Publications by authors named "Adrian Hayday"

127 Publications

OMIP-079: Cell cycle of CD4 and CD8 naïve/memory T cell subsets, and of Treg cells from mouse spleen.

Cytometry A 2021 Oct 19. Epub 2021 Oct 19.

Institute of Molecular Biology and Pathology, National Research Council of Italy (CNR), Rome, Italy.

A multicolor flow cytometry panel was designed and optimized to define the following nine mouse T cell subsets: Treg (CD3 CD4 CD8 FoxP3 ), CD4 T naïve (CD3 CD4 CD8 FoxP3 CD44 CD62L ), CD4 T central memory (CD3 CD4 CD8 FoxP3 CD44 CD62L ), CD4 T effector memory (CD3 CD4 CD8 FoxP3 CD44 CD62L ), CD4 T EMRA (CD3 CD4 CD8 FoxP3 CD44 CD62L ), CD8 T naïve (CD3 CD8 CD4 CD44 CD62L ), CD8 T central memory (CD3 CD8 CD4 CD44 CD62L ), CD8 T effector memory (CD3 CD8 CD4 CD44 CD62L ), and CD8 T EMRA (CD3 CD8 CD4 CD44 CD62L ). In each T cell subset, a dual staining for Ki-67 expression and DNA content was employed to distinguish the following cell cycle phases: G (Ki67 , with 2n DNA), G (Ki67 , with 2n DNA), and S-G /M (Ki67 , with 2n < DNA ≤ 4n). This panel was established for the analysis of mouse (C57BL/6J) spleen.
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http://dx.doi.org/10.1002/cyto.a.24509DOI Listing
October 2021

Corrigendum: To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis.

Front Immunol 2021 28;12:756641. Epub 2021 Sep 28.

Immunosurveillance Laboratory, The Francis Crick Institute, London, United Kingdom.

[This corrects the article DOI: 10.3389/fimmu.2021.653974.].
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http://dx.doi.org/10.3389/fimmu.2021.756641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507465PMC
September 2021

Comment on "Aberrant type 1 immunity drives susceptibility to mucosal fungal infections".

Science 2021 09 16;373(6561):eabi6235. Epub 2021 Sep 16.

Francis Crick Institute, London, UK.

[Figure: see text].
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http://dx.doi.org/10.1126/science.abi6235DOI Listing
September 2021

SARS-CoV-2 RNAemia and proteomic trajectories inform prognostication in COVID-19 patients admitted to intensive care.

Nat Commun 2021 06 7;12(1):3406. Epub 2021 Jun 7.

Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, King's College London, London, UK.

Prognostic characteristics inform risk stratification in intensive care unit (ICU) patients with coronavirus disease 2019 (COVID-19). We obtained blood samples (n = 474) from hospitalized COVID-19 patients (n = 123), non-COVID-19 ICU sepsis patients (n = 25) and healthy controls (n = 30). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was detected in plasma or serum (RNAemia) of COVID-19 ICU patients when neutralizing antibody response was low. RNAemia is associated with higher 28-day ICU mortality (hazard ratio [HR], 1.84 [95% CI, 1.22-2.77] adjusted for age and sex). RNAemia is comparable in performance to the best protein predictors. Mannose binding lectin 2 and pentraxin-3 (PTX3), two activators of the complement pathway of the innate immune system, are positively associated with mortality. Machine learning identified 'Age, RNAemia' and 'Age, PTX3' as the best binary signatures associated with 28-day ICU mortality. In longitudinal comparisons, COVID-19 ICU patients have a distinct proteomic trajectory associated with mortality, with recovery of many liver-derived proteins indicating survival. Finally, proteins of the complement system and galectin-3-binding protein (LGALS3BP) are identified as interaction partners of SARS-CoV-2 spike glycoprotein. LGALS3BP overexpression inhibits spike-pseudoparticle uptake and spike-induced cell-cell fusion in vitro.
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http://dx.doi.org/10.1038/s41467-021-23494-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184784PMC
June 2021

Safety and immunogenicity of one versus two doses of the COVID-19 vaccine BNT162b2 for patients with cancer: interim analysis of a prospective observational study.

