Search

In recent years, our understanding about the functional complexity of CD8

Supplementary Table 3. Expanded T-cell lines after first MHC-multimer pull down. Expanded T-cell lines after second MHC-multimer pull down.

Supplementary Table 2. MiHA validation scores

Supplementary Figure 2. Gene expression profiles of CLYBL (A) and TEP1 (B) were analyzed.

Purpose: T-cell recognition of minor histocompatibility antigens (MiHA) not only plays an important role in the beneficial graft-versus-leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) but also mediates serious GVH complications associated with allo-SCT. Using a reverse immunology approach, we aim to develop a method enabling the identification of T-cell responses directed against predefined antigens, with the goal to select those MiHAs that can be used clinically in combination with allo-SCT.Experimental Design: In this study, we used a recently developed MiHA selection algorithm to select candidate MiHAs within the HLA-presented ligandome of transformed B cells. From the HLA-presented ligandome that predominantly consisted of monomorphic peptides, 25 polymorphic peptides with a clinically relevant allele frequency were selected. By high-throughput screening, the availability of high-avidity T cells specific for these MiHA candidates in different healthy donors was analyzed.Results: With the use of MHC multimer enrichment, analyses of expanded T cells by combinatorial coding MHC multimer flow cytometry, and subsequent single-cell cloning, positive T-cell clones directed to two new MiHA: LB-CLYBL-1Y and LB-TEP1-1S could be demonstrated, indicating the immunogenicity of these two MiHAs.Conclusions: The biologic relevance of MiHA LB-CLYBL-1Y was demonstrated by the detection of LB-CLYBL-1Y–specific T cells in a patient suffering from acute myeloid leukemia (AML) that experienced an anti-leukemic response after treatment with allo-SCT. Clin Cancer Res; 21(9); 2177–86. ©2015 AACR.

Supplementary Table 1. Eluted published MiHA epitopes

Supplementary Figure 1. The HLA-binding affinity of 25 eluted highly potential MiHA candidates was analyzed by a binding assay that is based on HLA-recovery after UV-exchange monomer technology.

Complex three-dimensional in vitro organ-like models, or organoids, offer a unique biological tool with distinct advantages over two-dimensional cell culture systems, which can be too simplistic, and animal models, which can be too complex and may fail to recapitulate human physiology and pathology. Significant progress has been made in driving stem cells to differentiate into different organoid types, though several challenges remain. For example, many organoid models suffer from high heterogeneity, and it can be difficult to fully incorporate the complexity of in vivo tissue and organ development to faithfully reproduce human biology. Successfully addressing such limitations would increase the viability of organoids as models for drug development and preclinical testing. On April 3-6, 2022, experts in organoid development and biology convened at the Keystone Symposium "Organoids as Tools for Fundamental Discovery and Translation" to discuss recent advances and insights from this relatively new model system into human development and disease.

Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients for a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. HUB developed an innovative alternative, build on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient specific organoids are generated from healthy and malignant tissues and stored as biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. Since cancer progression and responses to immunotherapy are governed by immune cell interactions in the tumour microenvironment, we developed an assay in which various types of tumour organoids are co-cultured with T cells modified with chimeric antigen receptors (CAR T cells) to assess the cytotoxic potential. CAR-T cell therapy has been demonstrated to be efficacious against many hematological malignancies, however therapeutic application to treat solid cancers remains challenging. Our organoid-T cell co-culture assay offers a good platform to study the response of tumour organoids on the CAR T cells and will greatly contribute to our understanding of the critical factors that determine a successful CAR T cell therapy. In collaboration with GSK, HUB tested the cytotoxicity of GSK CAR T cells which target specific target antigen on human colorectal cancer (CRC) organoids. Six HUB CRC organoid models were selected based on differential expression levels of target antigen that was confirmed by FACS antibody staining. To evaluate GSK CAR T cell mediated organoid killing, caspase 3/7 expression was monitored at various timepoints and IFNγ secretion was measured by ELISA. For control, staurosporine and nonspecific-CAR T cells were included. Detected CRC-organoid killing demonstrated to be in line with antigen expression levels, indicating the specificity of the tested CAR T cells. Nevertheless, some CRC-organoid models demonstrated more resilient to CAR T cell mediated killing and further evaluation revealed upregulation of check point molecules such as PD-L1. This study reveals that tumour organoid-T cell co-culture assay hold value for preclinical development of CAR T-cell products and for evaluating tumour-evasion strategies. Moreover, this platform could potentially predict patient specific responses to CAR- T-cell therapy. Citation Format: Soura Mardpour, Mirella Georgouli, Ana Pereira Puri, Ravit Oren, Joshua Audu-Evans, Farzin Pourfarzad, Sara Brett, Pleun Hombrink, Sylvia F. Boj. Tumour organoid-t cell co-culture assay to evaluate CAR t cell cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 908.

Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients for a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy since conventional 2D cancer models hold low clinical predictive value. We developed an innovative alternative, built on the discovery that adult stem cells proliferate and organise into three-dimensional organotypic structures when they are embedded into extracellular matrix. Patient specific organoids are generated from healthy and malignant tissues and stored as biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. Since cancer progression and responses to immunotherapy are governed by immune cell interactions in the tumor microenvironment, we developed an assay in which colorectal (CRC) and non-small cell lung cancer (NSCLC) tumor organoids are co-cultured with simultaneously expanded paired tumor infiltrating lymphocytes (TIL) as a source of tumor reactive cells. Our system allows robust expansion of TIL with preserved T-cell receptor repertoires and has an establishment rate of 75%. Paired resources allow for screening of immune-modulatory therapies under physiologic conditions not depending on peptide-pulsing and allo-reactivity. For this purpose, we developed a organoid-TIL co-culture system which quantifies and characterizes tumor organoid apoptosis by combining image-based analysis with flow cytometry and cytokine release assays as measurements of T-cell cytotoxicity against tumor (and normal-matched) organoids. Our system offers a powerful tool for the development and validation of cancer immunotherapy and may help to stratify cancer patients for susceptibility of various types of immunotherapies. Citation Format: Soura Mardpour, Claudia Beaurivage, Jane Sun, Lorenz Jahn, Rene Overmeer, Lars-Eric Fielmich, Pleun Hombrink, Farzin Pourfarzad, Sylvia F. Boj. Autologous organoids and T cell co-cultures as a powerful personalized platform for immunotherapy development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 188.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2 specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T cell compartment, and indicative of vascular leakage. The observed disparate T and B cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients. This article is protected by copyright. All rights reserved.

The ability of the adaptive immune system to form memory is key to providing protection against secondary infections. Resident memory T cells (T

Tissue-resident memory T cells (TRM cells) reside in the epithelium and contribute to the first line defense against invading pathogens. Snyder et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20212059) now report that clonally expanded, recipient T cells persist as TRM cells in human lung allografts despite intensive immunosuppression. Their persistence may contribute to chronic allograft dysfunction.

Local T cell responses are required for optimal protection of the lungs against airborne pathogens. This localized protection is mediated by various immune cells, including resident memory T cells (TRM). While human lung CD8+ TRM line the epithelium and are enriched for recognition of respiratory viruses, we found CD4+ TRM to exhibit more heterogeneous localization patterns, surrounding airways, forming clusters in the lung parenchyma, and lining the epithelium. This heterogeneity was also reflected functionally, as lung CD4+ TRM were enriched for recognition of diverse classes of respiratory pathogens. Upon stimulation, lung CD4+ TRM expressed different polyfunctional cytokine profiles depending on the pathogen recognized. CD4+ TRM responding to respiratory viruses and bacteria were biased for production of IFN-γ and IL-17, respectively. Strikingly, pathogen-specific CD4+ TRM also represent a significant fraction in NSCLC tumors that remained polyfunctional despite high PD-1 expression. These findings are not only important for vaccine design, but also provide a rationale for reinvigorating anti-tumor immunity through triggering of polyfunctional pathogen-specific CD4+ tumor infiltrating lymphocytes.

