Brain-resident memory CD8+ T cells: roles in protection and disease

Thesis

Reference

Brain-resident memory CD8+ T cells: roles in protection and disease

VINCENTI, Ilena

Abstract

Memory T cells (TM) are important components of the immunological memory. Resident memory T cells (TRM) are a subset of TM that reside long-term at sites of previous infection.

This thesis investigated the functioning of virus-induced TRM in the brain. Brain TRM (bTRM) proved to be potent and autonomous defenders of the brain against a viral re-infection by rapidly acquiring proliferative and cytotoxic programs as well as by producing cytokines.

Thereby, bTRM protected this important and vulnerable organ against otherwise fatal immunopathological disease. In contrast to this beneficial role of bTRM, virus-induced bTRM that recognized a neo-self-antigen presented by CNS-resident cells triggered a local inflammation and an autoimmune-like disease. Accordingly, inflamed brain areas of patients suffering from the human autoimmune disease Multiple Sclerosis contained high numbers of bTRM. This work therefore highlighted a potential double-edged role of bTRM. While virus-specific bTRM are potent immune defenders of the CNS against re-infection, self-reactive bTRM likely contribute to brain autoimmune diseases.

VINCENTI, Ilena. Brain-resident memory CD8+ T cells: roles in protection and disease. Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5294

DOI : 10.13097/archive-ouverte/unige:113169 URN : urn:nbn:ch:unige-1131694

Available at:

http://archive-ouverte.unige.ch/unige:113169

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE

Section de Médecine Fondamentale FACULTÉ DE MÉDECINE

Département de Pathologie et Immunologie Professeur Doron Merkler

Section de Chimie et Biochimie FACULTÉ DES SCIENCES

Département de Biochimie Professeur Thierry Soldati

Brain-Resident Memory CD8+ T Cells:

Roles In Protection And Disease

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biochimie

par

Ilena VINCENTI

de Lancy (GE)

Thèse N°5294

GENÈVE

Atelier ReproMail

Table of Contents

Acknowledgements ... V List of publications ... VII List of abbreviations ... IX

I. Abstract ... 1

1. Version française ... 1

2. English version ... 3

II. Introduction ... 5

1. CD8⁺ T cell responses ... 6

1.1 CD8⁺ T cell maturation ... 6

1.2 Antigen-recognition and priming of naive CD8⁺ T cells ... 7

1.3 Effector CD8⁺ T cell responses to acute infection ... 8

1.4 Long-lived memory CD8⁺ T cell subsets ... 9

2. Tissue-resident memory CD8⁺ T cells ... 11

2.1 TRM generation and maintenance ... 11

2.2 Role of TRM during reinfection ... 16

2.3 TRM in humans ... 17

3. The central nervous system ... 17

3.1 CNS structure ... 18

3.2 The CNS as an immune-privileged organ ... 20

3.3 Anti-viral immune response in the CNS ... 21

3.4 Multiple sclerosis: autoimmune disease of the CNS ... 22

4. Lymphocytic choriomeningitis virus ... 24

4.1 Virus origin ... 24

4.2 Virus structure ... 25

III. Aims of the thesis ... 27

IV. Results ... 29

1. Brain-resident memory T cells represent an autonomous cytotoxic barrier to viral infection ... 29

2. Brain-resident memory T cells drive brain inflammation after self-antigen recognition on astrocytes cell surface ... 51

2.1 Tamoxifen administration induces expression of LCMV glycoprotein (LCMV-GP) in astrocytes from GFAP-CreERT2xSt-GP mice ... 51

2.2 Expression of LCMV-GP in astrocytes activates LCMV-GP33-41–specific bTRM cells without the prerequisite of the inflammatory microenvironment associated with an infection ... 53

2.3 Bona-fide bTRM release IFN-γ and TNF-α pro-inflammatory cytokines and acquire cytotoxic and proliferative potential upon specific-antigen recognition on astrocytes ... 56

2.4 BTRM drive brain inflammation upon recognition of LCMV-GP33-41-expressing astrocytes ... 58

2.5 BTRM effector mechanisms upon recognition of LCMV-GP33-41-expressing astrocytes result in astrocyte death and trigger disease ... 59

II

2.6 CD8⁺ cells with a bTRM phenotype are found in human MS lesions and accumulate with disease

progression... 61

V. Discussion and perspectives ... 63

1. Generation of two subsets of non-recirculating TRM cells after acute viral infection in the CNS ... 63

2. BTRM maintenance within the CNS is mediated by homeostatic proliferation and cell longevity ... 64

3. BTRM protect the CNS during viral challenge ... 65

4. BTRM reactivation towards astrocytes-expressing antigen drives brain inflammation, without the prerequisite of the inflammatory microenvironment generally associated with viral infections ... 67

