Publications by authors named "Dietmar Zehn"

73 Publications

Auto-aggressive CXCR6 CD8 T cells cause liver immune pathology in NASH.

Nature 2021 Mar 24. Epub 2021 Mar 24.

Institute of Molecular Immunology and Experimental Oncology, School of Medicine, Technical University of Munich (TUM), Munich, Germany.

Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer. The accumulation of metabolites leads to cell stress and inflammation in the liver, but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6 CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6 CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6 CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.
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http://dx.doi.org/10.1038/s41586-021-03233-8DOI Listing
March 2021

Loss of the orphan nuclear receptor NR2F6 enhances CD8 T-cell memory via IFN-γ.

Cell Death Dis 2021 Feb 15;12(2):187. Epub 2021 Feb 15.

Translational Cell Genetics, Institute of Pharmacology and Genetics, Medical University of Innsbruck, Innsbruck, Austria.

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127KLRG1) or memory precursors cells (MPECs, CD127KLRG1) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8 memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6 OT-I T-cells showed that the augmented memory formation is CD8 T-cell intrinsic. Although the relative difference between the Nr2f6 and Nr2f6 OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8 T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8 T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8 T cells in vivo.
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http://dx.doi.org/10.1038/s41419-021-03470-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884426PMC
February 2021

Tailoring the resolution of single-cell RNA sequencing for primary cytotoxic T cells.

Nat Commun 2021 01 25;12(1):569. Epub 2021 Jan 25.

Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, 85354, Freising, Germany.

Single-cell RNA sequencing in principle offers unique opportunities to improve the efficacy of contemporary T-cell based immunotherapy against cancer. The use of high-quality single-cell data will aid our incomplete understanding of molecular programs determining the differentiation and functional heterogeneity of cytotoxic T lymphocytes (CTLs), allowing for optimal therapeutic design. So far, a major obstacle to high depth single-cell analysis of CTLs is the minute amount of RNA available, leading to low capturing efficacy. Here, to overcome this, we tailor a droplet-based approach for high-throughput analysis (tDrop-seq) and a plate-based method for high-performance in-depth CTL analysis (tSCRB-seq). The latter gives, on average, a 15-fold higher number of captured transcripts per gene compared to droplet-based technologies. The improved dynamic range of gene detection gives tSCRB-seq an edge in resolution sensitive downstream applications such as graded high confidence gene expression measurements and cluster characterization. We demonstrate the power of tSCRB-seq by revealing the subpopulation-specific expression of co-inhibitory and co-stimulatory receptor targets of key importance for immunotherapy.
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http://dx.doi.org/10.1038/s41467-020-20751-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835213PMC
January 2021

Origin and fine-tuning of effector CD8 T cell subpopulations in chronic infection.

Curr Opin Virol 2021 Feb 1;46:27-35. Epub 2020 Nov 1.

Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, 85354 Freising, Germany. Electronic address:

Persisting stimulation can skew CD8 T cells towards a hypofunctional state commonly referred to as T cell exhaustion. This functional attenuation likely constitutes a mechanism which evolved to balance T cell mediated viral control versus overwhelming immunopathology. Here, we highlight the recent progress in defining the genetic mechanisms and factors shaping the differentiation of exhausted CD8 T cells. We review how the transcription factor Tox imposes an exhausted phenotype in the Tcf1+ progenitors and how CD4 help fine-tunes the effector subsets that emerge from this progenitor population. Both processes critically shape the spectrum of effector function performed by CD8 T cells and the level of resulting virus control. Finally, we discuss how these insights can be exploited to boost the immune response in chronic infection and cancer.
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http://dx.doi.org/10.1016/j.coviro.2020.10.003DOI Listing
February 2021

Two parallel worlds of memory T cells.

Nat Immunol 2020 12;21(12):1484-1485

Division of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich (TUM), Freising, Germany.

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http://dx.doi.org/10.1038/s41590-020-00815-yDOI Listing
December 2020

TOX defines the degree of CD8+ T cell dysfunction in distinct phases of chronic HBV infection.

Gut 2020 Oct 23. Epub 2020 Oct 23.