BM has been put forward as a major reservoir for memory CD8(+) T cells. In order to fulfill that function, BM should "store" memory CD8(+) T cells, which in biological terms would require these "stored" memory cells to be in disequilibrium with the circulatory pool. This issue is a matter of ongoing debate. Here, we unequivocally demonstrate that murine and human BM harbors a population of tissue-resident memory CD8(+) T (T-RM) cells. These cells develop against various pathogens, independently of BM infection or local antigen recognition. BM CD8(+) T-RM cells share a transcriptional program with resident lymphoid cells in other tissues; they are polyfunctional cytokine producers and dependent on IL-15, Blimp-1, and Hobit. CD8(+) T-RM cells reside in the BM parenchyma, but are in close contact with the circulation. Moreover, this pool of resident T cells is not size-restricted and expands upon peripheral antigenic re-challenge. This works extends the role of the BM in the maintenance of CD8(+) T cell memory to include the preservation of an expandable reservoir of functional, non-recirculating memory CD8(+) T cells, which develop in response to a large variety of peripheral antigens.

During acute viral infections in mice, IL-7Rα and KLRG1 together are used to distinguish the short-lived effector cells (SLEC; IL-7Rα lo KLRG hi ) from the precursors of persisting memory cells (MPEC; IL-7Rα hi KLRG1 lo ). We here show that these markers can be used to define distinct subsets in the circulation and lymph nodes during the acute phase and in “steady state” in humans. In contrast to the T cells in the circulation, T cells derived from lymph nodes hardly contain any KLRG1-expressing cells. The four populations defined by IL-7Rα and KLRG1 differ markedly in transcription factor, granzyme and chemokine receptor expression. When studying renal transplant recipients experiencing a primary hCMV and EBV infection, we also found that after viral control, during latency, Ki-67-negative SLEC can be found in the peripheral blood in considerable numbers. Thus, combined analyses of IL-7Rα and KLRG1 expression on human herpes virus-specific CD8 + T cells can be used to separate functionally distinct subsets in humans. As a noncycling IL-7Rα lo KLRG1 hi population is abundant in healthy humans, we conclude that this combination of markers not only defines short-lived effector cells during the acute response but also stable effector cells that are formed and remain present during latent herpes infections.

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).

Most tissues are populated by tissue-resident memory T cells (TRM cells), which are adapted to their niche and appear to be indispensable for local protection against pathogens. Here we show that human white matter-derived brain CD8+ T cells can be subsetted into CD103−CD69+ and CD103+CD69+ T cells both with a phenotypic and transcription factor profile consistent with TRM cells. Specifically, CD103 expression in brain CD8+ T cells correlates with reduced expression of differentiation markers, increased expression of tissue-homing chemokine receptors, intermediate and low expression of the transcription factors T-bet and eomes, increased expression of PD-1 and CTLA-4, and low expression of cytolytic enzymes with preserved polyfunctionality upon activation. Brain CD4+ T cells also display TRM cell-associated markers but have low CD103 expression. We conclude that the human brain is surveilled by TRM cells, providing protection against neurotropic virus reactivation, whilst being under tight control of key immune checkpoint molecules., Tissue-resident immune cells are important for local protections from pathogens. Here the authors show that brain tissue-resident memory T cells (TRM cells) can be further subsetted by CD103 expression, with higher CD103 correlates with increased chemokine receptor and exhaustion markers such as PD1 or CTLA4, but reduced differentiation markers.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:β2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

CD8 T cells acquire cytotoxic molecules including granzyme B during effector differentiation. Both tissue-resident memory CD8 T cells (Trm) and circulating CD45RA+ effector-type T cells (Temra) cells have the ability to retain granzyme B protein expression into the memory phase, but it is unclear how this persistence of cytolytic activity is regulated during steady state. Previously, we have described that the transcriptional regulators Hobit and Blimp-1 have overlapping target genes that include granzyme B, but their impact on the regulation of cytotoxicity in Trm and Temra cells during homeostasis has remained unclear. We examined the expression regulation of Hobit and Blimp-1 in murine and human CD8 T-cells to determine their timeframe of activity. While Blimp-1 mRNA was expressed throughout effector and memory T cells, Blimp-1 protein, was only transiently expressed during the effector stage. In contrast, Hobit mRNA and protein expression was stably maintained during quiescence, but downregulated after activation. Notably, Blimp-1 was required for expression of granzyme B in murine effector T cells and Trm, while Hobit specifically regulated granzyme B in murine Trm during the memory phase. These findings suggest that Blimp-1 initiates cytotoxic effector function and that Hobit maintains cytotoxicity in a deployment-ready modus in Trm.