5. BTRM contribution in the pathogenesis of multiple sclerosis ... 69

6. TRM in chronic inflammatory diseases ... 71

VI. Final conclusions ... 75

VII. Methods... 77

1. Media and buffers... 77

2. Solutions and solvents ... 78

3. Reagents ... 79

3.1 Antibiotics and antivirals ... 79

3.2 Enzymes ... 79

3.3 Chemicals ... 79

3.4 Kits ... 79

3.5 Flow cytometry antibodies ... 80

3.6 Flow cytometry tetramers ... 80

3.7 Flow cytometry viable dye ... 81

3.8 Primary antibodies for histology ... 81

3.9 Secondary antibodies for histology ... 81

3.10 Histology reagents ... 82

3.11 Other reagents ... 82

3.12 Consumables ... 83

4. Cell culture and bacterial methods ... 84

4.1 Cell line and bacteria ... 84

4.2 Cell culture ... 84

5. DNA and RNA related methods... 84

5.1 Transformation of E. Coli ... 84

5.2 Bacteria colony culture and miniprep ... 85

5.3 Mouse genotyping ... 85

5.4 Quantitative real-time PCR ... 85

6. Mice ... 87

6.1 Mice strains ... 87

6.2 Intravenous injections ... 87

6.3 Intracranial injections ... 87

6.4 Intraperitoneal injections ... 87

6.5 Tamoxifen administration ... 87

6.6 Adoptive transfers ... 87

6.7 Rotarod ... 88

6.8 Antibody-mediated in vivo depletion ... 88

6.9 Intravascular staining of CD8⁺ T cells ... 88

7. Human tissue ... 89

8. Virus ... 89

8.1 Virus strains and viral infections ... 89

8.2 LCMV-NPN400S virus rescue ... 89

8.3 Virus propagation... 90

8.4 Focus Forming Assay (FFA) ... 91

9. Flow cytometric samples and staining procedures ... 92

9.1 Tissue samples ... 92

9.2 Flow cytometry staining procedure ... 93

10. Histology ... 94

10.1 Frozen tissues ... 94

10.2 HOPE-fixed tissues ... 94

10.3 PFA-fixed tissues ... 95

10.4 Human tissue ... 96

10.5 Histological staining procedure... 96

10.6 Histological quantification ... 96

11. Instruments and softwares ... 96

VIII. References ... 99

Acknowledgements

I would like to begin by acknowledging my supervisor, the Pr. Doron Merkler, for giving me the opportunity to work in his lab. I really appreciated his professional and personal integrity. He cultured an amazing environment for young scientists to explore and grow. I thank him for his time, respect and education.

I also sincerely thank Pr. Thierry Soldati for his help, Pr. Daniel Pinschewer for his collaboration and elaboration of experiments, and Pr. Stéphanie Hugues for her availability. I thank them all for being members of my thesis committee and for judging this work.

A special thanks goes to Pr. Karin Steinbach, my “mini-boss” how I was used to call her. I am very grateful for her excellent guidance. She has always been very helpful and supportive. She has been an example for my scientific development, and it is thanks to her that I became the young scientist I am today. It has been a sincere pleasure to work with her throughout my thesis, despite that I did not achieve her speed working skills ;) I also enjoyed all our discussions, both scientific and private ones, and I thank her for having been such a great guide during my first time in Germany ;)

I additionally would like to thank all members of Doron’s lab. Ingrid, I will always be grateful for your personal support. You were my daily sunshine, always in a good mood and always thinking positively. You helped immensely with experiments and were always willing to give a hand whenever I needed help. Nico, thanks for your good advices, our discussions contributed to my personal development. I also would like to thank you for making me laugh so much, your jokes and funny stories were making days easier. Kristof, thanks for sharing your deep understanding of the brain, for teaching me new techniques, and for teaching me how to score EAE ;). Karim, it was a pleasure to share my desk with you, I really appreciated our daily discussions. Mario, I don’t know how I would have managed this adventure without your informatics skills, thanks for being always available. Bogna, I enjoyed our occasional after-work drinks, thanks for your technical help and for our “how to become a good PhD” discussions. Thank you also for the last minute help to proofread my thesis. Giovanni, why do people like mountains?  Thank you for being the encyclopedia of the lab, it was a pleasure to test your knowledge days after days. Thank you also for making me taste real Sicilian Cannoli, they are delicious!

I also would like to thank my family, Mom and Dad, who always supported me during this journey.

You have been there, trying to remind me why I was there. I also would like to thank my good friends, Virgi, Caro, Deli, Day, Marie, Amel, the Moreira-team and the “Loisinois”, for being very supportive, and biochemists colleagues, Benjamin and Nicolas, for being part of this adventure.

Finally, a particular thanks to my fiancé, Andrea. Thanks for always supporting me, motivating me, helping me and also many thanks for your patience. You have been an exceptional support without whom this journey would not have been possible.

List of publications

K. Steinbach, I. Vincenti, D. Merkler. “Resident-memory T cells in tissue-restricted immune responses: for better or worse?” Frontiers in Immunology, 2018, 9

G. Di Liberto, S. Pantelyushin, M. Kreutzfeldt, N. Page, S. Musardo, R. Coras, K. Steinbach, I.

Vincenti, B. Klimek, T. Lingner, G. Salinas, N. Lin-Marq, O. Staszewski, M. J. Costa Jordão, I.

Wagner, K. Egervari, M. Mack, C. Bellone, I. Blümcke, M. Prinz, D. D. Pinschewer, and D. Merkler.

“Neurons under T Cell Attack Coordinate Phagocyte-Mediated Synaptic Stripping.” Cell, 2018, 175(2)

N. Page, B. Klimek, M. De Roo, K. Steinbach, H. Soldati, S. Lemeille, I. Wagner, M. Kreutzfeldt, G.

Di Liberto, I. Vincenti, T. Lingner, G. Salinas, W. Brück, M. Simons, R. Murr, J. Kaye, D. Zehn, D. D.

Pinschewer, and D. Merkler. “Expression of the DNA-Binding Factor TOX Promotes the Encephalitogenic Potential of Microbe-Induced Autoreactive CD8+T Cells.” Immunity, 2018, 48(5) I. Vincenti, K. Steinbach, M. Kreutzfeldt, N. Page, A. Muschaweckh, I. Wagner, I. Drexler, D.

Pinschewer, T. Korn, and D. Merkler. “Brain-resident memory T cells represent an autonomous cytotoxic barrier to viral infection.” J. Exp. Med., 2016, 213(8)

S. Emamzadah, L. Tropia, I. Vincenti, B. Falquet, and T. D. Halazonetis. “Reversal of the DNA- binding induced loop L1 conformational switch in an engineered human p53 protein.” J. Mol.