Lancet Oncol 2021 06 27;22(6):765-778. Epub 2021 Apr 27.

Guy's and St Thomas' NHS Foundation Trust, London, UK.

Background: The efficacy and safety profiles of vaccines against SARS-CoV-2 in patients with cancer is unknown. We aimed to assess the safety and immunogenicity of the BNT162b2 (Pfizer-BioNTech) vaccine in patients with cancer.

Methods: For this prospective observational study, we recruited patients with cancer and healthy controls (mostly health-care workers) from three London hospitals between Dec 8, 2020, and Feb 18, 2021. Participants who were vaccinated between Dec 8 and Dec 29, 2020, received two 30 μg doses of BNT162b2 administered intramuscularly 21 days apart; patients vaccinated after this date received only one 30 μg dose with a planned follow-up boost at 12 weeks. Blood samples were taken before vaccination and at 3 weeks and 5 weeks after the first vaccination. Where possible, serial nasopharyngeal real-time RT-PCR (rRT-PCR) swab tests were done every 10 days or in cases of symptomatic COVID-19. The coprimary endpoints were seroconversion to SARS-CoV-2 spike (S) protein in patients with cancer following the first vaccination with the BNT162b2 vaccine and the effect of vaccine boosting after 21 days on seroconversion. All participants with available data were included in the safety and immunogenicity analyses. Ongoing follow-up is underway for further blood sampling after the delayed (12-week) vaccine boost. This study is registered with the NHS Health Research Authority and Health and Care Research Wales (REC ID 20/HRA/2031).

Findings: 151 patients with cancer (95 patients with solid cancer and 56 patients with haematological cancer) and 54 healthy controls were enrolled. For this interim data analysis of the safety and immunogenicity of vaccinated patients with cancer, samples and data obtained up to March 19, 2021, were analysed. After exclusion of 17 patients who had been exposed to SARS-CoV-2 (detected by either antibody seroconversion or a positive rRT-PCR COVID-19 swab test) from the immunogenicity analysis, the proportion of positive anti-S IgG titres at approximately 21 days following a single vaccine inoculum across the three cohorts were 32 (94%; 95% CI 81-98) of 34 healthy controls; 21 (38%; 26-51) of 56 patients with solid cancer, and eight (18%; 10-32) of 44 patients with haematological cancer. 16 healthy controls, 25 patients with solid cancer, and six patients with haematological cancer received a second dose on day 21. Of the patients with available blood samples 2 weeks following a 21-day vaccine boost, and excluding 17 participants with evidence of previous natural SARS-CoV-2 exposure, 18 (95%; 95% CI 75-99) of 19 patients with solid cancer, 12 (100%; 76-100) of 12 healthy controls, and three (60%; 23-88) of five patients with haematological cancers were seropositive, compared with ten (30%; 17-47) of 33, 18 (86%; 65-95) of 21, and four (11%; 4-25) of 36, respectively, who did not receive a boost. The vaccine was well tolerated; no toxicities were reported in 75 (54%) of 140 patients with cancer following the first dose of BNT162b2, and in 22 (71%) of 31 patients with cancer following the second dose. Similarly, no toxicities were reported in 15 (38%) of 40 healthy controls after the first dose and in five (31%) of 16 after the second dose. Injection-site pain within 7 days following the first dose was the most commonly reported local reaction (23 [35%] of 65 patients with cancer; 12 [48%] of 25 healthy controls). No vaccine-related deaths were reported.

Interpretation: In patients with cancer, one dose of the BNT162b2 vaccine yields poor efficacy. Immunogenicity increased significantly in patients with solid cancer within 2 weeks of a vaccine boost at day 21 after the first dose. These data support prioritisation of patients with cancer for an early (day 21) second dose of the BNT162b2 vaccine.

