with allergy. Objective How RV infections and chronic allergic diseases are linked and what role RV plays in the breaking of tolerance in regulatory T (Treg) cells is unknown. Therefore, this study aims to investigate the effects of RV on Treg cells. Methods Treg cells were isolated from subjects with asthma and controls after experimental infection with the RV-A16 (RV16) and analyzed with next-generation sequencing. Additionally, suppression assays, quantitative PCR assays, and protein quantifications were performed with Treg cells after in vitro RV16 infection. Results RV16 induced a strong antiviral response in Treg cells from subjects with asthma and controls, including the upregulation of IFI44L, MX1, ISG15, IRF7, and STAT1. In subjects with asthma, the inflammatory response was exaggerated and showed a dysregulated immune response compared with that in the controls. Furthermore, subjects with asthma failed to upregulate several immunosuppressive molecules such as CTLA4 and CD69, and they upregulated the inflammasome-related genes PYCARD and AIM2. Additionally, RV16 reduced the suppressive capacity of Treg cells from healthy subjects and subjects with asthma in vitro and increased TH2 cell–type cytokine production. Conclusions Treg cells from healthy subjects and subjects with asthma displayed an antiviral response after RV infection and showed reduced suppressive capacity. These data suggest that Treg cell function might be altered or impaired during RV infections, which might play an important role in the association between RV and the development of asthma and asthma exacerbations.

Recovery from COVID-19 is associated with production of anti-SARS-CoV-2 antibodies, but it is uncertain whether these confer immunity. We describe viral RNA shedding duration in hospitalized patients and identify patients with recurrent shedding. We sequenced viruses from two distinct episodes of symptomatic COVID-19 separated by 140 days in a single patient, to conclusively describe reinfection with a new strain harboring the spike variant D614G. With antibody and B cell analytics, we show correlates of adaptive immunity, including a differential response to D614G. Finally, we discuss implications for vaccine programs and begin to define benchmarks for protection against reinfection from SARS-CoV-2.

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals’ serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived.

A novel B cell subset that produces proangiogenic cytokines is increased in pathologies associated with angiogenesis., B cells contribute to immune responses through the production of immunoglobulins, antigen presentation, and cytokine production. Several B cell subsets with distinct functions and polarized cytokine profiles have been reported. In this study, we used transcriptomics analysis of immortalized B cell clones to identify an IgG4+ B cell subset with a unique function. These B cells are characterized by simultaneous expression of proangiogenic cytokines including VEGF, CYR61, ADM, FGF2, PDGFA, and MDK. Consequently, supernatants from these clones efficiently promote endothelial cell tube formation. We identified CD49b and CD73 as surface markers identifying proangiogenic B cells. Circulating CD49b+CD73+ B cells showed significantly increased frequency in patients with melanoma and eosinophilic esophagitis (EoE), two diseases associated with angiogenesis. In addition, tissue-infiltrating IgG4+CD49b+CD73+ B cells expressing proangiogenic cytokines were detected in patients with EoE and melanoma. Our results demonstrate a previously unidentified proangiogenic B cell subset characterized by expression of CD49b, CD73, and proangiogenic cytokines.

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BACKGROUND: Signal transducer and activator of transcription 3 hyper-IgE syndrome (STAT3-HIES) is caused by heterozygous mutations in the STAT3 gene and is associated with eczema, elevated serum IgE, and recurrent infections resembling severe atopic dermatitis, while clinically relevant specific IgE is almost absent. METHODS: To investigate the impact of STAT3 signaling on B-cell responses, we assessed lymph node and bone marrow, blood B and plasma cell subsets, somatic hypermutations in Ig genes, and in vitro proliferation and antibody production in STAT3-HIES patients and healthy controls. RESULTS: Lymph nodes of STAT3-HIES patients showed normal germinal center architecture and CD138+ plasma cells residing in the paracortex, which expressed IgE, IgG, and IgM but not IgA. IgE+ plasma cells were abundantly present in STAT3-HIES bone marrow. Proliferation of naive B cells upon stimulation with CD40L and IL-4 was similar in patients and controls, while patient cells showed reduced responses to IL-21. IgE, IgG1, IgG3 and IgA1 transcripts showed reduced somatic hypermutations. Peripheral blood IgE+ memory B-cell frequencies were increased in STAT3-HIES, while other memory B-cell frequencies except for IgG4+ cells were decreased. CONCLUSIONS: Despite impaired STAT3 signaling, STAT3-HIES patients can mount in vivo T-cell-dependent B-cell responses, while circulating memory B cells, except for those expressing IgG4 and IgE, were reduced. Reduced molecular maturation demonstrated the critical need of STAT3 signaling for optimal affinity maturation and B-cell differentiation, supporting the need for immunoglobulin substitution therapy and explaining the high IgE serum level in the majority with absent allergic symptoms.