Biol., 2014, 426(4)

List of abbreviations

α-DG α-dystroglycan

APC antigen presenting cells

BBB blood-brain barrier

BCSFB blood-cerebrospinal fluid barrier

bTRM brain tissue-resident memory CD8⁺ T cells

CCR chemokine receptor

CMV cytomegalovirus

CNS central nervous system

CSF cerebrospinal fluid

CTL cytotoxic T lymphocytes

DAPI 4’,6-diamidino-2-phenylindole

DAMPs damage associated molecular patterns

DCs dendritic cells

dLN draining lymph node

EBV Epstein-Barr virus

ERV endogenous retrovirus

ERVS endogenous retrovirus sequences

Fas-L Fas-ligand

GAS interferon-γ-activated sequences GFAP glial fibrillary acidic protein GKO interferon-γ-knock-out

gMGI geometric mean fluorescence intensity

GP glycoprotein

GzmB granzyme B

HBV hepatitis B virus

HCV hepatitis C virus

HERVs human endogenous retrovirus HHV-6 human herpes virus 6

HIV human immunodeficiency virus

HLA human leukocyte locus

HSV herpes simplex virus

i.c. intra-cranially

IELs intra-epithelial lymphocytes

i.p. intra-peritoneally

IFN interferon

IFNAR interferon α/β receptor

IFN-γ interferon-γ

IFNGR IFN-γ-receptor

IGR intergenic region

X ǀ

IL interleukin

IRES internal ribosomal entry site IRFs interferon regulatory factors ISGs interferon-stimulated genes

KLRG1 killer cell lectin-like receptor subfamily G member 1 LAMP-1 CD107a, lysosomal-associated membrane protein 1

LASV Lassa virus

LCMV lymphocytic choriomeningitis virus

LCMV-GP lymphocytic choriomeningitis virus glycoprotein

LCMVwt lymphocytic choriomeningitis virus wildtype, strain Armstrong L-CTCL leukemic cutaneous T cell lymphoma

LNs lymph nodes

NLT non-lymphoid tissues

NK natural killer

MBP myelin basic protein

MF mycosis fungoides

MHC major histocompatibility complex

MHC-I major histocompatibility complex class I

MS multiple sclerosis

MVA modified vaccinia virus

NMO neuromyelitis optica

NP nucleoprotein

OVA ovalbumine

PAMPs pathogen-associated molecular patterns

PFA paraformaldehyde

PKO perforin knock-out

PRR pattern recognition receptors

PP-MS primary-progressive multiple sclerosis PR-MS primary-relapsing multiple sclerosis pStat5 phosphorylated Stat-5

rLCMV recombinant LCMV, VSV-GP

rLCMV-lsGP recombinant LCMV, VSV-GP and the leader sequence of LCMV-GP RR-MS relapsing-remitting multiple sclerosis

S1P1 sphingosine-1-phosphate receptor-1

SEP sclérose en plaque

SLECs short-lived effector cells SLOs secondary lymphoid organs

SNC système nerveux central

SP-MS secondary-progressive multiple sclerosis

RABV Rabies virus

T1D type-I diabetes

TCM central memory CD8⁺ T cells

TCR T cell receptor

TEM effector memory CD8⁺ T cells TFs transcription factors

TIL tumor-infiltrating lymphocytes TGF-β transforming growth factor-β TNF-α tumor-necrosis-factor-α

TM memory CD8⁺ T cells

TReg regulatory CD4⁺ T cells

TRM tissue-resident memory CD8⁺ T cells

UTR untranslated region

VSV vesicular stomatitis virus

VSV-G vesicular stomatitis virus glycoprotein

WNV West Nile virus

YFP yellow fluorescent protein

I. Abstract

1. Version française

Le système nerveux central (SNC) est un organe essentiel au fonctionnement de notre organisme.

Cet organe est constitué de neurones, des cellules post-mitotiques avec une faible capacité de régénération, et il est donc particulièrement vulnérable aux dommages tissulaires observés lors d’une réponse inflammatoire accompagnant les infections ou les maladies auto-immunes. La réponse immunitaire induite lors d’une infection avec des virus neurotropiques peut engendrer des méningites (inflammation de l’enveloppe cérébrale) ou des encéphalites (inflammation du parenchyme cérébral), pouvant parfois conduire à la mort de l’individu. À ce titre, les cellules T représentent un élément clé dans la lutte contre les infections et génèrent une mémoire immunologique qui persiste à long terme et qui favorise une protection accrue en cas de réinfection avec un pathogène structurellement associé à celui précédemment rencontré. En revanche, lorsque les cellules T s’attaquent à des éléments du soi exprimés dans le SNC, ces dernières peuvent participer à la pathogenèse de maladies auto-immunes, comme par exemple la sclérose en plaques (SEP).

Un élément clé de la mémoire immunologique est la génération de cellules T CD8⁺ mémoires appelée « tissue-resident » (TRM), qui ont la propriété de résider dans les tissus précédemment infectés. Au cours d’une réinfection avec un pathogène apparenté, la localisation des cellules TRM

principalement aux sites d’entrée des pathogènes leur permet une détection rapide. De plus, ces cellules alertent le système immunitaire de la présence d’un pathogène et accélèrent l’élimination de ce dernier, contribuant ainsi à la protection de l’hôte. Ces cellules ont été identifiées au cours des dix dernières années et certains aspects de leur biologie restaient méconnus lorsque j’ai initié ce projet.

La première partie de cette thèse a consisté en l’étude de l’activité des TRM lors d’une réinfection dans le cerveau. L’infection intra-crâniale (i.c.) par le virus naturel des rongeurs LCMV, le virus de la choriomeningite lymphocytaires, est un modèle murin couramment utilisé pour l’étude d’infections du SNC. L’infection transitoire du cerveau par un LCMV recombinant atténué génère une population de cellules TRM qui constituent une barrière de défense contre une nouvelle infection avec la souche sauvage de LCMV (LCMVwt). Après la reconnaissance d’antigènes dérivés de LCMV, les cellules TRM acquièrent une capacité effectrice se manifestant par une prolifération, une production de cytokines pro-inflammatoires et une forte activité cytotoxique.

De ce fait, les cellules TRM ont été capables d’éliminer les cellules infectées et ont protégé les souris de l’immunopathologie fatale faisant suite à la choriomeningite. Cette activité protectrice a pu s’effectuer indépendamment des lymphocytes T nouvellement recrutés à partir de la périphérie. Cette partie de mon projet m’a donc conduite à émettre l’hypothèse que les cellules TRM représentent une barrière antivirale autonome du cerveau.