Immune responses in tissues are constrained by the physiological properties of the tissue involved. Tissue-resident memory T cells (TRM cells) are a recently discovered lineage of T cells specialized for life and function within tissues. Emerging evidence has shown that TRM cells have a special role in the control of solid tumors. A high frequency of TRM cells in tumors correlates with favorable disease progression in patients with cancer, and studies of mice have shown that TRM cells are necessary for optimal immunological control of solid tumors. Here we describe what defines TRM cells as a separate lineage and how these cells are generated. Furthermore, we discuss the properties that allow TRM cells to operate in normal and transformed tissues, as well as implications for the treatment of patients with cancer.

Resident memory T cells (TRM) reside in the lung epithelium and mediate protective immunity against respiratory pathogens. Although lung CD8(+) TRM have been extensively characterized, the properties of CD4(+) TRM remain unclear. Here we determined the transcriptional signature of CD4(+) TRM, identified by the expression of CD103, retrieved from human lung resection material. Various tissue homing molecules were specifically upregulated on CD4(+) TRM, whereas expression of tissue egress and lymph node homing molecules were low. CD103(+) TRM expressed low levels of T-bet, only a small portion expressed Eomesodermin (Eomes), and although the mRNA levels for Hobit were increased, protein expression was absent. On the other hand, the CD103(+) TRM showed a Notch signature. CD4(+)CD103(+) TRM constitutively expressed high transcript levels of numerous cytotoxic mediators that was functionally reflected by a fast recall response, magnitude of cytokine production, and a high degree of polyfunctionality. Interestingly, the superior cytokine production appears to be because of an accessible interferon-γ (IFNγ) locus and was partially because of rapid translation of preformed mRNA. Our studies provide a molecular understanding of the maintenance and potential function of CD4(+) TRM in the human lung. Understanding the specific properties of CD4(+) TRM is required to rationally improve vaccine design.Mucosal Immunology advance online publication 15 November 2017; doi:10.1038/mi.2017.94

Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients with a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy. Challenges range from lack of confidence in animal cancer models, inter-tumor heterogeneity and the absence of cellular microenvironment. Hubrecht Organoid Technology (HUB) offers an innovative alternative, build on the discovery that adult stem cells proliferate and organize into three-dimensional organotypic structures when they are embedded into Matrigel. HUBOrganoids™ are generated from healthy and disease tissues and stored as “living” biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. HUB has developed organoid-T-cell co-culture systems that allow a physiologic representation of the complexity of interactions in the tumor microenvironment, which is important for evaluating the effects of novel therapies. HUB offers an innovative platform combining image-based analysis with flow cytometry to visualize and quantify T-cell-tumor interactions as well as measurements of T-cell cytotoxicity against tumor (and normal-matched) organoids for colorectal and several other cancers. Our platform offers a powerful strategy for the development and validation of cancer immunotherapy and may help to screen the efficacy of immune checkpoint blockade or bispecific antibodies. In addition, cancer organoid-T-cell co-culture may also predict the functionality of tumor-infiltrating lymphocytes (TIL) and chimeric antigen receptor (CAR)-engineered T cells. As such, our platform offers opportunities in preclinical development and could become a diagnostic tool to predict individual patient response to immunotherapy. Citation Format: Pleun Hombrink, Soura Mardpour, Ewout Spaan, Jarmil Hanrath, Farzin Pourfarzad, Jasper Mullenders, Rene Overmeer, G.J Robert, Vries, F Sylvia, Boj. Organoid-T-cell co-cultures for preclinical testing of adoptive cell and cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2977.

