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Evidence to date supports regulatory T cell (Treg) alterations in endometriosis; however, the relationship remains unclear, and Tregs have not previously been investigated with respect to infertility in endometriosis. This prospective cross-sectional cohort study details circulating and endometrial tissue-specific disturbances in Tregs and broader gated populations in women of reproductive age with and without endometriosis (n = 57 and 29, respectively) using flow cytometry and immunohistochemistry. Participants were characterised by menstrual cycle phase, r-ASRM endometriosis disease stage and fertility status. In the endometrium of women with endometriosis, endometrial Tregs and CD4+ lymphocyte proportions did not change between the proliferative and secretory phases, while in women without the disease, they significantly decreased (p = 0.045 and p = 0.039, respectively). In women with endometriosis, endometrial Tregs were lower than in women without endometriosis overall (p = 0.050 as a proportion of all CD45+ immune cells). We have shown for the first time that proportions of CD4+ lymphocytes (p = 0.021), overall lymphocytes (p = 0.034) and non-granulocytes (p = 0.027) were significantly decreased in the endometrium of women with moderate-severe (r-ASRM stages III and IV) compared to minimal-mild (r-ASRM stages I and II) endometriosis. During the secretory phase, circulating Treg proportions were significantly increased in infertile compared to fertile women (p = 0.049). This study confirms differences in endometrial Tregs in women with endometriosis, with blunting of normal menstrual cyclical variations, reduced proportions during the proliferative phase and disease stage-specific relationships.

B cells play a major role in multiple sclerosis (MS), with many successful therapeutics capable of removing them from circulation. One such therapy, alemtuzumab, is thought to reset the immune system without the need for ongoing therapy in a proportion of patients. The exact cells contributing to disease pathogenesis and quiescence remain to be identified. We utilized mass cytometry to analyze B cells from the blood of patients with relapse-remitting MS (RRMS) before and after alemtuzumab treatment, and during relapse. A complementary RRMS cohort was analyzed by single-cell RNA sequencing. The R package "Spectre" was used to analyze these data, incorporating FlowSOM clustering, sparse partial least squares-discriminant analysis and permutational multivariate analysis of variance. Immunoglobulin (Ig)A

Virus-specific T-cells (VSTs) from third-party donors mediate short- and long-term antiviral effects in allogeneic hematopoietic stem cell transplant (HSCT) recipients with relapsed or refractory viral infections. We investigated early administration of third-party VSTs, together with antiviral therapy in patients requiring treatment for first cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. Thirty HSCT patients were treated with 1 to 4 VST infusions (2 × 107 cells/m2; CMV n=27, EBV n=3) at a median of 4 days after initiation of antiviral treatment. The overall viral response rate was 100%, with a complete response (CR) rate of 94%. Of the 28 patients who achieved a CR, 23 remained virus PCR negative (n=9) or below quantitation limit (n=14) for the duration of follow-up. Four patients had brief episodes of quantifiable reactivation not requiring additional therapy, and one required a second infusion after initial CR, remaining PCR negative thereafter. All 3 patients treated for EBV post-transplant lymphoproliferative disorder achieved sustained CR. Rates of aGVHD and cGVHD after infusion were 13% and 23%, respectively. There were no serious infusion-related adverse events. VST infusion was associated with rapid recovery of CD8+CD45RA−CD62L− and a slower recovery of CD4+CD45RA−CD62L− effector memory T-cells; CMV-specific T-cells comprised up to 13% of CD8+ cells. At 1 year post-transplant, non-relapse mortality was 10%, cumulative incidence of relapse was 7%, overall survival was 88% and 25 of 27 patients had ECOG status of 0 or 1. Early administration of third-party VSTs in conjunction with antiviral treatment appears safe and leads to excellent viral control and clinical outcomes. Registered on Australian New Zealand Clinical Trials Registry as #ACTRN12618000343202.

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).

Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4+ T cells with potent antifungal characteristics, including production of tumor necrosis factor α, interferon γ, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice–compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen–matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.