Dans un second temps, j’ai analysé si des cellules TRM autoréactives peuvent induire une réponse autodestructive dans le cerveau en l’absence de l’inflammation généralement associée à une

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infection. Afin d’aborder cette question, nous avons généré un modèle expérimental murin d’inflammation cérébrale induite par les cellules TRM. Dans ce dernier, des cellules TRM

autoréactives ont été générés par une infection i.c. avec un LCMV recombinant atténué exprimant un nouvel antigène du soi. Nous avons observé que l’induction de l’expression de l’antigène du soi par les astrocytes, une population spécifique du SNC, stimule l’activité effectrice des cellules TRM au même titre que lors d’une réinfection virale. Dans ce modèle, l’activation des TRM s’est accompagnée d’un recrutement de cellules T CD4⁺ et de cellules B dans le SNC ainsi qu’une accumulation d’astrocytes apoptotiques associé à des performances locomotrices réduites. La notion selon laquelle les cellules TRM acquièrent des fonctions effectrices lors de la simple expression de leur antigène par les astrocytes a donc apporté de nouveaux éléments mécanistiques sur leur mode d’activation.

En résumé, ce travail de thèse m’a permis de mettre en évidence que la présence de cellules TRM

dans le cerveau peut être à double tranchant. Bien que les cellules TRM participant à l’élimination de pathogènes constituent une source de protection pour le SNC, j’ai au contraire observé que des cellules TRM autoréactives peuvent s’avérer être des acteurs potentiels dans la pathogenèse de certaines maladies autoimmunes. À ce titre, la présence de lymphocytes avec un phénotype proche des TRM dans des lésions cérébrales de patients atteints de SEP supporte l’idée selon laquelle ce sous-type de lymphocyte T participe à l’initiation/exacerbation des lésions cérébrales qui caractérisent la SEP.

2. English version

The central nervous system (CNS) is a vital organ that contains post-mitotic neurons with only limited regenerative capacity. Therefore, this compartment is particularly vulnerable to inflammation-induced tissue damage, as observed in infectious and inflammatory diseases.

Infection with neurotropic viruses and the resulting immune response can lead to severe diseases, such as meningitis (inflammation of the brain coverings) or encephalitis (inflammation of the brain parenchyma), which sometimes even leads to the death of the host. T cells are often essential to successfully combat such infections and provide enhanced protection against reinfection with the same or a closely related pathogen for years afterwards, a process known as immunological memory. On the contrary, T cells recognizing CNS self-antigens can become drivers of autoimmune diseases, such as in Multiple Sclerosis (MS).

An important element of immunological memory is the generation of non-recirculating tissue- resident memory CD8⁺ T cells (TRM) at sites of previous infection. Being localized primarily at pathogen entry sites, these cells rapidly detect a re-invading pathogen, alert the immune system of its presence and contribute to its accelerated clearance in the immunized host. Since TRM were only identified during the last decade, key aspects of TRM biology were still unknown when I initiated this project.

In the first part of this thesis, I aimed to understand the contribution of TRM in conferring protection towards a re-encountered pathogen in the brain. Intracranial (i.c.) infection with lymphocytic choriomeningitis virus (LCMV), a natural rodent pathogen that can also infect humans, is an established experimental model to investigate transient and fatal brain infections.

Brain infection with attenuated recombinant LCMV (rLCMV) was used to generate brain TRM

(bTRM), and their protective capacity was assessed during viral challenge with wildtype LCMV (LCMVwt). Upon recognition of LCMV antigen, bTRM rapidly acquired proliferative and cytotoxic programs and produced cytokines. By that, bTRM were able to eliminate infected cells and thereby protected from otherwise fatal immunopathological disease even without the help of recruited T cells. This part of my work identified bTRM as potent and autonomous anti-viral defenders of the brain.

In the second part of my thesis, I aimed to investigate whether self-reactive bTRM could drive an autoimmune-like disease in the CNS in the absence of the inflammation generally associated with an infection. In order to answer this question, we generated an experimental mouse model of bTRM-induced cerebral inflammation. In this model, self-specific bTRM were generated by an i.c.

infection with an attenuated LCMV expressing a neo-self antigen. The timely controlled expression of the cognate self-antigen in astrocytes, a CNS-restricted cell population, stimulated the effector activity of bTRM in a similar manner as during a viral reinfection. BTRM activation was associated with the recruitment of CD4⁺ T cells and B cells to the CNS as well as an accumulation of dying astrocytes and clinical symptoms of locomotor impairment. This bTRM-driven brain inflammation occurring following recognition of the cognate antigen on astrocytes without the prerequisite of a preexisting local inflammation opens new insights into the requirements of bTRM

activation.

4 ǀ

Altogether, my thesis describes bTRM as a double-edged sword. While virus-specific bTRM are potent immune defenders of the CNS, I observed that self-reactive bTRM could be involved in the pathogenesis of brain autoimmune diseases. In line with this, I could demonstrate the presence of bTRM-like cells in brain samples of human MS patients, supporting the notion than bTRM

contribute to the initiation/exacerbation of cerebral lesions characteristic for MS.

II. Introduction

Our body is constantly exposed to numerous potentially deleterious microbes. Every day, the immune system fights against pathogens, in most cases in a clinically silent manner. A particular feature of our immune system is to remember intruders for years after the primary infection has been cleared. This enhanced protection is mediated by the generation of long-lived memory T cells and antibodies that remain in the host for years. Immunological memory is characterized by a more rapid and a more efficient response to a reencountered pathogen. A common application of this memory is vaccination, where administration of inactivated or attenuated pathogens, or simply their immunogenic components, results in the development of antibodies and more rarely of pathogen-specific memory T cells. Vaccines have been essential not only to protect vaccinated individuals, but also to limit the spread of a pathogen in a population, the most notable example being the eradication of the infectious smallpox virus from Europe¹. Today, most used vaccines induce the production of pathogen-specific antibodies. Unfortunately, the attempts to design effective vaccines against pathogens such as herpes simplex virus (HSV) or human immunodeficiency virus (HIV) have failed. For this reason, there is a growing effort in generating vaccines that favor the generation of memory T cells. Thus it became of particular interest to elucidate the mechanisms that regulate the generation of the different CD8⁺ T cell memory subsets, their maintenance as well as their contribution in conferring protection. Importantly, understanding the T cell biology of the recently identified tissue-resident memory T cells (TRM), a potent form of local immune memory T cells that reside within peripheral tissues and operate as first line of defense against frequently invading pathogens, is of huge interest.