Funding: King's College London, Cancer Research UK, Wellcome Trust, Rosetrees Trust, and Francis Crick Institute.
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http://dx.doi.org/10.1016/S1470-2045(21)00213-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078907PMC
June 2021

To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis.

Front Immunol 2021 9;12:653974. Epub 2021 Apr 9.

Immunosurveillance Laboratory, The Francis Crick Institute, London, United Kingdom.

This study discusses substantive advances in T cell proliferation analysis, with the aim to provoke a re-evaluation of the generally-held view that Ki-67 is a reliable proliferation marker , and to offer a more sensitive and effective method for T cell cycle analysis, with informative examples in mouse and human settings. We summarize recent experimental work from our labs showing that, by Ki-67/DNA dual staining and refined flow cytometric methods, we were able to identify T cells in the S-G/M phases of the cell-cycle in the peripheral blood (collectively termed "T Double S" for T cells in S-phase : in short "T" cells). Without our refinement, such cells may be excluded from conventional lymphocyte analyses. Specifically, we analyzed clonal expansion of antigen-specific CD8 T cells in vaccinated mice, and demonstrated the potential of T cells to reflect immune dynamics in human blood samples from healthy donors, and patients with type 1 diabetes, infectious mononucleosis, and COVID-19. The Ki-67/DNA dual staining, or T assay, provides a reliable approach by which human peripheral blood can be used to reflect the dynamics of human lymphocytes, rather than providing mere steady-state phenotypic snapshots. The method does not require highly sophisticated "-omics" capabilities, so it should be widely-applicable to health care in diverse settings. Furthermore, our results argue that the T assay can provide a window on immune dynamics in extra-lymphoid tissues, a long-sought potential of peripheral blood monitoring, for example in relation to organ-specific autoimmune diseases and infections, and cancer immunotherapy.
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http://dx.doi.org/10.3389/fimmu.2021.653974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062736PMC
May 2021

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant.

Immunity 2021 06 1;54(6):1276-1289.e6. Epub 2021 Apr 1.

Department of Infectious Diseases, School of Immunology & Microbial Sciences, King's College London, London, UK; Genotype-to-Phenotype UK National Virology Consortium. Electronic address:

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.
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http://dx.doi.org/10.1016/j.immuni.2021.03.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015430PMC
June 2021

S95021, a novel selective and pan-neutralizing anti interferon alpha (IFN-α) monoclonal antibody as a candidate treatment for selected autoimmune rheumatic diseases.

J Transl Autoimmun 2021 1;4:100093. Epub 2021 Mar 1.

Center for Therapeutic Innovation, Immuno-inflammatory Disease, Institut de Recherches Servier, 125 Chemin de Ronde, 78290, Croissy sur Seine, France.

Increased interferon-α (IFN-α) production is a critical component in the pathophysiology of systemic lupus erythematosus (SLE) and other rheumatic autoimmune diseases. Herein, we report the characterization of S95021, a fully human IgG1 anti-IFN-α monoclonal antibody (mAb) as a novel therapeutic candidate for targeted patient populations. S95021 was expressed in CHOZN GS-/- cells, purified by chromatography and characterized by using electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry. High purity S95021 was obtained as a monomeric entity comprising different charge variants mainly due to -glycosylation. Surface plasmon resonance kinetics experiments showed strong association rates with all IFN-α subtypes and estimated KDs below picomolar values. Pan-IFN-α-binding properties were confirmed by immunoprecipitation assays and neutralization capacity with reporter HEK-Blue IFN-α/β cells. S95021 was IFN-α-selective and exhibited superior potency and broader neutralization profile when compared with the benchmark anti-IFN-α mAbs rontalizumab and sifalimumab. STAT-1 phosphorylation and the type I IFN gene signature induced in human peripheral blood mononuclear cells by recombinant IFN-α subtypes or plasmas from selected autoimmune patients were efficiently reduced by S95021 in a dose-dependent manner. Together, our results show that S95021 is a new potent, selective and pan IFN-α-neutralizing mAb. It is currently further evaluated as a valid therapeutic candidate in selected autoimmune diseases in which the IFN-α pro-inflammatory pathway is dysregulated.
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http://dx.doi.org/10.1016/j.jtauto.2021.100093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7972961PMC
March 2021

Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS-CoV-2 Spike.

bioRxiv 2021 Feb 3. Epub 2021 Feb 3.

The interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, the N-terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike-reactive monoclonal antibodies from SARS-CoV-2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD-specific. None of the S2-specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD-specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD-specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.
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http://dx.doi.org/10.1101/2021.02.03.429355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872354PMC
February 2021

Acute Immune Signatures and Their Legacies in Severe Acute Respiratory Syndrome Coronavirus-2 Infected Cancer Patients.

Cancer Cell 2021 02 5;39(2):257-275.e6. Epub 2021 Jan 5.

Targeted Therapy Team, The Institute of Cancer Research, London, UK.

Given the immune system's importance for cancer surveillance and treatment, we have investigated how it may be affected by SARS-CoV-2 infection of cancer patients. Across some heterogeneity in tumor type, stage, and treatment, virus-exposed solid cancer patients display a dominant impact of SARS-CoV-2, apparent from the resemblance of their immune signatures to those for COVID-19 non-cancer patients. This is not the case for hematological malignancies, with virus-exposed patients collectively displaying heterogeneous humoral responses, an exhausted T cell phenotype and a high prevalence of prolonged virus shedding. Furthermore, while recovered solid cancer patients' immunophenotypes resemble those of non-virus-exposed cancer patients, recovered hematological cancer patients display distinct, lingering immunological legacies. Thus, while solid cancer patients, including those with advanced disease, seem no more at risk of SARS-CoV-2-associated immune dysregulation than the general population, hematological cancer patients show complex immunological consequences of SARS-CoV-2 exposure that might usefully inform their care.
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http://dx.doi.org/10.1016/j.ccell.2021.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833668PMC
February 2021

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

Nat Commun 2020 12 11;11(1):6372. Epub 2020 Dec 11.

Epithelial Stem Cell Biology & Regenerative Medicine laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.
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http://dx.doi.org/10.1038/s41467-020-20082-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732825PMC
December 2020

Longitudinal proteomic profiling reveals increased early inflammation and sustained apoptosis proteins in severe COVID-19.

Sci Rep 2020 11 25;10(1):20533. Epub 2020 Nov 25.

Molecular Pathology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia.

SARS-CoV-2 infection has a risk to develop into life-threatening COVID-19 disease. Whereas age, hypertension, and chronic inflammatory conditions are risk factors, underlying host factors and markers for disease severity, e.g. requiring intensive care unit (ICU) treatment, remain poorly defined. To this end, we longitudinally profiled blood inflammation markers, antibodies, and 101 plasma proteins of hospitalized COVID-19 patients who did or did not require ICU admission. While essentially all patients displayed SARS-CoV-2-specific antibodies and virus-neutralization capacity within 12-15 days, a rapid, mostly transient upregulation of selective inflammatory markers including IL-6, CXCL10, CXCL11, IFNγ, IL-10, and monocyte-attracting CCL2, CCL7 and CCL8, was particularly evident in ICU patients. In addition, there was consistent and sustained upregulation of apoptosis-associated proteins CASP8, TNFSF14, HGF, and TGFB1, with HGF discriminating between ICU and non-ICU cohorts. Thus, COVID-19 is associated with a selective inflammatory milieu within which the apoptotic pathway is a cardinal feature with potential to aid risk-based patient stratification.
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http://dx.doi.org/10.1038/s41598-020-77525-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689507PMC
November 2020

Wendy Havran: Scientist, mentor, advocate.

Immunol Rev 2020 11 16;298(1):289-291. Epub 2020 Oct 16.

Department of Biological Sciences, California State University San Marcos, San Marcos, CA, USA.

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http://dx.doi.org/10.1111/imr.12921DOI Listing
November 2020

Human γδ T cells recognize CD1b by two distinct mechanisms.