Melanoma, as for many other cancers, undergoes a selection process during progression that limits many innate and adaptive tumor control mechanisms. Immunotherapy with immune checkpoint blockade overcomes one of the escape mechanisms but if the tumor is not eliminated other escape mechanisms evolve that require new approaches for tumor control. Some of the innate mechanisms that have evolved against infections with microorganisms and viruses are proving to be active against cancer cells but require better understanding of how they are activated and what inhibitory mechanisms may need to be targeted. This is particularly so for inflammasomes which have evolved against many different organisms and which recruit a number of cytotoxic mechanisms that remain poorly understood. Equally important is understanding of where these mechanisms will fit into existing treatment strategies and whether existing strategies already involve the innate killing mechanisms.

Myeloid lineage cells present in human peripheral blood include dendritic cells (DC) and monocytes. The DC are identified phenotypically as HLA-DR+ cells that lack major cell surface lineage markers for T cells (CD3), B cells (CD19, CD20), NK cells (CD56), red blood cells (CD235a), hematopoietic stem cells (CD34), and Mo that express CD14. Both DC and Mo can be phenotypically divided into subsets. DC are divided into plasmacytoid DC, which are CD11c−, CD304+, CD85g+, and myeloid DC that are CD11c+. The CD11c+ DC are readily classified as CD1c+DC and CD141+ DC. Monocytes are broadly divided into the CD14+CD16− (classical) and CD14dimCD16+ subsets (nonclassical). A population of myeloid-derived cells that have DC characteristics, that is, HLA-DR+ and lacking lineage markers including CD14, but express CD16 are generally clustered with CD14dimCD16+ monocytes. We used high-dimensional clustering analyses of fluorescence and mass cytometry data, to delineate CD14+ monocytes, CD14dimCD16+ monocytes (CD16+Mo), and CD14− CD16+DC (CD16+DC). We sought to identify the functional and kinetic relationship of CD16+DC to CD16+Mo. We demonstrate that differentiation of CD16+DC and CD16+Mo during activation with IFNγ in vitro and as a result of an allo-hematopoietic cell transplant (HCT) in vivo resulted in distinct populations. Recovery of blood CD16+DC in both auto- and allo-(HCT) patients after myeloablative conditioning showed similar reconstitution and activation kinetics to CD16+Mo. Finally, we show that expression of the cell surface markers CD300c, CCR5, and CLEC5a can distinguish the cell populations phenotypically paving the way for functional differentiation as new reagents become available.

BackgroundShort-chain fatty acids (SCFAs) produced by the gut microbiota have beneficial anti-inflammatory and gut homeostasis effects and prevent type 1 diabetes (T1D) in mice. Reduced SCFA production indicates a loss of beneficial bacteria, commonly associated with chronic autoimmune and inflammatory diseases, including T1D and type 2 diabetes. Here we addressed whether a metabolite-based dietary supplement has an impact on humans with T1D. We conducted a single-arm pilot-and-feasibility trial with high-amylose maize resistant starch modified with acetate and butyrate (HAMSAB) to assess safety, while monitoring changes in the microbiota in alignment with modulation of the immune system status.ResultsHAMSAB supplement was administered for six weeks with follow-up at 12 weeks in adults with long-standing T1D. Increased concentrations of SCFA acetate, propionate, and butyrate in stools and plasma were in concert with a shift in the composition and function of the gut microbiota. While glucose control and insulin requirements did not change, subjects with the highest SCFA concentrations exhibited the best glycemic control. Bifidobacterium longum, Bifidobacterium adolescentis, and vitamin B7 production correlated with lower HbA1c and basal insulin requirements. Circulating B and T cells developed a more regulatory phenotype post-intervention.ConclusionChanges in gut microbiota composition, function, and immune profile following six weeks of HAMSAB supplementation were associated with increased SCFAs in stools and plasma. The persistence of these effects suggests that targeting dietary SCFAs may be a mechanism to alter immune profiles, promote immune tolerance and improve glycemic control for the treatment of T1D.Trial registrationACTRN12618001391268. Registered 20 August 2018, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=375792