Although pathogen-specific memory T cells can play a beneficial role in protecting individuals from recurrent infections, they can also become detrimental if they recognize and target self-structures, such as autoreactive T cells do in autoimmune diseases. More than 80 autoimmune diseases have been identified, few examples being rheumatoid arthritis, systemic lupus erythematous and multiple sclerosis (MS). In MS, immune cells infiltrate the central nervous system (CNS) and autoreactive T cells probably target the myelin sheath that insulates neurons. Perturbed neuronal transmission and accumulating neurodegeneration are the consequences, and can cause severe disability. Animal models have played a crucial role in elucidating key features of MS and in suggesting pharmacological interventions, yet the mechanisms of disease initiation and progression remain incompletely understood.

The brain is a particular structure that is anatomically separated from the rest of the body and is very vulnerable to infection, immunopathology and autoimmune diseases since its regenerative capacity is very limited. A better understanding of immune reactions in the brain and in particular the behavior of cytotoxic TRM in protection and disease of the CNS is thus of fundamental implications.

6 ǀ

1. CD8

T cell responses

The first prerequisite for successful immune defense against an invading pathogen is its detection.

Our innate immune system ensures immune-surveillance and homeostasis via expression of germ- line encoded pattern recognition receptors (PRR) that sense conserved molecular structures known as pathogen-associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). The sensing of foreign antigens by the innate immune system is essential to limit the initial spread of the infection and to alert the adaptive immune system. The adaptive immune system relies on genetically-remodeled and educated receptors that display specific and directed responses against distinct infectious pathogens. An important arm of the adaptive immune system is the CD8⁺ T cell, a key player in the cytotoxic elimination of cells infected with intracellular pathogens such as viruses.

1.1 CD8

T cell maturation

Naïve T cells arise from bone marrow–derived precursors and undergo selection in the thymus, where T cell precursors mature and differentiate. Thymic maturation initiates by somatic recombination of genes coding for the T cell receptor (TCR) and the expression of TCR co-receptors (CD3, CD4, CD8). In a next step, CD4⁺CD8⁺ double-positive T cell precursors undergo positive selection, in which their TCR needs to recognize self-antigens in the form of a complex consisting of a short peptide (typically 8-11 amino acids) bound to major histocompatibility complex (MHC) molecules with low-affinity, which serves as an indication that their TCR is principally functional. It is during this step that decision of keeping the CD4 or CD8 co-receptor is taken, depending on which one is engaged in TCR recognition. T cell maturation continues with a negative selection, in which T cell clones that recognize self-antigen-MHC complexes on antigen presenting cells (APC) with high affinity and that could potentially give rise to autoreactive T cell response are eliminated. This process of purging almost all strongly autoreactive T cells from the repertoire is called “central tolerance”. The thymic maturation produces both CD4⁺ and CD8⁺ naïve T cells with a large TCR repertoire, ideally responding to foreign-antigen-MHC complexes with high affinity, but not to self- antigen-MHC complexes. However, central tolerance is incomplete and some T cells might also recognize to some extend self-structures. Autoreactive T cells arise either because they are positively selected by recognizing self-antigen-MHC complexes or/and because not all self-antigens are expressed in the thymus. When mature, naïve T cells exit the thymus and circulate through the blood to lymph nodes (LNs) and the spleen, the so-called “secondary lymphoid organs” (SLOs), where they screen for an encounter with their cognate antigen. Naïve CD8⁺ T cells are long-lived and their survival and self-renewal is dependent on exogenous signals under the form of cytokines.

The key homeostatic cytokines that are crucial for naïve T cells are interleukin (IL)-7 and IL-15. The size of the naïve T cell pool is thus determined by the availability of these cytokines.

1.2 Antigen-recognition and priming of naive CD8

T cells

All nucleated cells digest intracellularly expressed antigens in the proteasome and present them as short peptides in complex with MHC class I (MHC-I) molecules on the cell surface. During an infection in non-lymphoid tissues (NLT), activation of pattern recognition receptors (PRR), which sense conserved molecular structures including pathogen-associated molecular pattern (PAMPs), on dendritic cells (DCs) leads to their maturation and migration to the draining lymph nodes (dLN).

Free soluble antigens can either directly access to local SLOs via lymphatic vessels or reach the bloodstream and enter the spleen but, in both cases, specialized macrophages in SLOs capture them^2,3. Mature DCs and macrophages are professional APC that present processed antigens to naive T cells in SLOs, in which they localize along the stromal cell network^4,5. Activated professional APC produce chemoattractant molecules that guide naïve T cells migration in SLOs and promote T cell-APC contacts, increasing the chance that a naïve T cell encounters its cognate antigen during an infection⁶. During antigen recognition, both TCR affinity for the peptide-MHC-I complex as well as levels of antigen presented by the APC control the contact duration between the T cell and the APC and strongly influence TCR signaling and T cell activation⁶. Upon activation, professional APC also express co-stimulatory molecules, such as CD80, CD86 and CD40L that interact with CD28, CTLA-4 and CD40, respectively, on the surface of T lymphocytes. The involvement of co-stimulatory molecules increases the strength of the interaction between the peptide-MHC-I complex on the APC and the TCR during cognate antigen recognition, allowing naïve CD8⁺ T cells to receive the necessary activating signals. In order to be fully activated, a naïve T cell requires to recognize its cognate antigen with high-affinity on an APC, or, to undergo multiple intermediate-affinity interactions and accumulate sufficient activation signals to reach the activation threshold⁶.