Tissue-resident memory CD8+ T (TRM) cells that develop in the epithelia at portals of pathogen entry are important for improved protection against re-infection. CD8+ TRM cells within the skin and the small intestine are long-lived and maintained independently of circulating memory CD8+ T cells. In contrast to CD8+ TRM cells at these sites, CD8+ TRM cells that arise after influenza virus infection within the lungs display high turnover and require constant recruitment from the circulating memory pool for long-term persistence. The distinct characteristics of CD8+ TRM cell maintenance within the lungs may suggest a unique program of transcriptional regulation of influenza-specific CD8+ TRM cells. We have previously demonstrated that the transcription factors Hobit and Blimp-1 are essential for the formation of CD8+ TRM cells across several tissues, including skin, liver, kidneys, and the small intestine. Here, we addressed the roles of Hobit and Blimp-1 in CD8+ TRM cell differentiation in the lungs after influenza infection using mice deficient for these transcription factors. Hobit was not required for the formation of influenza-specific CD8+ TRM cells in the lungs. In contrast, Blimp-1 was essential for the differentiation of lung CD8+ TRM cells and inhibited the differentiation of central memory CD8+ T (TCM) cells. We conclude that Blimp-1 rather than Hobit mediates the formation of CD8+ TRM cells in the lungs, potentially through control of the lineage choice between TCM and TRM cells during the differentiation of influenza-specific CD8+ T cells.

BM has been put forward as a major reservoir for memory CD8

In recent years, our understanding about the functional complexity of CD8+ T-cell populations has increased tremendously. The immunology field is now facing challenges to translate these insights into phenotypic definitions that correlate reliably with distinct functional traits. This is key to adequately monitor and understand compound immune responses including vaccination and immunotherapy regimens. Here we will summarize our understanding of the current state in the human CD8+ T-cell subset characterization field. We will address how reliably the currently used cell surface markers are connected to differentiation status and function of particular subsets. By restricting ourselves to CD8+ αβ T cells, we will focus mostly on major histocompatibility complex (MHC) class I-restricted virus- and tumor-specific T cells. This comes with a major advantage as fluorescently labeled peptide-loaded MHC class I multimers have been widely used to identify and characterize these cells.

Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1β (IL-1β) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

Over the past decade, it has become clear that there is an important subset of memory T cells that resides in tissues — tissue resident memory T cells (TRM). There is an emerging understanding that TRM have a role in human tissue specific immune and inflammatory diseases. Furthermore, the nature of the molecular signals that maintain TRM in tissues is the subject of much investigation. In addition while it is logical for TRM to be located in barrier tissues at interfaces with the environment in human and mouse, TRM have also been found in brain, kidney, joint, and other non-barrier tissues in both species. Their biology and behavior make it likely that they play a role in chronic relapsing and remitting diseases of both barrier and non-barrier tissues. This review will discuss recent understandings of the biology of TRM with a particular focus on their role in disease.

Contains fulltext : 200117.pdf (Publisher’s version ) (Open Access) Resident memory T cells (TRM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103(+) TRM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103(+)CD8(+) tumor infiltrating lymphocytes (TILs), with TRM properties, are a positive prognostic marker. To better understand the role of TRM in tumors, we performed a detailed characterization of CD8(+) and CD4(+) TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8(+) and CD4(+) T cell infiltrates in tumors were comparable, but we observed a sharp contrast in TRM ratios compared to surrounding lung tissue. The majority of both CD4(+) and CD8(+) TILs expressed CD69 and a subset also expressed CD103, both hallmarks of TRM. While CD103(+)CD8(+) T cells were enriched in tumors, CD103(+)CD4(+) T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103(+)CD4(+) and CD103(+)CD8(+) TILs showed multiple characteristics of TRM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8(+) and CD4(+) TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103(+) TILs. Strikingly, CD103(+)CD4(+) TILs were the most potent producers of TNF-alpha and IFN-gamma, while other TIL subsets lacked such cytokine production. Whereas, CD103(+)CD4(+)PD-1(low) TILs produced the most effector cytokines, CD103(+)CD4(+)PD-1(++) and CD69(+)CD4(+)PD-1(++) TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung TRM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103(+)CD4(+) and CD69(+)CD8(+) TILs. Our findings thus provide a rationale to target CD103(+)CD4(+) TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.