Human cytomegalovirus reactivation is a major opportunistic infection after allogeneic haematopoietic stem cell transplantation and has a complex relationship with post-transplant immune reconstitution. Here, we use mass cytometry to define patterns of innate and adaptive immune cell reconstitution at key phases of human cytomegalovirus reactivation in the first 100 days post haematopoietic stem cell transplantation. Human cytomegalovirus reactivation is associated with the development of activated, memory T-cell profiles, with faster effector-memory CD4+ T-cell recovery in patients with low-level versus high-level human cytomegalovirus DNAemia. Mucosal-associated invariant T cell levels at the initial detection of human cytomegalovirus DNAemia are significantly lower in patients who subsequently develop high-level versus low-level human cytomegalovirus reactivation. Our data describe distinct immune signatures that emerged with human cytomegalovirus reactivation after haematopoietic stem cell transplantation, and highlight Mucosal-associated invariant T cell levels at the first detection of reactivation as a marker that may be useful to anticipate the magnitude of human cytomegalovirus DNAemia.

SummaryHuman cytomegalovirus (HCMV) reactivation is a major opportunistic infection after allogeneic haematopoietic stem cell transplantation and has a complex relationship with post-transplant immune reconstitution. Here, we used mass cytometry to comprehensively define global patterns of innate and adaptive immune cell reconstitution at key phases of HCMV reactivation (before detection, initial detection, peak and near resolution) in the first 100 days post-transplant. In addition to identifying patterns of immune reconstitution in those with or without HCMV reactivation, we found mucosal-associated invariant T (MAIT) cell levels at the initial detection of HCMV DNAemia distinguished patients who subsequently developed low-level versus high-level HCMV reactivation. In addition, early recovery of effector-memory CD4+ T cells distinguished low-level and high-level reactivation. Our data describe distinct immune signatures that emerged with HCMV reactivation post-HSCT, and highlight MAIT cell levels at the initial detection of reactivation as a potential prognostic marker to guide clinical decisions regarding pre-emptive therapy.

Diabetic neuropathic pain is a common and devastating complication of type 1 diabetes, but the mechanism by which it develops and persists is yet to be fully elucidated. This study utilised high-dimensional suspension mass cytometry in a pilot cohort to investigate differences in peripheral blood immunophenotypes between type 1 diabetes patients with (n ​= ​9) and without (n ​= ​9) peripheral neuropathic pain. The abundance and activation of several leukocyte subsets were investigated with unsupervised clustering approaches FlowSOM and SPADE, as well as by manual gating. Major findings included a proportional increase in CD4+ central memory T cells and an absolute increase in classical monocytes, non-classical monocytes, and mature natural killer cells in type 1 diabetes patients with pain compared to those without pain. The expression of CD27, CD127, and CD39 was upregulated on select T cell populations, and the phosphorylated form of pro-inflammatory transcription factor MK2 was upregulated across most populations. These results provide evidence that distinct immunological signatures are associated with painful neuropathy in type 1 diabetes patients. Further research may link these changes to mechanisms by which pain in type 1 diabetes is initiated and maintained, paving the way for much needed targeted treatments.

T cell infiltration of tumors plays an important role in determining colorectal cancer disease progression and has been incorporated into the Immunoscore prognostic tool. In this study, mass cytometry was used to demonstrate a significant increase in the frequency of both conventional CD25+FOXP3+CD127lo regulatory T cells (Tregs) as well as BLIMP-1+ Tregs in the tumor compared with nontumor bowel (NTB) of the same patients. Network cluster analyses using SCAFFoLD, VorteX, and CITRUS revealed that an increase in BLIMP-1+ Tregs was a single distinguishing feature of the tumor tissue compared with NTB. BLIMP-1+ Tregs represented the most significantly enriched T cell population in the tumor compared with NTB. The enrichment of ICOS, CD45RO, PD-1, PDL-1, LAG-3, CTLA-4, and TIM-3 on BLIMP-1+ Tregs suggests that BLIMP-1+ Tregs have a more activated phenotype than conventional Tregs and may play a role in antitumor immune responses.