In order to favor long-lasting interaction with APC, CD8⁺ T cells transiently express CD69 early after activation. CD69 induces internalization of the egress receptor sphingosine-1-phosphate receptor- 1 (S1P1) from the cell surface, favoring CD8⁺ T cell retention within SLOs by preventing CD8⁺ T cells from exiting by following a sphingosine-1-phosphate gradient^7,8. Additionally, professional APC produce inflammatory cytokines, such as type-I interferons (IFN) and IL-12. Furthermore, depending on the infected tissue they egressed from, DC–derived signals induce expression of specific cell surface adhesion molecules and receptors on activated CD8⁺ T cells that confer the ability to leave SLOs and migrate preferentially to the infected tissue⁹. Although the precise signals are not yet completely discovered, this tissue-specific imprinting for migration occurs for a variety of tissues, if not all. For example, during brain infection, priming of effector CD8⁺ T cells induces expression of brain-homing receptors such as the α4β1 integrin (VLA-4), that allows them to specifically cross the endothelium of brain vessels¹⁰.

Activation of self-reactive T cells is prevented by several mechanisms, which together are called

"peripheral tolerance". First, without an infection, APC do not mature and do not express co- stimulatory signals. As a result, self-reactive T cells do not get activated and undergo a state of long- term repression of TCR signaling that is called anergy¹¹. Then, lymph node stromal cells express peripheral-tissue-restricted antigens and can induce the deletion of self-reactive T cells, probably via expression of inhibitory receptors¹². Additionally, so-called regulatory T cells (TReg) can suppress activated autoreactive T cells¹³.

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Thus, efficient priming of naïve CD8⁺ T cells requires the combination of three signals: recognition of the cognate antigen by the TCR (signal 1), co-stimulation by co-stimulatory molecules (signal 2) and cytokine stimulation (signal 3). If these three signals are provided, they act together to prime CD8⁺ T cells. CD8⁺ T cell priming initiates a cellular reprogramming that results in rapid cell proliferation and differentiation in a heterogeneous population of fully functional effector CD8⁺ T cells with acquisition of migratory capacities and effector functions (Figure 1). During acquisition of an effector phenotype, effector cells downregulate the chemokine receptor (CCR)-7 as well as L- selectin (CD62L)¹⁴, two homing molecules necessary for trafficking within SLOs, thereby facilitating their exit from SLOs and migration to infected site.

Figure 1. CD8⁺ T cell immune response to acute infection. Priming occurs in secondary lymphoid organs (SLOs), where naïve CD8⁺ T cells encounter mature professional antigen-presenting cells (APC) displaying their cognate antigen as a complex bound to MHC-I surface molecules. In the presence of co-stimulation and inflammatory cytokines, an efficient immune response is mounted and naïve CD8⁺ T cells undergo clonal expansion and differentiation, giving rise to a heterogeneous population of short-lived effector cells (SLECs) and memory precursors. At the same time, professional APC instruct CD8⁺ T cells to express a specific pattern of adhesion molecules and receptors in order to migrate into the inflamed tissue. SLECs secrete effector cytokines (IFN-γ, TNF-α, IL-2) and cytotoxic molecules (granzymes, perforins) resulting in viral clearance. Thereafter, there is a contraction phase where the majority of effector cells die by apoptosis.

A small subset of memory precursors survives and generates long-lived memory CD8⁺ T cells that further differentiate into central memory T cells (TCM), effector memory T cells (TEM) and tissue-resident memory T cells (TRM), each subset having particular migratory, proliferative and effector potential. These long-lived memory CD8⁺ T cells can provide for years protective immunity to reinfection.

1.3 Effector CD8

T cell responses to acute infection

An efficient CD8⁺ T cell priming gives rise to various subsets of effector CD8⁺ T cells. The hallmark of effector cells is their ability to induce the death (apoptosis) of infected cells, which they can accomplish by various effector mechanisms¹⁵. For this reason, they are often referred to as cytotoxic T lymphocytes (CTL). Once in the infected tissue, effector cells can mediate cytolytic and non- cytolytic virus clearance. Effector T cells that recognize their cognate antigen presented on MHC-I on an infected cell can release cytotoxic granules. The directed degranulation of so called perforins towards the infected cell permeabilizes its membrane and the subsequent secretion of granzymes

leads to apoptosis-mediated cell death. In addition, effector cells express Fas-ligand (Fas-L) that induces receptor-mediated apoptosis in cells expressing the death receptor Fas. Furthermore, effector CD8⁺ T cells produce pro-inflammatory cytokines such as interferon-γ (IFN-γ) and tumor- necrosis-factor-α (TNF-α). Both cytokines exert their effect by acting on a large spectrum of cells expressing appropriate receptors, in which they induce proteins that inhibit viral replication, the production of other cytokines and under certain circumstances even cell death¹⁶.

After resolution of the infection, the presence of highly cytotoxic effector cells within the peripheral tissue is not required anymore. Most effector CD8⁺ T cells are terminally-differentiated cells with limited pro-survival and proliferative capacity (so-called short-lived effector cells, SLECs), which undergo apoptosis after antigen clearance, leaving behind few surviving cells (Figure 1). These remaining cells are memory precursors, which further differentiate into long-lived memory cells¹⁷, probably giving rise to all long-lived CD8⁺ T cell subsets (Figure 1). SLECs and memory precursor CD8⁺ T cells can be distinguished by differential expression of the surface molecules killer cell lectin-like receptor subfamily G member 1 (KLRG1) and the α-chain of IL-7 survival factor receptor (IL-7Rα, CD127). SLECs are characterized by their KLRG1⁺CD127^- phenotype¹⁸. KLRG1 expression is associated with a terminally differentiated state and the lack of CD127 expression is in part responsible for SLECs short-lived phenotype, since they are not sensitive to IL-7–derived survival signals. In contrary to SLECs, memory precursors are characterized by a KLRG1^-CD127⁺ phenotype¹⁸, allowing memory precursors survival, differentiation, and memory CD8⁺ T cells homeostasis¹⁹. Importantly, memory precursors cells express the IL-2Rβ (CD122), shared by the IL-2R and IL-15R¹⁹. The cytokine IL-2 is involved in memory cell proliferation¹⁹. IL-15 is involved in the maintenance of memory precursor CD8⁺ T cells, since CD8⁺ memory precursors adoptively transferred in IL-15 deficient mice fail to survive¹⁹. Furthermore, it was shown that IL-7 and IL-15-dependent longevity was maintaining memory T cells by homeostatic proliferation through Stat5 signaling^20,21. Activation of Stat5 probably mediates memory precursors and memory cell survival via expression of anti- apoptotic molecules such as Bcl-2¹⁹.