CD8

CD8+ T cells clear primary infections with intracellular pathogens and provide long-term immunity against reinfection. Two different types of CD8+ T cells are responsible for these functions: short-lived effector T cells and memory T cells. The cellular relationship between these two types of CD8+ T cells has been subject to much investigation. Both cell types can derive from a single naive CD8+ T cell precursor. Their generation requires a fate choice early during a T cell response. As a result, two populations of T cells emerge. One of these consists of terminally differentiated short-lived effector T cells. The other contains cells able to develop into long-lived memory T cells. A foundation for development of these two populations may be laid during the first division of an activated naive T cell precursor, as a consequence of asymmetric segregation of fate-determining factors into the daughter cells. Nonetheless, the binary choice between the two lineages is strongly influenced by signals, which ensure that the differentiation process is matched with the needs posed by the infection. Here, we will discuss the genetic and metabolic programs governing differentiation of these two lineages as well as the processes leading to their induction and consolidation to create bistability. These processes involve extensive lateral inhibition between the programs as well as positive feedback between the genetic programs and the signaling pathways responsible for their induction. These features will be highlighted by discussing the role of the Notch signaling pathway in guiding the decision between the two lineages.

Immunotherapy of B-cell malignancies using CD19-targeted chimeric antigen receptor-transduced T cells or CD20-targeted therapeutic monoclonal antibodies has shown clinical efficacy. However, refractory disease and the emergence of antigen-loss tumor escape variants after treatment demonstrate the need to target additional antigens. Here we aimed to target the B-cell receptor-associated protein CD79b by a T-cell receptor (TCR)-based approach. Because thymic selection depletes high-avidity T cells recognizing CD79b-derived peptides presented in self-HLA molecules, we aimed to isolate T cells recognizing these peptides presented in allogeneic HLA. Peptide-HLA tetramers composed of CD79b peptides bound to either HLA-A2 or HLA-B7 were used to isolate T-cell clones from HLA-A*0201 and B*0702-negative individuals. For 3 distinct T-cell clones, CD79b specificity was confirmed through CD79b gene transduction and CD79b-specific shRNA knockdown. The CD79b-specific T-cell clones were highly reactive against CD79b-expressing primary B-cell malignancies, whereas no recognition of nonhematopoietic cells was observed. Although lacking CD79b-cell surface expression, intermediate reactivity toward monocytes, hematopoietic progenitor cells, and T-cells was observed. Quantitative reverse transcriptase polymerase chain reaction revealed low CD79b gene expression in these cell types. Therefore, aberrant gene expression must be taken into consideration when selecting common, apparently lineage-specific self-antigens as targets for TCR-based immunotherapies.

Immunotherapy for hematological malignancies or solid tumors by administration of monoclonal antibodies or T cells engineered to express chimeric antigen receptors or T-cell receptors (TCRs) has demonstrated clinical efficacy. However, antigen-loss tumor escape variants and the absence of currently targeted antigens on several malignancies hamper the widespread application of immunotherapy. We have isolated a TCR targeting a peptide of the intracellular B cell-specific transcription factor BOB1 presented in the context of HLA-B*07:02. TCR gene transfer installed BOB1 specificity and reactivity onto recipient T cells. TCR-transduced T cells efficiently lysed primary B-cell leukemia, mantle cell lymphoma, and multiple myeloma in vitro. We also observed recognition and lysis of healthy BOB1-expressing B cells. In addition, strong BOB1-specific proliferation could be demonstrated for TCR-modified T cells upon antigen encounter. Furthermore, clear in vivo antitumor reactivity was observed of BOB1-specific TCR-engineered T cells in a xenograft mouse model of established multiple myeloma. Absence of reactivity toward a broad panel of BOB1- but HLA-B*07:02+ nonhematopoietic and hematopoietic cells indicated no off-target toxicity. Therefore, administration of BOB1-specific TCR-engineered T cells may provide novel cellular treatment options to patients with B-cell malignancies, including multiple myeloma.