Proc Natl Acad Sci U S A 2020 09 31;117(37):22944-22952. Epub 2020 Aug 31.

Division of Rheumatology, Inflammation, and Immunity, Brigham and Women's Hospital, Boston, MA 02115;

γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1 TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αβ TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids.
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http://dx.doi.org/10.1073/pnas.2010545117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502712PMC
September 2020

β2 Integrins differentially regulate γδ T cell subset thymic development and peripheral maintenance.

Proc Natl Acad Sci U S A 2020 09 26;117(36):22367-22377. Epub 2020 Aug 26.

Institute of Infection, Immunity & Inflammation, University of Glasgow, G12 8TA Glasgow, United Kingdom;

The γδ T cells reside predominantly at barrier sites and play essential roles in immune protection against infection and cancer. Despite recent advances in the development of γδ T cell immunotherapy, our understanding of the basic biology of these cells, including how their numbers are regulated in vivo, remains poor. This is particularly true for tissue-resident γδ T cells. We have identified the β family of integrins as regulators of γδ T cells. β-integrin-deficient mice displayed a striking increase in numbers of IL-17-producing Vγ6Vδ1 γδ T cells in the lungs, uterus, and circulation. Thymic development of this population was normal. However, single-cell RNA sequencing revealed the enrichment of genes associated with T cell survival and proliferation specifically in β-integrin-deficient IL-17 cells compared to their wild-type counterparts. Indeed, β-integrin-deficient Vγ6 cells from the lungs showed reduced apoptosis ex vivo, suggesting that increased survival contributes to the accumulation of these cells in β-integrin-deficient tissues. Furthermore, our data revealed an unexpected role for β integrins in promoting the thymic development of the IFNγ-producing CD27 Vγ4 γδ T cell subset. Together, our data reveal that β integrins are important regulators of γδ T cell homeostasis, inhibiting the survival of IL-17-producing Vγ6Vδ1 cells and promoting the thymic development of the IFNγ-producing Vγ4 subset. Our study introduces unprecedented mechanisms of control for γδ T cell subsets.
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http://dx.doi.org/10.1073/pnas.1921930117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486781PMC
September 2020

A dynamic COVID-19 immune signature includes associations with poor prognosis.

Nat Med 2020 10 17;26(10):1623-1635. Epub 2020 Aug 17.

Molecular Pathology Research Group, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia.

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.
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http://dx.doi.org/10.1038/s41591-020-1038-6DOI Listing
October 2020

Butyrophilin-like proteins display combinatorial diversity in selecting and maintaining signature intraepithelial γδ T cell compartments.

Nat Commun 2020 07 28;11(1):3769. Epub 2020 Jul 28.

Immunosurveillance Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW11AT, UK.

Butyrophilin-like (Btnl) genes are emerging as major epithelial determinants of tissue-associated γδ T cell compartments. Thus, the development of signature, murine TCRγδ intraepithelial lymphocytes (IEL) in gut and skin depends on Btnl family members, Btnl1 and Skint1, respectively. In seeking mechanisms underlying these profound effects, we now show that normal gut and skin γδ IEL development additionally requires Btnl6 and Skint2, respectively, and furthermore that different Btnl heteromers can seemingly shape different intestinal γδ IEL repertoires. This formal genetic evidence for the importance of Btnl heteromers also applied to the steady-state, since sustained Btnl expression is required to maintain the signature TCR.Vγ7 IEL phenotype, including specific responsiveness to Btnl proteins. In sum, Btnl proteins are required to select and to maintain the phenotypes of tissue-protective γδ IEL compartments, with combinatorially diverse heteromers having differential impacts on different IEL subsets.
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http://dx.doi.org/10.1038/s41467-020-17557-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387338PMC
July 2020

Tracking immunodynamics by identification of S-G/M-phase T cells in human peripheral blood.

J Autoimmun 2020 08 12;112:102466. Epub 2020 May 12.