The regulation of effector versus memory CD8⁺ T cell differentiation is controlled by various transcription factors (TFs) such as Eomes, Bcl-6, T-bet and Blimp-1²². High expression of T-bet and Blimp-1 expression rather give rise to terminally differentiated effector cells, while Eomes and Bcl- 6 expression in early effectors favor memory CD8⁺ T cell differentiation²². During an acute infection, the expression of these TFs is in part modulated via transient signaling through the inhibitory receptor PD-1 early after CD8⁺ T cell activation, likely by directly interfering with the extent of TCR signaling²³. SLECs are important to successfully terminate an acute infection and the generation of long-lived memory CD8⁺ T cells provides long-term protection against reinfection.

1.4 Long-lived memory CD8

T cell subsets

Upon reinfection, the presence of long-lived memory cells results in a faster and a more efficient immune response. For more than one decade, CD8⁺ memory T cells were divided into two subsets depending of their migratory capacities²⁴. These populations were termed central memory (TCM) and effector memory (TEM) T cells. While TCM mainly locate to SLOs, TEM patrol NLT (Figure 1).

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Over the last 15 years, it has been discovered that an additional population of memory CD8⁺ T cell persists at site of previously resolved infection^25,26, indicating that the composition of the memory pool is more complex than originally thought. This memory subset is referred to as tissue-resident memory CD8⁺ T cells (TRM) (Figure 1), and is a critical player involved in the early defense against a secondary infection.

1.1.1. Central memory CD8⁺ T cells (TCM)

TCM express the chemokine receptor CCR7 as well as L-selectin (CD62L), which are homing molecules necessary to extravasate via high endothelial venules to enter SLOs^14,24. Therefore, TCM are mainly localized in lymphoid tissues and have a reduced circulating capacity. Upon stimulation, TCM do not exert immediate effector functions but rapidly proliferate and give rise to new effector T cells²⁴. As such, TCM produce and secrete high amounts of IL-2¹⁴, a cytokine promoting T cell proliferation and differentiation. In fact, TCM have been considered the T memory "stem" cells, since they can give rise to effectors and TEM upon reactivation²⁷, thereby conferring high protection for clearance of intracellular pathogens.

1.1.2. Effector memory CD8⁺ T cells

TEM do not express CCR7 and CD62L molecules and are thus mostly excluded from lymphoid tissues²⁸. Instead, they express homing receptors for migration and trafficking into NLT²⁴ and are thus predominantly detected in peripheral compartments²⁹. TEM constantly recirculate through the blood and peripheral tissues, which is their predominant characteristic³⁰.

During reactivation by their cognate antigen, TEM can exert immediate effector function²⁴ leading to fast pathogen control. This rapid activation limits pathogen spread during the time a new effector T cell pool is generated by TCM in SLOs.

1.1.3. Tissue-resident memory CD8⁺ T cells

In 2001, David Masopust described for the first time that CD62L^- memory CD8⁺ T cells that populate various NLT after resolution of a viral or bacterial systemic infection exhibit higher cytotoxic activity and faster cytokine production ex vivo compared to splenic CD62L^- TEM29. This was the first indication that memory cells residing in peripheral tissues were functionally distinct from those located within SLOs and that they might constitute a different pool of memory T cells. It is in 2009 that graft experiments highlighted that CD62L^- memory cells found in the skin after herpes simplex virus (HSV) infection were not in equilibrium with the peripheral circulating memory T cells (TCM and TEM) and instead were exclusively residing within this tissue²⁵. Parabiosis experiments provided further evidence that this memory subset was not recirculating, since, in contrast to peripheral TCM and TEM

memory cell subsets, these cells did not equilibrate between surgically joined mice that shared a common blood circulation^26,31. This distinctive non-migratory phenotype and their stable persistence without input from circulating T cells gave rise to their name “tissue-resident” memory CD8⁺ T cells (TRM).

2. Tissue-resident memory CD8

T cells

Since their initial description within the skin, TRM have been reported in many different tissues after clearance of numerous infectious agents. Long-lived TRM have been observed in the gut after mucosal infection where they developed as non-migratory intra-epithelial lymphocyte (IELs)²⁶. The generation of TRM cells was also described following brain infection with vesicular stomatitis virus (VSV)³². Furthermore, TRM were described in other tissues such as salivary glands, female reproductive tract, stomach, kidney, pancreas, heart, lung, tonsils, thymus and liver^33–37. Interestingly, these cells were also observed at entry points of SLOs³⁸, indicating that dLNs might be part of the localized immune protection related to TRM.

The positioning of this specialized memory T cell population at sites of previous infection has been associated with an accelerated and more efficient immune response in case of a reinfection when compared to hosts that contained only TEM and TCM. In particular, TRM presence in confined sites was shown to play a critical role in the host defense against local reinfection by immediately reducing the dissemination of the re-invading pathogen25,31,33,39,40.

2.1 T

generation and maintenance

2.1.1 TRM origin and transcriptional signature

TRM generation initiates at early stages of infection, when effector cells migrate into inflamed tissues and receive local signals that program TRM differentiation. Indeed, effector T cells administrated intra-tracheally into infected lung airways adopt a non-migratory phenotype that gives rise to TRM

cells⁴¹. TRM mostly arise from CD127⁺KLRG1^- memory precursor cells, similarly to peripheral memory T cells25,26,37,42. However, a recent study indicated that early CD127⁺KLRG1^- TRM precursors within NLT are transcriptionally distinct from memory precursors cells from SLOs that give rise to TCM

cells⁴³. This suggests that local stimuli in NLT trigger gene expression modifications in TRM precursor cells already early on. While the precise nature and the source of these signals are not fully elucidated, it is well accepted that the requirements for TRM differentiation are organ-specific and vary between different NLT. Gene array analyses of TRM generated in the skin, the gut, the lung, the brain and the kidney by diverse inflammatory contexts revealed that TRM from different tissues share a core transcriptional signature, although expression of some survival factors and effector- associated genes are also differentially expressed^37,43–45. These analyses also confirmed that despite originating from similar precursor cells, TRM are a distinct memory subset and have a unique differentiation program when compared to circulating memory populations. A list of selected transcripts defining the core signature of TRM is shown in Table 1.