Summary Background There is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses. Methods We describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape. Findings We found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape. Interpretation Our results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered. Funding The work was partially funded by The Saban Research Institute at Children’s Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.

Background The involvement of B cells in allergen tolerance induction remains largely unexplored. This study investigates the role of B cells in this process, by comparing B-cell responses in allergic patients before and during allergen immunotherapy (AIT) and naturally exposed healthy beekeepers before and during the beekeeping season. Methods Circulating B cells were characterized by flow cytometry. Phospholipase A2 (PLA)-specific B cells were identified using dual-color staining with fluorescently labeled PLA. Expression of regulatory B-cell-associated surface markers, interleukin-10, chemokine receptors, and immunoglobulin heavy-chain isotypes, was measured. Specific and total IgG1, IgG4, IgA, and IgE from plasma as well as culture supernatants of PLA-specific cells were measured by ELISA. Results Strikingly, similar responses were observed in allergic patients and beekeepers after venom exposure. Both groups showed increased frequencies of plasmablasts, PLA-specific memory B cells, and IL-10-secreting CD73- CD25+ CD71+ BR 1 cells. Phospholipase A2-specific IgG4-switched memory B cells expanded after bee venom exposure. Interestingly, PLA-specific B cells showed increased CCR5 expression after high-dose allergen exposure while CXCR4, CXCR5, CCR6, and CCR7 expression remained unaffected. Conclusions This study provides the first detailed characterization of allergen-specific B cells before and after bee venom tolerance induction. The observed B-cell responses in both venom immunotherapy-treated patients and naturally exposed beekeepers suggest a similar functional immunoregulatory role for B cells in allergen tolerance in both groups. These findings can be investigated in other AIT models to determine their potential as biomarkers of early and successful AIT responses.

BACKGROUND: Human rhinoviruses (HRV) are one of the main causes of virus induced asthma exacerbations. Infiltration of B lymphocytes into the subepithelial tissue of the lungs has been demonstrated during rhinovirus infection in allergic individuals. However, the mechanisms through which HRVs modulate the immune responses of monocytes and lymphocytes are not yet well described. OBJECTIVE: To study the dynamics of virus uptake by monocytes and lymphocytes, and the ability of HRVs to induce activation of in vitro cultured human peripheral blood mononuclear cells. METHODS: Flow cytometry was used for the enumeration and characterization of lymphocytes. Proliferation was estimated using (3) H-thymidine or CFSE labelling and ICAM-1 blocking. We used bead based multiplex assays and quantitative PCR for cytokine quantification. HRV accumulation and replication inside B lymphocytes was detected by a combination of in situ hybridation (ISH), immunofluorescence and with PCR for positive strand and negative strand viral RNA. Cell images were acquired with imaging flow cytometry. RESULTS: By means of imaging flow cytometry, we demonstrate a strong and quick binding of HRV types 16 and 1B to monocytes, and slower interaction of these HRVs with CD4+ T cells, CD8+ T cells and CD19+ B cells. Importantly, we show that HRVs induce the proliferation of B cells while addition of anti-ICAM-1-antibody partially reduces this proliferation for HRV16. We prove with ISH that HRVs can enter B cells, form their viral replication centers and the newly formed virions are able to infect HeLa cells. In addition, we demonstrate that similarly to epithelial cells, HRVs induce the production of pro-inflammatory cytokines in PBMCs. CONCLUSION: Our results demonstrate for the first time that HRVs enter and form viral replication centers in B lymphocytes and induce the proliferation of B cells. Newly formed virions have the capacity to infect other cells (HeLa). These findings indicate that the regulation of human rhinovirus induced B cell responses could be a novel approach to develop therapeutics to treat the virus-induced exacerbation of asthma. This article is protected by copyright. All rights reserved.