High‐dimensional cytometry represents an exciting new era of immunology research, enabling the discovery of new cells and prediction of patient responses to therapy. A plethora of analysis and visualization tools and programs are now available for both new and experienced users; however, the transition from low‐ to high‐dimensional cytometry requires a change in the way users think about experimental design and data analysis. Data from high‐dimensional cytometry experiments are often underutilized, because of both the size of the data and the number of possible combinations of markers, as well as to a lack of understanding of the processes required to generate meaningful data. In this article, we explain the concepts behind designing high‐dimensional cytometry experiments and provide considerations for new and experienced users to design and carry out high‐dimensional experiments to maximize quality data collection., High‐dimensional cytometry represents an exciting new era of immunology research; however, the transition from low‐ to high‐dimensional cytometry requires a change in the way users think about experimental design and data analysis. We explain the concepts behind designing high‐dimensional cytometry experiments and provide considerations for new and experienced users to design and carry out high‐dimensional experiments. ​

We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

Background/Introduction Inflammation is now a well-established component of the pathophysiology of coronary artery disease (CAD), but it is unknown whether atherosclerosis is associated with a distinct circulating immune cell profile. Mass cytometry time-of-flight (CYTOF) is a new precision technology which can be used to assess leukocyte populations comprehensively. Purpose To determine if patients with calcified and non-calcified (soft) coronary plaque have distinct circulating immune cell profiles when compared to healthy controls, and to assess whether this could be used to detect sub-clinical CAD. Methods Patients referred for a CT coronary angiogram were recruited; blood samples were collected and peripheral blood mononuclear cells (PBMCs) were isolated. Imaging data was analysed using a modified Gensini scoring system which incorporated plaque composition, with higher weighting given to soft plaque. The modified Gensini scores were then used to further segregate into calcified-predominant and soft-predominant disease groups. CYTOF analysis was performed on the PBMCs, with groups as outlined in Table 1. Results Age was significantly higher in the CAD+ group, but all other demographic features and risk factors did not differ between groups. Patients with predominantly calcified disease showed an increase in memory CD8 T cells (p=0.004), an increase in CD 39+ CD4 T cells (p=0.028), and a decrease in naïve CD8 T cells (p=0.005), which suggests an accumulated memory response in more quiescent disease. Patients with predominantly soft-plaque disease have higher pro-inflammatory monocyte populations (p=0.013) and proliferative CD4 T cell populations (p=0.011), suggesting acute innate and adaptive responses to biologically active plaque. Conclusions This pilot study has shown that further study should be pursued into the utility of CYTOF to identify sub-clinical CAD through differences in peripheral circulating immune cell profiles. Figure 1 Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): National Health and Medical Research Council of Australia, Heart Research Australia

Objective Adoptive immunotherapy with ex vivo expanded tumor‐specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP‐compliant manufacturing method for PRAME‐specific T cells from healthy donors for adoptive immunotherapy. Methods Mononuclear cells were pulsed with PRAME 15‐mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137neg fraction in medium supplemented with interleukin (IL)‐2, IL‐7 and IL‐15. Cultured T cells were restimulated with antigen‐pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re‐exposure were assessed with flow cytometry, enzyme‐linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T‐cell receptor (TCR) beta clonality studies were performed on selected cultures. Results PRAME‐stimulated cultures (n = 10) had mean expansion of 2500‐fold at day 18. Mean CD3+ percentage was 96% with CD4:CD8 ratio of 4:1. Re‐exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)‐γ (mean: 12.2%) and TNF‐α (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype (CXCR3+/CCR4neg/CCR6neg/Tbet+, mean: 73%) and cytokine production including IL‐2, IL‐4, IL‐8, IL‐13 and GM‐CSF (2%, 6%, 8%, 4% and 11%, respectively). Conclusion PRAME‐specific T cells for adoptive immunotherapy were enriched from healthy donor mononuclear cells. The products were oligoclonal, exhibited Th1 phenotype and produced multiple cytokines., Adoptive immunotherapy with ex vivo expanded tumor‐specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP‐compliant manufacturing method for PRAME‐specific T cells from healthy donors for adoptive immunotherapy.