Internal Medicine II, Faculty of Medicine, Freiburg University Hospital, Freiburg, Germany

Objective: Chronic hepatitis B virus (HBV) infection is characterised by HBV-specific CD8+ T cell dysfunction that has been linked to Tcell exhaustion, a distinct differentiation programme associated with persisting antigen recognition. Recently, Thymocyte Selection-Associated High Mobility Group Box (TOX) was identified as master regulator of CD8+ T cell exhaustion. Here, we addressed the role of TOX in HBV-specific CD8+ T cell dysfunction associated with different clinical phases of infection.

Design: We investigated TOX expression in HBV-specific CD8+ T cells from 53 HLA-A*01:01, HLA-A*11:01 and HLA-A*02:01 positive patients from different HBV infection phases and compared it to hepatitis C virus (HCV)-specific, cytomegalovirus (CMV)-specific, Epstein-Barr virus (EBV)-specific and influenza virus (FLU)-specific CD8+ T cells. Phenotypic and functional analyses of virus-specific CD8+ T cells were performed after peptide-loaded tetramer-enrichment and peptide-specific expansion.

Results: Our results show that TOX expression in HBV-specific CD8+ T cells is linked to chronic antigen stimulation, correlates with viral load and is associated with phenotypic and functional characteristics of T-cell exhaustion. In contrast, similar TOX expression in EBV-specific and CMV-specific CD8+ T cells is not linked to T-cell dysfunction suggesting different underlying programmes. TOX expression in HBV-specific CD8+ T cells is also affected by targeted antigens, for example, core versus polymerase. In HBV-specific CD8+ T cells, TOX expression is maintained after spontaneous or therapy-mediated viral control in chronic but not self-limiting acute HBV infection indicating a permanent molecular imprint after chronic but not temporary stimulation.

Conclusion: Our data highlight TOX as biomarker specific for dysfunctional virus-specific CD8+ T cells in the context of an actively persisting infection.
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http://dx.doi.org/10.1136/gutjnl-2020-322404DOI Listing
October 2020

SARS-CoV-2 infections in cancer outpatients-Most infected patients are asymptomatic carriers without impact on chemotherapy.

Cancer Med 2020 11 6;9(21):8020-8028. Epub 2020 Oct 6.

Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Munich, Germany.

Oncologic patients are regarded as the population most at risk of developing a severe course of COVID-19 due to the fact that malignant diseases and chemotherapy often weaken the immune system. In the face of the ongoing SARS-CoV-2 pandemic, how particular patients deal with this infection remains an important question. In the period between the 15 and 26 April 2020, a total of 1227 patients were tested in one of seven oncologic outpatient clinics for SARS-CoV-2, regardless of symptoms, employing RT-qPCR. Of 1227 patients, 78 (6.4%) were tested positive of SARS-CoV-2. Only one of the patients who tested positive developed a severe form of COVID-19 with pneumonia (CURB-65 score of 2), and two patients showed mild symptoms. Fourteen of 75 asymptomatic but positively tested patients received chemotherapy or chemo-immunotherapy according to their regular therapy algorithm (±4 weeks of SARS-CoV-2 test), and 48 of 78 (61.5%) positive-tested patients received glucocorticoids as co-medication. None of the asymptomatic infected patients showed unexpected complications due to the SARS-CoV-2 infection during the cancer treatment. These data clearly contrast the view that patients with an oncologic disease are particularly vulnerable to SARS-CoV-2 and suggest that compromising therapies could be continued or started despite the ongoing pandemic. Moreover the relatively low appearance of symptoms due to COVID-19 among patients on chemotherapy and other immunosuppressive co-medication like glucocorticoids indicate that suppressing the response capacity of the immune system reduces disease severity.
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http://dx.doi.org/10.1002/cam4.3435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643635PMC
November 2020

PTPN2 Deficiency Enhances Programmed T Cell Expansion and Survival Capacity of Activated T Cells.