Background Long-term follow-up of allergen-specific B cells in terms of immunoglobulin isotype expression, plasmablast differentiation, and regulatory B (Breg) cell development during allergen-specific immunotherapy (AIT) has not been reported. Objective Allergen-specific B-cell responses during 2 years of house dust mite AIT were compared between responder and nonresponder patients. Methods B cells specific for Der p 1 were detected by using the fluorochrome-labeled allergen method. The frequency of IgA-, IgG1- and IgG4-switched Der p 1–specific B cells, plasmablasts, and IL-10– and IL-1 receptor antagonist (IL-1RA)–producing Breg cells were investigated and correlated to clinical response to AIT. Results Sixteen of 25 patients completed the 2-year study. Eleven responder patients showed a successful response to AIT, as measured by a decrease in symptom-medication scores from 13.23 ± 0.28 to 2.45 ± 0.24 (P = .001) and a decrease in skin prick test reactivity to house dust mite from 7.0 ± 1.3 to 2.7 ± 0.5 mm (P = .001). IgG4+ and IgA+ Der p 1–specific B cells showed a significant increase after AIT, with a significantly greater frequency in responders compared with nonresponders in the IgG4+ but not the IgA+ fraction. The frequency of plasmablasts and IL-10– and/or IL-1RA–producing Breg cells was greater among responders compared with nonresponders after 2 years. The increased frequency of Der p 1–specific IgG4+ B cells, plasmablasts, and IL-10+ and dual-positive IL-10+IL-1RA+ Breg cells significantly correlated with improved clinical symptoms over the course of AIT. Conclusion Allergen-specific B cells in patients responding to AIT are characterized by increased numbers of IgA- and IgG4-expressing Der p 1–specific B cells, plasmablasts, and IL-10+ and/or IL-1RA+ Breg cells.

The escape of anti-self B cells from tolerance mechanisms like clonal deletion, receptor editing, and anergy results in the production of autoantibodies, which is a hallmark of many autoimmune disorders. In this study, we demonstrate that both germline sequences and somatic mutations contribute to autospecificity of B cell clones. For this issue, we investigated the development of antinuclear autoantibodies (ANAs) and their repertoire in two different mouse models. First, in aging mice that were shown to gain several autoimmune features over time including ANAs. Second, in mice undergoing a chronic graft-versus-host disease (GVHD), thereby developing systemic lupus erythematosus-like symptoms. Detailed repertoire analysis revealed that somatic hypermutations (SHM) were present in all Vh and practically all Vl regions of ANAs generated in these two models. The ANA B cell repertoire in aging mice was restricted, dominated by clonally related Vh1-26/Vk4-74 antibodies. In the collection of GVHD-derived ANAs, the repertoire was less restricted, but the usage of the Vh1-26/Vk4-74 combination was still apparent. Germline conversion showed that the SHM in the 4-74 light chain are deterministic for autoreactivity. Detailed analysis revealed that antinuclear reactivity of these antibodies could be induced by a single amino acid substitution in the CDR1 of the Vk4-74. In both aging B6 and young GVHD mice, conversion of the somatic mutations in the Vh and Vl regions of non Vh1-26/Vk4-74 using antibodies showed that B cells with a germline-encoded V gene could also contribute to the ANA-reactive B cell repertoire. These findings indicate that two distinct pathways generate ANA-producing B cells in both model systems. In one pathway, they are generated by Vh1-26/Vk4-74 expressing B cells in the course of immune responses to an antigen that is neither a nuclear antigen nor any other self-antigen. In the other pathway, ANA-producing B cells are derived from progenitors in the bone marrow that express B cell receptors (BCRs), which bind to nuclear antigens and that escape tolerance induction, possibly as a result of crosslinking of their BCRs by multivalent determinants of nuclear antigens.

Allergen-specific immunotherapy (AIT) has been used for more than 100 years as a clinical tolerance-inducing and immune tolerance-inducing therapy for allergic diseases and represents a potentially curative method of treatment. AIT functions through multiple mechanisms including early desensitization of basophils and mast cells, regulating T-cell and B-cell responses, changing antibody isotypes, and decreasing activation, mediator release and affected tissue migration of eosinophils, basophils, and mast cells. Similar molecular and cellular mechanisms have been observed in subcutaneous AIT, sublingual AIT and peptide immunotherapy as well as natural tolerance to high doses of allergen exposure in beekeepers and cat owners.

Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8-12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4(+) T-cell dependent, since old (40-50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age-related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4(+) T cells that promote ANA production were radioresistant. Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T-cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T-cell homeostasis may drive the onset of some autoimmune features.