Immunosurveillance Laboratory, The Francis Crick Institute, London, UK; Peter Gorer Department of Immunobiology, King's College London, London, UK; Institute of Molecular Biology and Pathology, National Research Council of Italy (CNR), Rome, Italy. Electronic address:

The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G/M phases of the cell-cycle (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "T" cells). Without protocol refinement, T could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of T cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV carriers; and recently-diagnosed T1D patients. T were reproducibly more abundant among CD8 T cells and a defined subset of T-regulatory CD4 T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive T cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking T cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful.
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http://dx.doi.org/10.1016/j.jaut.2020.102466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527781PMC
August 2020

The Innate Biologies of Adaptive Antigen Receptors.

Annu Rev Immunol 2020 04 4;38:487-510. Epub 2020 Feb 4.

Peter Gorer Department of Immunobiology, King's College, London, SE1 9RT, United Kingdom; email:

Nonclonal innate immune responses mediated by germ line-encoded receptors, such as Toll-like receptors or natural killer receptors, are commonly contrasted with diverse, clonotypic adaptive responses of lymphocyte antigen receptors generated by somatic recombination. However, the Variable (V) regions of antigen receptors include germ line-encoded motifs unaltered by somatic recombination, and theoretically available to mediate nonclonal, innate responses, that are independent of or largely override clonotypic responses. Recent evidence demonstrates that such responses exist, underpinning the associations of particular γδ T cell receptors (TCRs) with specific anatomical sites. Thus, TCRγδ can make innate and adaptive responses with distinct functional outcomes. Given that αβ T cells and B cells can also make nonclonal responses, we consider that innate responses of antigen receptor V-regions may be more widespread, for example, inducing states of preparedness from which adaptive clones are better selected. We likewise consider that potent, nonclonal T cell responses to microbial superantigens may reflect subversion of physiologic innate responses of TCRα/β chains.
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http://dx.doi.org/10.1146/annurev-immunol-102819-023144DOI Listing
April 2020

High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation.

Nat Immunol 2020 01 16;21(1):86-100. Epub 2019 Dec 16.

Wellcome Sanger Institute, Hinxton, UK.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.
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http://dx.doi.org/10.1038/s41590-019-0549-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338221PMC
January 2020

Butyrophilin-like 3 Directly Binds a Human Vγ4 T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen.

Immunity 2019 11 15;51(5):813-825.e4. Epub 2019 Oct 15.

Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK; Cancer Immunology and Immunotherapy Centre, University of Birmingham, Birmingham, UK. Electronic address:

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4 TCR, "LES" with an affinity (∼15-25 μM) comparable to many αβ TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined.
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http://dx.doi.org/10.1016/j.immuni.2019.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868513PMC
November 2019

An innate-like Vδ1 γδ T cell compartment in the human breast is associated with remission in triple-negative breast cancer.

Sci Transl Med 2019 10;11(513)

Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King's College London, London SE1 9RT, UK.

Innate-like tissue-resident γδ T cell compartments capable of protecting against carcinogenesis are well established in mice. Conversely, the degree to which they exist in humans, their potential properties, and their contributions to host benefit are mostly unresolved. Here, we demonstrate that healthy human breast harbors a distinct γδ T cell compartment, primarily expressing T cell receptor (TCR) Vδ1 chains, by comparison to Vδ2 chains that predominate in peripheral blood. Breast-resident Vδ1 cells were functionally skewed toward cytolysis and IFN-γ production, but not IL-17, which has been linked with inflammatory pathologies. Breast-resident Vδ1 cells could be activated innately via the NKG2D receptor, whereas neighboring CD8 αβ T cells required TCR signaling. A comparable population of Vδ1 cells was found in human breast tumors, and when paired tumor and nonmalignant samples from 11 patients with triple-negative breast cancer were analyzed, progression-free and overall survival correlated with Vδ1 cell representation, but not with either total γδ T cells or Vδ2 T cells. As expected, progression-free survival also correlated with αβ TCRs. However, whereas in most cases TCRαβ repertoires focused, typical of antigen-specific responses, this was not observed for Vδ1 cells, consistent with their innate-like responsiveness. Thus, maximal patient benefit may accrue from the collaboration of innate-like responses mounted by tissue-resident Vδ1 compartments and adaptive responses mounted by αβ T cells.
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http://dx.doi.org/10.1126/scitranslmed.aax9364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877350PMC
October 2019

γδ T Cell Update: Adaptate Orchestrators of Immune Surveillance.