Cell Rep 2020 07;32(4):107957

Division of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich (TUM), Freising, Germany; Division of Immunology and Allergy, Department of Medicine, Lausanne University Hospital, Lausanne, Switzerland. Electronic address:

Manipulating molecules that impact T cell receptor (TCR) or cytokine signaling, such as the protein tyrosine phosphatase non-receptor type 2 (PTPN2), has significant potential for advancing T cell-based immunotherapies. Nonetheless, it remains unclear how PTPN2 impacts the activation, survival, and memory formation of T cells. We find that PTPN2 deficiency renders cells in vivo and in vitro less dependent on survival-promoting cytokines, such as interleukin (IL)-2 and IL-15. Remarkably, briefly ex vivo-activated PTPN2-deficient T cells accumulate in 3- to 11-fold higher numbers following transfer into unmanipulated, antigen-free mice. Moreover, the absence of PTPN2 augments the survival of short-lived effector T cells and allows them to robustly re-expand upon secondary challenge. Importantly, we find no evidence for impaired effector function or memory formation. Mechanistically, PTPN2 deficiency causes broad changes in the expression and phosphorylation of T cell expansion and survival-associated proteins. Altogether, our data underline the therapeutic potential of targeting PTPN2 in T cell-based therapies to augment the number and survival capacity of antigen-specific T cells.
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http://dx.doi.org/10.1016/j.celrep.2020.107957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408006PMC
July 2020

Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes.

J Hepatol 2020 12 26;73(6):1347-1359. Epub 2020 Jun 26.

Institute of Molecular Immunology and Experimental Oncology, University Hospital München rechts der Isar; Technical University of Munich, Munich, Germany.

Background & Aims: Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism.

Methods: We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests.

Results: We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation.

Conclusion: Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition.

Lay Summary: The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.
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http://dx.doi.org/10.1016/j.jhep.2020.06.026DOI Listing
December 2020

Charting the Roadmap of T Cell Exhaustion.

Immunity 2020 05;52(5):724-726

Division of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany. Electronic address:

Exposure to chronic infection or malignant tumors triggers the differentiation of functionally impaired, so-called exhausted T cells. In this issue of Immunity, Beltra et al. reveal a multi-step developmental process that defines how T cells transition from proliferating progenitors into terminally differentiated exhausted T cells.
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http://dx.doi.org/10.1016/j.immuni.2020.04.019DOI Listing
May 2020

Beta cell-specific CD8 T cells maintain stem cell memory-associated epigenetic programs during type 1 diabetes.

Nat Immunol 2020 05 30;21(5):578-587. Epub 2020 Mar 30.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA.

The pool of beta cell-specific CD8 T cells in type 1 diabetes (T1D) sustains an autoreactive potential despite having access to a constant source of antigen. To investigate the long-lived nature of these cells, we established a DNA methylation-based T cell 'multipotency index' and found that beta cell-specific CD8 T cells retained a stem-like epigenetic multipotency score. Single-cell assay for transposase-accessible chromatin using sequencing confirmed the coexistence of naive and effector-associated epigenetic programs in individual beta cell-specific CD8 T cells. Assessment of beta cell-specific CD8 T cell anatomical distribution and the establishment of stem-associated epigenetic programs revealed that self-reactive CD8 T cells isolated from murine lymphoid tissue retained developmentally plastic phenotypic and epigenetic profiles relative to the same cells isolated from the pancreas. Collectively, these data provide new insight into the longevity of beta cell-specific CD8 T cell responses and document the use of this methylation-based multipotency index for investigating human and mouse CD8 T cell differentiation.
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http://dx.doi.org/10.1038/s41590-020-0633-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7183435PMC
May 2020

Cell-intrinsic adrenergic signaling controls the adaptive NK cell response to viral infection.

J Exp Med 2020 04;217(4)

Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY.

Natural killer (NK) cells are innate lymphocytes that exhibit adaptive features, such as clonal expansion and memory, during viral infection. Although activating receptor engagement and proinflammatory cytokines are required to drive NK cell clonal expansion, additional stimulatory signals controlling their proliferation remain to be discovered. Here, we describe one such signal that is provided by the adrenergic nervous system, and demonstrate that cell-intrinsic adrenergic signaling is required for optimal adaptive NK cell responses. Early during mouse cytomegalovirus (MCMV) infection, NK cells up-regulated Adrb2 (which encodes the β2-adrenergic receptor), a process dependent on IL-12 and STAT4 signaling. NK cell-specific deletion of Adrb2 resulted in impaired NK cell expansion and memory during MCMV challenge, in part due to a diminished proliferative capacity. As a result, NK cell-intrinsic adrenergic signaling was required for protection against MCMV. Taken together, we propose a novel role for the adrenergic nervous system in regulating circulating lymphocyte responses to viral infection.
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http://dx.doi.org/10.1084/jem.20190549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144534PMC
April 2020

Dynamics of the Coreceptor-LCK Interactions during T Cell Development Shape the Self-Reactivity of Peripheral CD4 and CD8 T Cells.