Immune tolerance to both self-antigens and innocuous non–self-antigens is essential to protect the host against chronic inflammatory diseases and tissue damage. A wide range of cell types and suppressive molecules are involved in induction and maintenance of tolerance. In addition to their key function in the production of immunoglobulins, B cells can regulate immune responses through their surface molecules and secretion of cytokines. Regulatory B (Breg) cells are characterized by their immunosuppressive capacity, which is often mediated through IL-10 secretion. However, IL-35 and TGF-β have also been associated with B cell–mediated immunosuppression. Several types of murine and human Breg cells have been described, such as mouse CD5 + CD1d hi B10 cells, CD21 hi CD23 hi CD24 hi transitional stage 2–like B cells, and CD138 + plasma cells and plasmablasts. Human Breg cell types include CD27 + CD24 high B10 cells, CD24 hi CD38 hi immature transitional B cells, and CD73 − CD25 + CD71 + B R 1 cells and a subset of plasma cells. Support for the in vivo existence of allergen-specific human Breg cells comes from direct detection of their increase during the course of allergen-specific immunotherapy, as well as their increased expression in nonallergic but high-dose allergen–exposed beekeepers. Human B R 1 cells selectively upregulate IgG 4 antibodies on differentiation to plasma cells. This suggests an additional immune regulatory role because of the noninflammatory and blocking antibody function of IgG 4 . Taken together, Breg cells appear to be involved in mediating allergen tolerance, but many open questions remain to be answered.

BACKGROUND Interleukin-22 is produced by certain T helper cells subsets (Th17, Th22) and at lower levels by γ-δ T cells, NKT and innate lymphoid cells. Th22 cells are unique immune cells that regulate tissue responses by IL-22 production. The exact discrimination between Th17 cells that co-produce IL-22 and single IL-22-producing Th22 cells has not been possible until the present study. Isolation of pure Th22 cells without co-expression of cytokines of other T-cell subsets is essential to better understand their function in humans. The aim of this study is the isolation and characterization of viable, human IL-22-producing CD4+ T cells that do not produce IL-17A. METHODS Isolation of viable Th22 cells was performed with the combination of two cytokine secretion assays detecting IL-17A- and IL-22-producing cells in a single purification step. RESULTS The newly developed cytokine secretion assay consists of anti-IL-22 and anti-IL-17A catch antibodies, which via biotin-streptavidin interaction are bound to the biotinylated surface of the target cell, and anti-IL-22 and IL-17A detection antibody labelled with a fluorescent dye, which detects cytokines bound to these catch antibodies. A unique population of human Th22 cells, which do not produce IL-17A, was sorted, and cytokine expression pattern was confirmed by quantitative PCR analysis and ELISA. The presented technique allows the detection and isolation of pure human Th22 cells. CONCLUSIONS This technique may allow the purification of any single cytokine-producing cell subset, and the combination of several different cytokine secretion assays can be used to purify and characterize novel and unique cell subsets.

There have been extensive developments on cellular and molecular mechanisms of immune regulation in allergy, asthma, autoimmune diseases, tumor development, organ transplantation, and chronic infections during the last few years. Better understanding the functions, reciprocal regulation, and counterbalance of subsets of immune and inflammatory cells that interact through interleukins, interferons, TNF-α, and TGF- β offer opportunities for immune interventions and novel treatment modalities in the era of development of biological immune response modifiers particularly targeting these molecules or their receptors. More than 60 cytokines have been designated as interleukins since the initial discoveries of monocyte and lymphocyte interleukins (called IL-1 and IL-2, respectively). Studies of transgenic or gene- deficient mice with altered expression of these cytokines or their receptors and analyses of mutations and polymorphisms in human genes that encode these products have provided essential information about their functions. Here we review recent developments on IL-1 to IL-38, TNF-α, TGF-β, and interferons. We highlight recent advances during the last few years in this area and extensively discuss their cellular sources, targets, receptors, signaling pathways, and roles in immune regulation in patients with allergy and asthma and other inflammatory diseases.

Background Childhood exposure to a farm environment has been shown to protect against the development of inflammatory diseases, such as allergy, asthma, and inflammatory bowel disease. Objective We sought to investigate whether both exposure to microbes and exposure to structures of nonmicrobial origin, such as the sialic acid N-glycolylneuraminic acid (Neu5Gc), might play a significant role. Methods Exposure to Neu5Gc was evaluated by quantifying anti-Neu5Gc antibody levels in sera of children enrolled in 2 farm studies: the Prevention of Allergy Risk factors for Sensitization in Children Related to Farming and Anthroposophic Lifestyle (PARSIFAL) study (n = 299) and the Protection Against Allergy Study in Rural Environments (PASTURE) birth cohort (cord blood [n = 836], 1 year [n = 734], 4.5 years [n = 700], and 6 years [n = 728]), and we associated them with asthma and wheeze. The effect of Neu5Gc was examined in murine airway inflammation and colitis models, and the role of Neu5Gc in regulating immune activation was assessed based on helper T-cell and regulatory T-cell activation in mice. Results In children anti-Neu5Gc IgG levels correlated positively with living on a farm and increased peripheral blood forkhead box protein 3 expression and correlated inversely with wheezing and asthma in nonatopic subjects. Exposure to Neu5Gc in mice resulted in reduced airway hyperresponsiveness and inflammatory cell recruitment to the lung. Furthermore, Neu5Gc administration to mice reduced the severity of a colitis model. Mechanistically, we found that Neu5Gc exposure reduced IL-17+ T-cell numbers and supported differentiation of regulatory T cells. Conclusions In addition to microbial exposure, increased exposure to non–microbial-derived Neu5Gc might contribute to the protective effects associated with the farm environment., published version, peerReviewed