Summary In healthy individuals and in patients with invasive aspergillosis, Aspergillus -specific T-cells in peripheral blood display mainly a Thelper1 phenotype. Although in other fungal infections Thelper17 immunity is important, it was suggested that in aspergillus infection Thelper17 cells do not play a role or may even be detrimental. Objectives To compare the cytokine profiles of Aspergillus -specific CD4+ T-cells in peripheral blood and in the lung. To investigate the Thelper phenotype at the primary location of A. fumigatus exposure. Methods Lung-derived T-cells and peripheral blood T-cells from COPD-patients were stimulated with overlapping peptides of 6 A. fumigatus proteins. Aspergillus -specific T-cells were identified on the basis of the activation marker CD154 and production of TNFα. In addition, production of the cytokines IFNγ, IL-17, IL-4 and IL-5 by the Aspergillus -specific T-cells was measured. Results The majority of lung-derived Aspergillus -specific T-cells displayed a Thelper17 phenotype, and only low percentages of cells produced IFNγ. In contrast, in the peripheral blood of COPD patients Aspergillus -specific T-cells displayed a Thelper1 phenotype, similar as peripheral blood-derived Aspergillus -specific T-cells from healthy individuals. Conclusions These data demonstrate that in A. fumigatus infection, similar as in other fungal infections, Thelper17 cells may play a more important role in the immune response than was appreciated until now.

The T cell lineage is commonly divided into CD4-expressing helper T cells that polarize immune responses through cytokine secretion and CD8-expressing cytotoxic T cells that eliminate infected target cells by virtue of the release of cytotoxic molecules. Recently, a population of CD4(+) T cells that conforms to the phenotype of cytotoxic CD8(+) T cells has received increased recognition. These cytotoxic CD4(+) T cells display constitutive expression of granzyme B and perforin at the protein level and mediate HLA class II-dependent killing of target cells. In humans, this cytotoxic profile is found within the human cytomegalovirus (hCMV)-specific, but not within the influenza- or Epstein-Barr virus-specific CD4(+) T cell populations, suggesting that, in particular, hCMV infection induces the formation of cytotoxic CD4(+) T cells. We have previously described that the transcription factor Homolog of Blimp-1 in T cells (Hobit) is specifically upregulated in CD45RA(+) effector CD8(+) T cells that arise after hCMV infection. Here, we describe the expression pattern of Hobit in human CD4(+) T cells. We found Hobit expression in cytotoxic CD4(+) T cells and accumulation of Hobit(+) CD4(+) T cells after primary hCMV infection. The Hobit(+) CD4(+) T cells displayed highly overlapping characteristics with Hobit(+) CD8(+) T cells, including the expression of cytotoxic molecules, T-bet, and CX3CR1. Interestingly, γδ(+) T cells that arise after hCMV infection also upregulate Hobit expression and display a similar effector phenotype as cytotoxic CD4(+) and CD8(+) T cells. These findings suggest a shared differentiation pathway in CD4(+), CD8(+), and γδ(+) T cells that may involve Hobit-driven acquisition of long-lived cytotoxic effector function

Tissue-resident memory T cells (TRM cells) in the airways mediate protection against respiratory infection. We characterized TRM cells expressing integrin αE (CD103) that reside within the epithelial barrier of human lungs. These cells had specialized profiles of chemokine receptors and adhesion molecules, consistent with their unique localization. Lung TRM cells were poised for rapid responsiveness by constitutive expression of deployment-ready mRNA encoding effector molecules, but they also expressed many inhibitory regulators, suggestive of programmed restraint. A distinct set of transcription factors was active in CD103(+) TRM cells, including Notch. Genetic and pharmacological experiments with mice revealed that Notch activity was required for the maintenance of CD103(+) TRM cells. We have thus identified specialized programs underlying the residence, persistence, vigilance and tight control of human lung TRM cells.