Objectives Cytomegalovirus (CMV) is known to have a significant impact on immune recovery post‐allogeneic haemopoietic stem cell transplant (HSCT). Adoptive therapy with donor‐derived or third‐party virus‐specific T cells (VST) can restore CMV immunity leading to clinical benefit in prevention and treatment of post‐HSCT infection. We developed a mass cytometry approach to study natural immune recovery post‐HSCT and assess the mechanisms underlying the clinical benefits observed in recipients of VST. Methods A mass cytometry panel of 38 antibodies was utilised for global immune assessment (72 canonical innate and adaptive immune subsets) in HSCT recipients undergoing natural post‐HSCT recovery (n = 13) and HSCT recipients who received third‐party donor‐derived CMV‐VST as salvage for unresponsive CMV reactivation (n = 8). Results Mass cytometry identified distinct immune signatures associated with CMV characterised by a predominance of innate cells (monocytes and NK) seen early and an adaptive signature with activated CD8+ T cells seen later. All CMV‐VST recipients had failed standard antiviral pharmacotherapy as a criterion for trial involvement; 5/8 had failed to develop the adaptive immune signature by study enrolment despite significant CMV antigen exposure. Of these, VST administration resulted in development of the adaptive signature in association with CMV control in three patients. Failure to respond to CMV‐VST in one patient was associated with persistent absence of the adaptive immune signature. Conclusion The clinical benefit of CMV‐VST may be mediated by the recovery of an adaptive immune signature characterised by activated CD8+ T cells., In this study, immune profiling with mass cytometry identified immune signatures associated with cytomegalovirus (CMV) reactivation after allogeneic stem cell transplant in patients undergoing natural immune recovery. Failure to control CMV was associated with failure to develop an adaptive immune signature that could be reversed with third‐party CMV virus‐specific T cells in some recipients.

Background CD4 T cells that express the chemokine receptor, CCR5, are the most important target of HIV-1 infection, but their functions, phenotypes and anatomical locations are poorly understood. We aimed to use multiparameter flow cytometry to better define the full breadth of these cells. Methods High-parameter fluorescence flow and mass cytometry were optimized to analyse subsets of CCR5 memory CD4 T cells, including CD25CD127 Tregs, CXCR3CCR6- Th1-like, CCR6CD161CXCR3- Th17-like, integrins α4s7 gut-homing, CCR4 skin-homing, CD62L lymph node-homing, CD38HLA-DR activated cells, and CD27-CD28- cytotoxic T lymphocytes, in a total of 22 samples of peripheral blood, ultrasound-guided fine needle biopsies of lymph nodes and excised tonsils. CCR5 antigen-specific CD4 T cells were studied using the OX40 flow-based assay. Results 10-20% of CCR5 memory CD4 T cells were Tregs, 10-30% were gut-homing, 10-30% were skin-homing, 20-40% were lymph node-homing, 20-50% were Th1-like and 20-40% were Th17-like cells. Up to 30% were cytotoxic T lymphocytes in CMV-seropositive donors, including cells that were either CCR5Granzyme K or CCR5Granzyme B. When all possible phenotypes were exhaustively analysed, more than 150 different functional and trafficking subsets of CCR5 CD4 T cells were seen. Moreover, a small population of resident CD69Granzyme KCCR5 CD4 T cells was found in lymphoid tissues. CMV- and Mycobacterium tuberculosis-specific CD4 T cells were predominantly CCR5. Conclusion These results reveal for the first time the prodigious heterogeneity of function and trafficking of CCR5 CD4 T cells in blood and in lymphoid tissue, with significant implications for rational approaches to prophylaxis for HIV-1 infection and for purging of the HIV-1 reservoir in those participants already infected.

Objectives Disease‐modifying therapies (DMTs) targeting B cells are amongst the most effective for preventing multiple sclerosis (MS) progression. IgG3 antibodies and their uncharacterised B‐cell clones are predicted to play a pathogenic role in MS. Identifying subsets of IgG3 + B cells involved in MS progression could improve diagnosis, could inform timely disease intervention and may lead to new DMTs that target B cells more specifically. Methods We designed a 31‐parameter B‐cell‐focused mass cytometry panel to interrogate the role of peripheral blood IgG3 + B cells in MS progression of two different patient cohorts: one to investigate the B‐cell subsets involved in conversion from clinically isolated syndrome (CIS) to MS; and another to compare MS patients with inactive or active stages of disease. Each independent cohort included a group of non‐MS controls. Results Nine distinct CD20+IgD−IgG3 + B‐cell subsets were identified. Significant changes in the proportion of CD21+CD24+CD27−CD38− and CD27+CD38hiCD71hi memory B‐cell subsets correlated with changes in serum IgG3 levels and time to conversion from CIS to MS. The same CD38− double‐negative B‐cell subset was significantly elevated in MS patients with active forms of the disease. A third CD21+CD24+CD27+CD38− subset was elevated in patients with active MS, whilst narrowband UVB significantly reduced the proportion of this switched‐memory B‐cell subset. Conclusion We have identified previously uncharacterised subsets of IgG3 + B cells and shown them to correlate with autoimmune attacks on the central nervous system (CNS). These results highlight the potential for therapies that specifically target IgG3 + B cells to impact MS progression., Mass cytometry has allowed us to identify nine unique IgG3+ B‐cell subsets. Using two independent cohorts of multiple sclerosis (MS) patients, we show that a number of these IgG3+ subsets are not only associated with MS progression but also affected by disease‐modifying therapies. These studies highlight the potential for therapies that specifically target IgG3+ B cells to impact MS progression.