Background B cells play a central role in IgE-mediated allergies. In damaged airway epithelium, they are exposed directly to aeroallergens. We aimed to assess whether direct exposure of B cells to pollen constituents affects allergic sensitization. Methods B cells from murine splenocytes and from blood samples of healthy donors were incubated for 8 days under Th2-like conditions with aqueous ragweed pollen extracts (Amb-APE) or its constituents. Secreted total IgM, IgG, and IgE was quantified by ELISA. Additionally, birch, grass, or pine-pollen extracts were tested. The number of viable cells was evaluated by ATP measurements. B-cell proliferation was measured by CFSE staining. IgE class switch was analyzed by quantitation of class switch transcripts. In an OVA/Alum i.p.-sensitization mouse model, Amb-APE was intranasally instilled for 11 consecutive days. Results Upon Th2 priming of murine B cells, ragweed pollen extract caused a dose-dependent increase in IgE production, while IgG and IgM were not affected. The low-molecular-weight fraction and phytoprostane E1 (PPE1) increased IgE production, while Amb a 1 did not. PPE1 enhanced IgE also in human memory B cells. Under Th1 conditions, Amb-APE did not influence immunoglobulin secretion. The IgE elevation was not ragweed specific. It correlated with proliferation of viable B cells, but not with IgE class switch. In vivo, Amb-APE increased total IgE and showed adjuvant activity in allergic airway inflammation. Conclusions Aqueous pollen extracts, the protein-free fraction of Amb-APE, and the pollen-contained substance PPE1 specifically enhance IgE production in Th2-primed B cells. Thus, pollen-derived nonallergenic substances might be responsible for B-cell-dependent aggravation of IgE-mediated allergies.

Background Distinct human IL-10–producing B-cell subsets with immunoregulatory properties have been described. However, the broader spectrum of their direct cellular targets and suppressive mechanisms has not been extensively studied, particularly in relation to direct and indirect IL-10–mediated functions. Objective The aim of the study was to investigate the effects of IL-10 overexpression on the phenotype and immunoregulatory capacity of B cells. Methods Primary human B cells were transfected with hIL-10, and IL-10–overexpressing B cells were characterized for cytokine and immunoglobulin production by means of specific ELISA and bead-based assays. Antigen presentation, costimulation capacity, and transcription factor signatures were analyzed by means of flow cytometry and quantitative RT-PCR. Effects of IL-10–overexpresing B cells on Toll-like receptor–triggered cytokine release from PBMCs, LPS-triggered maturation of monocyte-derived dendritic cells, and tetanus toxoid–induced PBMC proliferation were assessed in autologous cocultures. Results IL-10–overexpressing B cells acquired a prominent immunoregulatory profile comprising upregulation of suppressor of cytokine signaling 3 (SOCS3), glycoprotein A repetitions predominant (GARP), the IL-2 receptor α chain (CD25), and programmed cell death 1 ligand 1 (PD-L1). Concurrently, their secretion profile was characterized by a significant reduction in levels of proinflammatory cytokines (TNF-α, IL-8, and macrophage inflammatory protein 1α) and augmented production of anti-inflammatory IL-1 receptor antagonist and vascular endothelial growth factor. Furthermore, IL-10 overexpression was associated with a decrease in costimulatory potential. IL-10–overexpressing B cells secreted less IgE and potently suppressed proinflammatory cytokines in PBMCs, maturation of monocyte-derived dendritic cells (rendering their profile to regulatory phenotype), and antigen-specific proliferation in vitro . Conclusion Our data demonstrate an essential role for IL-10 in inducing an immunoregulatory phenotype in B cells that exerts substantial anti-inflammatory and immunosuppressive functions.