Authors:
Adrian C Hayday

J Immunol 2019 07;203(2):311-320

Peter Gorer Department of Immunobiology, King's College London, London SE1 9RT, United Kingdom; and Francis Crick Institute, London NW1 1AT, United Kingdom

As interest in γδ T cells grows rapidly, what key points are emerging, and where is caution warranted? γδ T cells fulfill critical functions, as reflected in associations with vaccine responsiveness and cancer survival in humans and ever more phenotypes of γδ T cell-deficient mice, including basic physiological deficiencies. Such phenotypes reflect activities of distinct γδ T cell subsets, whose origins offer interesting insights into lymphocyte development but whose variable evolutionary conservation can obfuscate translation of knowledge from mice to humans. By contrast, an emerging and conserved feature of γδ T cells is their "adaptate" biology: an integration of adaptive clonally-restricted specificities, innate tissue-sensing, and unconventional recall responses that collectively strengthen host resistance to myriad challenges. Central to adaptate biology are butyrophilins and other γδ cell regulators, the study of which should greatly enhance our understanding of tissue immunogenicity and immunosurveillance and guide intensifying clinical interest in γδ cells and other unconventional lymphocytes.
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http://dx.doi.org/10.4049/jimmunol.1800934DOI Listing
July 2019

Response to comment on 'AIRE-deficient patients harbor unique high-affinity disease-ameliorating autoantibodies'.

Elife 2019 06 27;8. Epub 2019 Jun 27.

Peter Gorer Department of Immunobiology, King's College London, London, United Kingdom.

In 2016, we reported four substantial observations of APECED/APS1 patients, who are deficient in AIRE, a major regulator of central T cell tolerance (Meyer et al., 2016). Two of those observations have been challenged. Specifically, 'private' autoantibody reactivities shared by only a few patients but collectively targeting >1000 autoantigens have been attributed to false positives (Landegren, 2019). While acknowledging this risk, our study-design included follow-up validation, permitting us to adopt statistical approaches to also limit false negatives. Importantly, many such private specificities have now been validated by multiple, independent means including the autoantibodies' molecular cloning and expression. Second, a significant correlation of antibody-mediated IFNα neutralization with an absence of disease in patients highly disposed to Type I diabetes has been challenged because of a claimed failure to replicate our findings (Landegren, 2019). However, flaws in design and implementation invalidate this challenge. Thus, our results present robust, insightful, independently validated depictions of APECED/APS1, that have spawned productive follow-up studies.
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http://dx.doi.org/10.7554/eLife.45826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597236PMC
June 2019

The γδTCR combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness.

Nat Immunol 2018 12 12;19(12):1352-1365. Epub 2018 Nov 12.

Peter Gorer Department of Immunobiology, School of Immunology & Microbial Sciences, King's College London, London, UK.

T lymphocytes expressing γδ T cell antigen receptors (TCRs) comprise evolutionarily conserved cells with paradoxical features. On the one hand, clonally expanded γδ T cells with unique specificities typify adaptive immunity. Conversely, large compartments of γδTCR intraepithelial lymphocytes (γδ IELs) exhibit limited TCR diversity and effect rapid, innate-like tissue surveillance. The development of several γδ IEL compartments depends on epithelial expression of genes encoding butyrophilin-like (Btnl (mouse) or BTNL (human)) members of the B7 superfamily of T cell co-stimulators. Here we found that responsiveness to Btnl or BTNL proteins was mediated by germline-encoded motifs within the cognate TCR variable γ-chains (V chains) of mouse and human γδ IELs. This was in contrast to diverse antigen recognition by clonally restricted complementarity-determining regions CDR1-CDR3 of the same γδTCRs. Hence, the γδTCR intrinsically combines innate immunity and adaptive immunity by using spatially distinct regions to discriminate non-clonal agonist-selecting elements from clone-specific ligands. The broader implications for antigen-receptor biology are considered.
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http://dx.doi.org/10.1038/s41590-018-0253-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874498PMC
December 2018