// Lorenz Jahn 1 , Renate S. Hagedoorn 1 , Dirk M. van der Steen 1 , Pleun Hombrink 1, 2 , Michel G.D. Kester 1 , Marjolein P. Schoonakker 1 , Danielle de Ridder 1 , Peter A. van Veelen 3 , J.H. Frederik Falkenburg 1 , Mirjam H.M. Heemskerk 1 1 Department of Hematology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands 2 Department of Hematopoiesis, Sanquin Research, 1006 AD Amsterdam, The Netherlands 3 Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands Correspondence to: Lorenz Jahn, email: l.jahn@lumc.nl Mirjam H.M. Heemskerk, email: m.h.m.heemskerk@lumc.nl Keywords: CD22, TCR gene transfer, immunotherapy, acute lymphoblastic leukemia, allogeneic HLA Received: June 13, 2016 Accepted: September 19, 2016 Published: September 26, 2016 ABSTRACT CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22 RPF ) presented in human leukocyte antigen (HLA)-B * 07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22 RPF -specific T-cell clone 9D4 was isolated from a healthy HLA-B * 07:02 neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22 neg hematopoietic cell subsets but weakly lysed CD22 low -expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy.

// Lorenz Jahn 1 , Dirk M. van der Steen 1 , Renate S. Hagedoorn 1 , Pleun Hombrink 1, 2 , Michel G.D. Kester 1 , Marjolein P. Schoonakker 1 , Danielle de Ridder 1 , Peter A. van Veelen 3, 4 , J.H. Frederik Falkenburg 1 , Mirjam H.M. Heemskerk 1 1 Department of Hematology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands 2 Department of Hematopoiesis, Sanquin Research, 1006 AD Amsterdam, The Netherlands 3 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, The Netherlands 4 Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands Correspondence to: Lorenz Jahn, email: l.jahn@lumc.nl Mirjam H.M. Heemskerk, email: m.h.m.heemskerk@lumc.nl Keywords: CD20, TCR gene transfer, monoclonal antibodies, immunotherapy, B-cell leukemia and lymphoma Received: August 20, 2016 Accepted: October 13, 2016 Published: October 20, 2016 ABSTRACT Immunotherapy of B-cell leukemia and lymphoma with CD20-targeting monoclonal antibodies (mAbs) has demonstrated clinical efficacy. However, the emergence of unresponsive disease due to low or absent cell surface CD20 urges the need to develop additional strategies. In contrast to mAbs, T-cells via their T-cell receptor (TCR) can recognize not only extracellular but also intracellular antigens in the context of HLA molecules. We hypothesized that T-cells equipped with high affinity CD20-targeting TCRs would be able to recognize B-cell malignancies even in the absence of extracellular CD20. We isolated CD8 + T-cell clones binding to peptide-MHC-tetramers composed of HLA-A*02:01 and CD20-derived peptide SLFLGILSV (CD20 SLF ) from HLA-A*02:01 neg healthy individuals to overcome tolerance towards self-antigens such as CD20. High avidity T-cell clones were identified that readily recognized and lysed primary HLA-A2 pos B-cell leukemia and lymphoma in the absence of reactivity against CD20-negative but HLA-A2 pos healthy hematopoietic and nonhematopoietic cells. The T-cell clone with highest avidity efficiently lysed malignant cell-lines that had insufficient extracellular CD20 to be targeted by CD20 mAbs. Transfer of this TCR installed potent CD20-specificity onto recipient T-cells and led to lysis of CD20 low malignant cell-lines. Moreover, our approach facilitates the generation of an off-the-shelf TCR library with broad applicability by targeting various HLA alleles. Using the same methodology, we isolated a T-cell clone that efficiently lysed primary HLA-B*07:02 pos B-cell malignancies by targeting another CD20-derived peptide. TCR gene transfer of high affinity CD20-specific TCRs can be a valuable addition to current treatment options for patients suffering from CD20 low B-cell malignancies.