Cell Rep 2020 02;30(5):1504-1514.e7

Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, 14220 Prague, Czech Republic. Electronic address:

Overtly self-reactive T cells are removed during thymic selection. However, it has been recently established that T cell self-reactivity promotes protective immune responses. Apparently, the level of self-reactivity of mature T cells must be tightly balanced. Our mathematical model and experimental data show that the dynamic regulation of CD4- and CD8-LCK coupling establish the self-reactivity of the peripheral T cell pool. The stoichiometry of the interaction between CD8 and LCK, but not between CD4 and LCK, substantially increases upon T cell maturation. As a result, peripheral CD8 T cells are more self-reactive than CD4 T cells. The different levels of self-reactivity of mature CD8 and CD4 T cells likely reflect the unique roles of these subsets in immunity. These results indicate that the evolutionary selection pressure tuned the CD4-LCK and CD8-LCK stoichiometries, as they represent the unique parts of the proximal T cell receptor (TCR) signaling pathway, which differ between CD4 and CD8 T cells.
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http://dx.doi.org/10.1016/j.celrep.2020.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003063PMC
February 2020

Lymphatic endothelial cells prime naïve CD8 T cells into memory cells under steady-state conditions.

Nat Commun 2020 Jan 27;11(1):538. Epub 2020 Jan 27.

Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Lymphatic endothelial cells (LECs) chemoattract naïve T cells and promote their survival in the lymph nodes, and can cross-present antigens to naïve CD8 T cells to drive their proliferation despite lacking key costimulatory molecules. However, the functional consequence of LEC priming of CD8 T cells is unknown. Here, we show that while many proliferating LEC-educated T cells enter early apoptosis, the remainders comprise a long-lived memory subset, with transcriptional, metabolic, and phenotypic features of central memory and stem cell-like memory T cells. In vivo, these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall, and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8 T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.
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http://dx.doi.org/10.1038/s41467-019-14127-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985113PMC
January 2020

MiR-23~27~24-mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation.

Sci Adv 2019 12 11;5(12):eaaw1715. Epub 2019 Dec 11.

Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.

Follicular helper T (T) cells are essential for generating protective humoral immunity. To date, microRNAs (miRNAs) have emerged as important players in regulating T cell biology. Here, we show that loss of miR-23~27~24 clusters in T cells resulted in elevated T cell frequencies upon different immune challenges, whereas overexpression of this miRNA family led to reduced T cell responses. Mechanistically, miR-23~27~24 clusters coordinately control T cells through targeting a network of genes that are crucial for T cell biology. Among them, thymocyte selection-associated HMG-box protein (TOX) was identified as a central transcription regulator in T cell development. TOX is highly up-regulated in both mouse and human T cells in a BCL6-dependent manner. In turn, TOX promotes the expression of multiple molecules that play critical roles in T cell differentiation and function. Collectively, our results establish a key miRNA regulon that maintains optimal T cell responses for resultant humoral immunity.
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http://dx.doi.org/10.1126/sciadv.aaw1715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905868PMC
December 2019

Precursor exhausted T cells: key to successful immunotherapy?

Nat Rev Immunol 2020 02 7;20(2):128-136. Epub 2019 Oct 7.

Department of Microbiology & Immunology Melbourne, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria, Australia.

Cytotoxic T cell immunity in response to chronic infections and tumours is maintained by a specialized population of CD8 T cells that exhibit hallmarks of both exhausted and memory cells and give rise to terminally differentiated exhausted effector cells that contribute to viral or tumour control. Importantly, recent work suggests these cells, which we refer to as 'precursor exhausted' T (T) cells, are responsible for the proliferative burst that generates effector T cells in response to immune checkpoint blockade targeting programmed cell death 1 (PD1), and increased T cell frequencies have recently been linked to increased patient survival. We believe the recent discovery of T cells not only represents a paradigm shift in our understanding of the mechanisms that maintain CD8 T cell responses in chronic infections and tumours but also opens up unexpected avenues for the development of new and innovative therapeutic approaches. In this Opinion article, we discuss the differentiation and function of T cells and suggest that targeting these cells may be key for successful immunotherapy.
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http://dx.doi.org/10.1038/s41577-019-0223-7DOI Listing
February 2020

Defining 'T cell exhaustion'.