Low-Density Lipoprotein Uptake Inhibits the Activation and Antitumor Functions of Human Vγ9Vδ2 T Cells.

Cancer Immunol Res 2018 04 22;6(4):448-457. Epub 2018 Jan 22.

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal.

Vγ9Vδ2 T cells, the main subset of γδ T lymphocytes in human peripheral blood, are endowed with antitumor functions such as cytotoxicity and IFNγ production. These functions are triggered upon T-cell receptor-dependent activation by non-peptidic prenyl pyrophosphates ("phosphoantigens") that are selective agonists of Vγ9Vδ2 T cells, and which have been evaluated in clinical studies. Because phosphoantigens have shown interindividual variation in Vγ9Vδ2 T-cell activities, we asked whether metabolic resources, namely lipids such as cholesterol, could affect phosphoantigen-mediated Vγ9Vδ2 T-cell activation and function. We show here that Vγ9Vδ2 T cells express the LDL receptor upon activation and take up LDL cholesterol. Resulting changes, such as decreased mitochondrial mass and reduced ATP production, correlate with downregulation of Vγ9Vδ2 T-cell activation and functionality. In particular, the expression of IFNγ, NKG2D, and DNAM-1 were reduced upon LDL cholesterol treatment of phosphoantigen-expanded Vγ9Vδ2 T cells. As a result, their capacity to target breast cancer cells was compromised both and in an xenograft mouse model. Thus, this study describes the role of LDL cholesterol as an inhibitor of the antitumor functions of phosphoantigen-activated Vγ9Vδ2 T cells. Our observations have implications for therapeutic applications dependent on Vγ9Vδ2 T cells. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0327DOI Listing
April 2018

Heteromeric interactions regulate butyrophilin (BTN) and BTN-like molecules governing γδ T cell biology.

Proc Natl Acad Sci U S A 2018 01 16;115(5):1039-1044. Epub 2018 Jan 16.

Peter Gorer Department of Immunobiology, King's College London, London SE1 9RT, United Kingdom;

The long-held view that gamma delta (γδ) T cells in mice and humans are fundamentally dissimilar, as are γδ cells in blood and peripheral tissues, has been challenged by emerging evidence of the cells' regulation by butyrophilin (BTN) and butyrophilin-like (BTNL) molecules. Thus, murine and the related gene, , mediate T cell receptor (TCR)-dependent selection of murine intraepithelial γδ T cell repertoires in gut and skin, respectively; and are TCR-dependent regulators of human gut γδ cells; and is essential for TCR-dependent activation of human peripheral blood Vγ9Vδ2 T cells. However, some observations concerning BTN/Btnl molecules continue to question the extent of mechanistic conservation. In particular, murine and human gut γδ cell regulation depends on pairings of Btnl1 and Btnl6 and BTNL3 and BTNL8, respectively, whereas blood γδ cells are reported to be regulated by BTN3A1 independent of other BTNs. Addressing this paradox, we show that BTN3A2 regulates the subcellular localization of BTN3A1, including functionally important associations with the endoplasmic reticulum (ER), and is specifically required for optimal BTN3A1-mediated activation of Vγ9Vδ2 T cells. Evidence that BTNL3/BTNL8 and Btnl1/Btnl6 likewise associate with the ER reinforces the prospect of broadly conserved mechanisms underpinning the selection and activation of γδ cells in mice and humans, and in blood and extralymphoid sites.
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http://dx.doi.org/10.1073/pnas.1701237115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798315PMC
January 2018
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