Effective treatment or prevention of immune side effects associated with checkpoint inhibitor therapy of cancer is an important goal in this new era of immunotherapy. Hepatitis due to immunotherapy with antibodies against PD-1 is uncommon and generally of low severity. We present an unusually severe case arising in a melanoma patient after more than 6 months uncomplicated treatment with anti-PD-1 in an adjuvant setting. The hepatitis rapidly developed resistance to high-dose steroids, requiring anti-thymocyte globulin (ATG) to achieve control. Mass cytometry allowed comprehensive phenotyping of circulating lymphocytes and revealed that CD4+ T cells were profoundly depleted by ATG, while CD8+ T cells, B cells, NK cells and monocytes were relatively spared. Multiple abnormalities in CD4+ T cell phenotype were stably present in the patient before disease onset. These included a population of CCR4−CCR6− effector/memory CD4+ T cells expressing intermediate levels of the Th1-related chemokine receptor CXCR3 and abnormally high multi-drug resistance type 1 transporter (MDR1) activity as assessed by a rhodamine 123 excretion assay. Expression of MDR1 has been implicated in steroid resistance and may have contributed to the severity and lack of a sustained steroid response in this patient. The number of CD4+ rhodamine 123-excreting cells was reduced > 3.5-fold after steroid and ATG treatment. This case illustrates the need to consider this form of steroid resistance in patients failing treatment with corticosteroids. It also highlights the need for both better identification of patients at risk and the development of treatments that involve more specific immune suppression. Electronic supplementary material The online version of this article (10.1007/s00262-017-2107-7) contains supplementary material, which is available to authorized users.

CD83 is a member of the Ig gene superfamily, first identified in activated lymphocytes. Since then, CD83 has become an important marker for defining activated human dendritic cells (DC). Several potential CD83 mRNA isoforms have been described, including a soluble form detected in human serum, which may have an immunosuppressive function. To further understand the biology of CD83, we examined its expression in different human immune cell types before and after activation using a panel of mouse and human anti-human CD83 mAb. The mouse anti-human CD83 mAbs, HB15a and HB15e, and the human anti-human CD83 mAb, 3C12C, were selected to examine cytoplasmic and surface CD83 expression, based on their different binding characteristics. Glycosylation of CD83, the CD83 mRNA isoforms, and soluble CD83 released differed among blood DC, monocytes, and monocyte-derived DC, and other immune cell types. A small T cell population expressing surface CD83 was identified upon T cell stimulation and during allogeneic MLR. This subpopulation appeared specifically during viral Ag challenge. We did not observe human CD83 on unstimulated human natural regulatory T cells (Treg), in contrast to reports describing expression of CD83 on mouse Treg. CD83 expression was increased on CD4+, CD8+ T, and Treg cells in association with clinical acute graft-versus-host disease in allogeneic hematopoietic cell transplant recipients. The differential expression and function of CD83 on human immune cells reveal potential new roles for this molecule as a target of therapeutic manipulation in transplantation, inflammation, and autoimmune diseases.