Nat Rev Immunol 2019 11 30;19(11):665-674. Epub 2019 Sep 30.

Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany.

'T cell exhaustion' is a broad term that has been used to describe the response of T cells to chronic antigen stimulation, first in the setting of chronic viral infection but more recently in response to tumours. Understanding the features of and pathways to exhaustion has crucial implications for the success of checkpoint blockade and adoptive T cell transfer therapies. In this Viewpoint article, 18 experts in the field tell us what exhaustion means to them, ranging from complete lack of effector function to altered functionality to prevent immunopathology, with potential differences between cancer and chronic infection. Their responses highlight the dichotomy between terminally differentiated exhausted T cells that are TCF1 and the self-renewing TCF1 population from which they derive. These TCF1 cells are considered by some to have stem cell-like properties akin to memory T cell populations, but the developmental relationships are unclear at present. Recent studies have also highlighted an important role for the transcriptional regulator TOX in driving the epigenetic enforcement of exhaustion, but key questions remain about the potential to reverse the epigenetic programme of exhaustion and how this might affect the persistence of T cell populations.
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http://dx.doi.org/10.1038/s41577-019-0221-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286441PMC
November 2019

Proliferation-competent Tcf1+ CD8 T cells in dysfunctional populations are CD4 T cell help independent.

Proc Natl Acad Sci U S A 2019 10 17;116(40):20070-20076. Epub 2019 Sep 17.

Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, 85354 Freising, Germany;

T cell maintenance in chronic infection and cancer follows a hierarchical order. Short-lived effector CD8 T cells are constitutively replaced from a proliferation-competent Tcf1-expressing progenitor population. This occurs spontaneously at low levels and increases in magnitude upon blocking PD-1 signaling. We explore how CD4 T cell help controls transition and survival of the progenitors and their progeny by utilizing single-cell RNA sequencing. Unexpectedly, absence of CD4 help caused reductions in cell numbers only among terminally differentiated cells while proliferation-competent progenitor cells remained unaffected with regard to their numbers and their overall phenotype. In fact, upon restoration of a functional CD4 compartment, the progenitors began to regenerate the effector CD8 T cells. Thus, unlike memory T cells for which secondary expansion requires CD4 T cell help, this is not a necessity for proliferation-competent progenitor cells in dysfunctional populations. Our data therefore reveals that proliferation-competent cells in dysfunctional populations show a previously unrecognized uncoupling of CD4 T cell help that is otherwise required by conventional memory T cells.
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http://dx.doi.org/10.1073/pnas.1902701116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778176PMC
October 2019

Attenuation of chronic antiviral T-cell responses through constitutive COX2-dependent prostanoid synthesis by lymph node fibroblasts.

PLoS Biol 2019 07 15;17(7):e3000072. Epub 2019 Jul 15.

Center for Immunity and Infection Lausanne, Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.

Lymphoid T-zone fibroblastic reticular cells (FRCs) actively promote T-cell trafficking, homeostasis, and expansion but can also attenuate excessive T-cell responses via inducible nitric oxide (NO) and constitutive prostanoid release. It remains unclear how these FRC-derived mediators dampen T-cell responses and whether this occurs in vivo. Here, we confirm that murine lymph node (LN) FRCs produce prostaglandin E2 (PGE2) in a cyclooxygenase-2 (COX2)-dependent and inflammation-independent fashion. We show that this COX2/PGE2 pathway is active during both strong and weak T-cell responses, in contrast to NO, which only comes into play during strong T-cell responses. During chronic infections in vivo, PGE2-receptor signaling in virus-specific cluster of differentiation (CD)8 cytotoxic T cells was shown by others to suppress T-cell survival and function. Using COX2flox/flox mice crossed to mice expressing Cre recombinase expression under control of the CC chemokine ligand (CCL19) promoter (CCL19cre), we now identify CCL19+ FRC as the critical source of this COX2-dependent suppressive factor, suggesting PGE2-expressing FRCs within lymphoid tissues are an interesting therapeutic target to improve T-cell-mediated pathogen control during chronic infection.
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http://dx.doi.org/10.1371/journal.pbio.3000072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657915PMC
July 2019

TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.

Nature 2019 07 17;571(7764):265-269. Epub 2019 Jun 17.

Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany.

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential. This process, known as T cell exhaustion or dysfunction, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1). The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1 self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.
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http://dx.doi.org/10.1038/s41586-019-1326-9DOI Listing
July 2019

Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation.

Cell Metab 2019 08 23;30(2):352-363.e8. Epub 2019 May 23.

Max Planck Institute of Immunobiology and Epigenetics, Freiburg 79108, Germany. Electronic address:

How cells adapt metabolism to meet demands is an active area of interest across biology. Among a broad range of functions, the polyamine spermidine is needed to hypusinate the translation factor eukaryotic initiation factor 5A (eIF5A). We show here that hypusinated eIF5A (eIF5A) promotes the efficient expression of a subset of mitochondrial proteins involved in the TCA cycle and oxidative phosphorylation (OXPHOS). Several of these proteins have mitochondrial targeting sequences (MTSs) that in part confer an increased dependency on eIF5AH. In macrophages, metabolic switching between OXPHOS and glycolysis supports divergent functional fates stimulated by activation signals. In these cells, hypusination of eIF5A appears to be dynamically regulated after activation. Using in vivo and in vitro models, we show that acute inhibition of this pathway blunts OXPHOS-dependent alternative activation, while leaving aerobic glycolysis-dependent classical activation intact. These results might have implications for therapeutically controlling macrophage activation by targeting the polyamine-eIF5A-hypusine axis.
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http://dx.doi.org/10.1016/j.cmet.2019.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688828PMC
August 2019

Acetate Promotes T Cell Effector Function during Glucose Restriction.

Cell Rep 2019 05;27(7):2063-2074.e5

Max Planck Institute of Immunobiology and Epigenetics, Freiburg 79108, Germany. Electronic address:

Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8 T cells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Ex vivo acetate treatment increases IFN-γ production by exhausted T cells, whereas reducing ACSS expression in T cells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote T cell function during cancer.
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http://dx.doi.org/10.1016/j.celrep.2019.04.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544383PMC
May 2019

MicroRNA-155 Expression Is Enhanced by T-cell Receptor Stimulation Strength and Correlates with Improved Tumor Control in Melanoma.

Cancer Immunol Res 2019 06 1;7(6):1013-1024. Epub 2019 May 1.

Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland.

microRNAs are short noncoding RNAs that regulate protein expression posttranscriptionally. We previously showed that miR-155 promotes effector CD8 T-cell responses. However, little is known about the regulation of miR-155 expression. Here, we report that antigen affinity and dose determine miR-155 expression in CD8 T cells. In B16 tumors expressing a low-affinity antigen ligand, tumor-specific infiltrating CD8 T cells showed variable miR-155 expression, whereby high miR-155 expression was associated with more cytokine-producing cells and tumor control. Moreover, anti-PD-1 treatment led to both increased miR-155 expression and tumor control by specific CD8 T cells. In addition, miR-155 overexpression enhanced exhausted CD8 T-cell persistence in the LCMV cl13 chronic viral infection model. In agreement with these observations in mouse models, miR-155 expression in human effector memory CD8 T cells positively correlated with their frequencies in tumor-infiltrated lymph nodes of melanoma patients. Low miR-155 target gene signature in tumors was associated with prolonged overall survival in melanoma patients. Altogether, these results raise the possibility that high miR-155 expression in CD8 tumor-infiltrating T cells may be a surrogate marker of the relative potency of antigen-specific CD8 T-cell responses.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0504DOI Listing
June 2019

Leveraging TCR Affinity in Adoptive Immunotherapy against Shared Tumor/Self-Antigens.

Cancer Immunol Res 2019 01 27;7(1):40-49. Epub 2018 Nov 27.