Blockade of the PD-1/PD-L1 pathway with targeted monoclonal antibodies has demonstrated encouraging anti-tumour activity in multiple cancer types. We present the case of a patient with BRAF-negative stage IVC anaplastic thyroid cancer (ATC) treated with the anti-PD-1 monoclonal antibody, pembrolizumab, following radiographic progression on chemoradiation. Blood samples were collected prior to and at four time points during treatment with pembrolizumab. Mass cytometry was used to determine expression of relevant biomarkers by peripheral blood mononuclear cells. Faecal samples were collected at baseline and 4 weeks following treatment initiation; taxonomic profiling using 16S ribosomal RNA (rRNA) gene sequencing was performed. Following treatment, a marked expansion in CD20+ B cell, CD16+ CD56lo NK cell and CD45RO+ CCR7+ central memory CD4+ T-cell populations was observed in the peripheral blood. Proportions of cells expressing the co-receptors TIGIT, OX40 and CD86 also increased during treatment. A high abundance of bacteria of the order Bacteroidales, specifically from the Bacteroidaceae and Rikenellaceae families, was identified in the faecal microbiota. Moreover, the patient’s microbiome was enriched in Clostridiales order members Ruminococcaceae, Veillonellaceae and Lachnospiraceae. Alpha diversity of the gut microbiome was significantly higher following initiation of checkpoint therapy as assessed by the Shannon and Simpson index. Our results suggest that treatment with pembrolizumab promotes expansion of T-, B- and NK cell populations in the peripheral blood at the time of tumour regression and have the potential to be implemented as predictive biomarkers in the context of checkpoint blockade therapy. Larger studies to confirm these findings are warranted.

Mass cytometry (MC) is a powerful research tool enabling high-dimensional analysis of single cells in suspension and within tissue sections following laser ablation. Here we describe the procedure of titrating metal-conjugated antibodies, to ensure that optimal levels of staining are achieved while minimizing nonspecific signals that may occur at high concentrations.

Mass cytometry is a multi-parametric technique that offers insight into functional and biological systems at a single cell level (Tanner et al., Cancer Immunol Immunother 62:955-965, 2013). One of the major advantages of mass cytometry is the ability to measure multiple intracellular markers, including phosphorylated proteins that are part of major signaling pathways, such as NF-κB, JAK/STAT, and ERK/MAPK. Here we describe an optimized mass cytometry protocol for staining human clinical blood samples with panels that include phosphorylated antibodies.

CD96 has recently been shown to be a potent immune checkpoint molecule in\ud mice, but a similar role in humans is not known. In this study, we provide a\ud detailed map of CD96 expression across human lymphocyte lineages, the\ud kinetics of CD96 regulation on T-cell activation and co-expression with other\ud conventional and emerging immune checkpoint molecules. We show that\ud CD96 is predominantly expressed by T cells and has a unique lymphocyte\ud expression profile. CD96\ud high\ud T cells exhibited distinct effector functions on\ud activation. Of note, CD96 expression was highly correlated with T-cell markers\ud in primary and metastatic human tumors and was elevated on antigen-\ud experienced T cells and tumor-infiltrating lymphocytes. Collectively, these data\ud demonstrate that CD96 may be a promising immune checkpoint to enhance\ud T-cell function against human cancer and infectious disease

CD4+ T helper (Th) cells that express the gut homing chemokine receptor CCR9 are increased in the peripheral blood of patients with inflammatory bowel disease and Sjögren's syndrome and in the inflamed lesions of autoimmune diseases that affect the accessory organs of the digestive system. However, despite the important role of the GIT in both immunity and autoimmunity, the nature of CCR9-expressing cells in GIT lymphoid organs and their role in chronic inflammatory diseases remains unknown. In this study, we analyzed the characteristics of CCR9+ Th and T follicular helper (Tfh) cells in GIT associated lymphoid tissues in health, chronic inflammation and autoimmunity. Our findings reveal an association between the transcriptome and phenotype of CCR9+ Th in the pancreas and CCR9+ Tfh cells from GIT-associated lymphoid tissues. GIT CCR9+ Tfh cells exhibited characteristics, including a Th17-like transcriptome and production of effector cytokines, which indicated a microenvironment-specific signature. Both CCR9+ Tfh cells and CCR9+ Th cells from GIT-associated lymphoid tissues migrated to the pancreas. The expression of CCR9 was important for migration of both subsets to the pancreas, but Tfh cells that accumulated in the pancreas had downmodulated expression of CXCR5. Taken together, the findings provide evidence that CCR9+ Tfh cells and Th cells from the GIT exhibit plasticity and can accumulate in distal accessory organs of the digestive system where they may participate in autoimmunity.