Laboratory of Cellular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, California.

Adoptive cellular therapy (ACT) using T-cell receptor (TCR)-engineered lymphocytes holds promise for eradication of disseminated tumors but also an inherent risk of pathologic autoimmunity if targeted antigens or antigenic mimics are expressed by normal tissues. We evaluated whether modulating TCR affinity could allow CD8 T cells to control tumor outgrowth without inducing concomitant autoimmunity in a preclinical murine model of ACT. RIP-mOVA mice express a membrane-bound form of chicken ovalbumin (mOVA) as a self-antigen in kidney and pancreas. Such mice were implanted with OVA-expressing ID8 ovarian carcinoma cells and subsequently treated with CD8 T lymphocytes (CTL) expressing either a high-affinity (OT-I) or low-affinity (OT-3) OVA-specific TCR. The effects on tumor growth versus organ-specific autoimmunity were subsequently monitored. High-affinity OT-I CTLs underwent activation and proliferation in both tumor-draining and pancreatic lymph nodes, leading to both rapid eradication of ID8-OVA tumors and autoimmune diabetes in all treated mice. Remarkably, the low-affinity OT-3 T cells were activated only by tumor-derived antigen and mediated transient regression of ID8-OVA tumors without concomitant autoimmunity. The OT-3 cells eventually upregulated inhibitory receptors PD-1, TIM-3, and LAG-3 and became functionally unresponsive, however, allowing the tumors in treated mice to reestablish progressive growth. Antibody-mediated blockade of the inhibitory receptors prevented exhaustion and allowed tumor clearance, but these mice also developed autoimmune diabetes. The findings reveal that low-affinity TCRs can mediate tumor regression and that functional avidity can discriminate between tumor-derived and endogenous antigen, while highlighting the risks involved in immune-checkpoint blockade on endogenous self-reactive T cells.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793606PMC
January 2019

PD-1 Blockade Unleashes Effector Potential of Both High- and Low-Affinity Tumor-Infiltrating T Cells.

J Immunol 2018 07 6;201(2):792-803. Epub 2018 Jun 6.

Department of Fundamental Oncology, Faculty of Biology and Medicine, University of Lausanne, 1066 Epalinges, Switzerland; and

Antitumor T cell responses involve CD8 T cells with high affinity for mutated self-antigen and low affinity for nonmutated tumor-associated Ag. Because of the highly individual nature of nonsynonymous somatic mutations in tumors, however, immunotherapy relies often on an effective engagement of low-affinity T cells. In this study, we studied the role of T cell affinity during peripheral priming with single-peptide vaccines and during the effector phase in the tumor. To that end, we compared the antitumor responses after OVA (N4) peptide vaccination of CD8 T cells carrying TCRs with high (OT-1) and low (OT-3) avidity for the N4 peptide in B16.N4 tumor-bearing C57BL/6 mice. Additionally, we assessed the response of OT-1 cells to either high-affinity (B16.N4) or low-affinity (B16.T4) Ag-expressing tumors after high-affinity (N4) or low-affinity (T4) peptide vaccination. We noticed that although low-affinity tumor-specific T cells expand less than high-affinity T cells, they express lower levels of inhibitory receptors and produce more cytokines. Interestingly, tumor-infiltrating CD8 T cells show similar in vivo re-expansion capacity to their counterparts in secondary lymphoid organs when transferred to tumor-free hosts, suggesting that T cells in tumors may be rekindled upon relief of tumor immunosuppression. Moreover, our results show that αPD-1 treatment enhances tumor control of high- and low-affinity ligand-expressing tumors, suggesting that combination of high-affinity peripheral priming by altered peptide ligands and checkpoint blockade may enable tumor control upon low-affinity Ag recognition in the tumor.
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http://dx.doi.org/10.4049/jimmunol.1701644DOI Listing
July 2018

Expression of the DNA-Binding Factor TOX Promotes the Encephalitogenic Potential of Microbe-Induced Autoreactive CD8 T Cells.

Immunity 2018 05;48(5):937-950.e8

Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland; Division of Clinical Pathology, Geneva University Hospital, Geneva, Switzerland. Electronic address:

Infections are thought to trigger CD8 cytotoxic T lymphocyte (CTL) responses during autoimmunity. However, the transcriptional programs governing the tissue-destructive potential of CTLs remain poorly defined. In a model of central nervous system (CNS) inflammation, we found that infection with lymphocytic choriomeningitis virus (LCMV), but not Listeria monocytogenes (Lm), drove autoimmunity. The DNA-binding factor TOX was induced in CTLs during LCMV infection and was essential for their encephalitogenic properties, and its expression was inhibited by interleukin-12 during Lm infection. TOX repressed the activity of several transcription factors (including Id2, TCF-1, and Notch) that are known to drive CTL differentiation. TOX also reduced immune checkpoint sensitivity by restraining the expression of the inhibitory checkpoint receptor CD244 on the surface of CTLs, leading to increased CTL-mediated damage in the CNS. Our results identify TOX as a transcriptional regulator of tissue-destructive CTLs in autoimmunity, offering a potential mechanistic link to microbial triggers.
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http://dx.doi.org/10.1016/j.immuni.2018.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040915PMC
May 2018

Strong homeostatic TCR signals induce formation of self-tolerant virtual memory CD8 T cells.

EMBO J 2018 07 11;37(14). Epub 2018 May 11.

Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic

Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute 10-20% of all peripheral CD8 T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen-experienced memory T cells are incompletely understood. By analyzing T-cell receptor repertoires and using retrogenic monoclonal T-cell populations, we demonstrate that the virtual memory T-cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self-reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T cells. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity.
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http://dx.doi.org/10.15252/embj.201798518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6043851PMC
July 2018

Shp-2 Is Dispensable for Establishing T Cell Exhaustion and for PD-1 Signaling In Vivo.

Cell Rep 2018 04;23(1):39-49

Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland; Institute for Research in Biomedicine, Università della Svizzera italiana, 6500 Bellinzona, Switzerland. Electronic address:

In chronic infection and cancer, T cells acquire a dysfunctional state characterized by the expression of inhibitory receptors. In vitro studies implicated the phosphatase Shp-2 downstream of these receptors, including PD-1. However, whether Shp-2 is responsible in vivo for such dysfunctional responses remains elusive. To address this, we generated T cell-specific Shp-2-deficient mice. These mice did not show differences in controlling chronic viral infections. In this context, Shp-2-deleted CD8 T lymphocytes expanded moderately better but were less polyfunctional than control cells. Mice with Shp-2-deficient T cells also showed no significant improvement in controlling immunogenic tumors and responded similarly to controls to α-PD-1 treatment. We therefore showed that Shp-2 is dispensable in T cells for globally establishing exhaustion and for PD-1 signaling in vivo. These results reveal the existence of redundant mechanisms downstream of inhibitory receptors and represent the foundation for defining these relevant molecular events.
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http://dx.doi.org/10.1016/j.celrep.2018.03.026DOI Listing
April 2018

Transcription Factor IRF4 Promotes CD8 T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

Immunity 2017 12 12;47(6):1129-1141.e5. Epub 2017 Dec 12.

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; Department of Microbiology and Immunology, The University of Melbourne, Parkville, VIC 3010, Australia; The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia. Electronic address:

During chronic stimulation, CD8 T cells acquire an exhausted phenotype characterized by expression of inhibitory receptors, down-modulation of effector function, and metabolic impairments. T cell exhaustion protects from excessive immunopathology but limits clearance of virus-infected or tumor cells. We transcriptionally profiled antigen-specific T cells from mice infected with lymphocytic choriomeningitis virus strains that cause acute or chronic disease. T cell exhaustion during chronic infection was driven by high amounts of T cell receptor (TCR)-induced transcription factors IRF4, BATF, and NFATc1. These regulators promoted expression of inhibitory receptors, including PD-1, and mediated impaired cellular metabolism. Furthermore, they repressed the expression of TCF1, a transcription factor required for memory T cell differentiation. Reducing IRF4 expression restored the functional and metabolic properties of antigen-specific T cells and promoted memory-like T cell development. These findings indicate that IRF4 functions as a central node in a TCR-responsive transcriptional circuit that establishes and sustains T cell exhaustion during chronic infection.
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http://dx.doi.org/10.1016/j.immuni.2017.11.021DOI Listing
December 2017