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Gene Product Expression

Bcl-6 TF associated with memory fate high

Blimp-1 TF associated with terminally differentiated effector fate high CD36 Lipid scavenger receptor (internalization of exogenous lipids) high CD44 C-lectin containing glycoprotein (hyaluronic acid binding) high CD69 Transmembrane C-type lectin protein (antagonizes S1P1) high

CD244 CD244 natural cell killer cell receptor 2B4 high

Cdh-1 Cadherin-1 high

Ctla4 Cytotoxic T lymphocyte-associated protein 4 high

E-Cdh E-cadherin high

GzmB Granzyme B (cytotoxic molecule) high

Hobit Homolog of BLIMP-1 in T cells; TF involved in tissue retention high

Icos Inducible T cell costimulator high

Irf4 TF associated with terminally differentiated effector fate high Itgae Integrin αE, CD103 (integrin αEβ7; epithelium associated) high Itga1 Integrin α1; CD49a (integrin α1β1; VLA-1) high ItgaL Integrin αL; CD11a (integrin αLβ2; LFA-1) high Litaf Lipopolysaccharide-induced tumor-necrosis factor high Lpl Lipoprotein lipase (internalization of triglycerides) high Notch TF associated with terminally differentiated effector fate high

Nr4a1 Orphan nuclear receptor TF; Nur77 high

Rgs1+Rgs2 Regulator of G protein signaling 1 and 2 high

Runx3 TF involved in tissue retention high

Eomes TF associated with memory fate low

Klf2 Krüppel-like factor 2; TF involved in tissue egress low Klre1 Killer cell lectin-like receptor family E member 1 low

S1pr1 Sphingosine 1-phosphate receptor 1 low

S1pr5 Sphingosine 1-phosphate receptor 5 low

T-bet TF associated with terminally differentiated effector fate low

Tcf-1 TF associated with memory fate low

Table 1. Part of TRM core-signature in the skin, gut, lung, brain and kidney. Gene transcripts upregulated (high expression) or downregulated (low expression) in TRM isolated from the skin, gut, lung, brain and kidney in comparison to TCM and TEM cells isolated from the spleen.

Interestingly, TRM cells utilize a differentiation program involving (TFs) associated with both effector and memory cell formation^22,45. For example, TRM establishment requires Blimp-1 expression but is repressed by high levels of T-bet, two TFs promoting terminally differentiated effector cell fate in circulating T cells. Similarly, Nr4a1 is necessary for TRM formation while Eomes suppresses it, although both TFs are necessary for TCM differentiation. TRM cells also phenotypically resemble a mixture of effector and memory cells among various NLT. TRM express typical markers related to effector functions, such as granzyme-B and IFN-γ as well as inhibitory molecules^43,46,47 (CD244, CTLA- 4, CD101, PD-1, Tim-3, Lag3 and Icos), likely to prevent aberrant activation. Additionally, TRM cells generally retain high proliferative capacity, similarly to TCM.

2.1.2 TRM retention and maintenance

TRM in different tissues are characterized by their surface expression of CD69 and sometimes combined expression of the integrin αE chain (CD103), that forms the integrin αEβ725,26,32,33. By contrast, other memory subsets express low levels of CD103 and CD69. CD103 and CD69 expression are regulated by the TFs Runx3 and Hobit⁴⁵ (Figure 2). In addition, these two TFs have been implicated in promoting TRM tissue-residency by inducing other adhesion molecules expression and by inhibiting tissue egress⁴⁵. The expression of CD69 on TRM is considered to antagonize the tissue egress receptor S1P1, inhibiting tissue egress already early on during TRM differentiation⁸. Further, TRM in multiple tissues downregulate the TF KLF2, which in turn induces downregulation of S1P1, CD62L and CCR7, a requirement for long-term TRM tissue retention^37,44,48 (Figure 2). CD103 expression is thought to anchor TRM within the epithelium of epithelial tissues via binding to E- cadherin^25,49. Indeed, CD103-deficient cells fail to localize to the epithelium although no defect in the initial tissue migration is observed^34,37. Thus, localization of CD103⁺ TRM within barrier tissues likely occurs in distinct sites with an apparent preference for epithelial layers. Besides CD103, other adhesion molecules are also involved in TRM retention within tissues. TRM from various NLT express variable levels of integrin α1 (CD49a), forming the α1β1 integrin heterodimer (VLA-1) that binds to extracellular matrix components such as collagens and that is also involved in migrating along the collagen^25,33,50. Additionally, TRM from diverse tissues often express E-cadherin and/or integrin αLβ2

(LFA-1)^33,37. The combination of adhesion molecules expressed by a particular TRM subset is probably involved in its location within that tissue and its specific retention.

In addition to the retention and inhibition of tissue egress, the establishment and maintenance of a stable population of bona fide non-migratory TRM also requires their long-term survival and self- renewal within tissue. In order to be maintained independently from circulating cells, TRM need to have access to local survival signals present in their environment. However, each tissue has a distinct microenvironment, due to differential structures, cell populations and expression of local factors.

Thus, the long-term maintenance and distribution of TRM in different tissues are influenced by the availability of these local survival signals and by the localization of the cells producing them.

Thereby, TRM in various tissues preferentially localize in tissue-specific microenvironmental niches⁵³, revealing an adaptation to signals of their tissue of residence. Accordingly, TRM from different tissues express variable expression of markers associated with anti-apoptotic signaling, such as Bcl-2, and homeostatic proliferation, such as Ki-67 and CD127 (IL-7Rα).

The maintenance of TRM independently from the circulation is described for TRM from various tissues, with the exception of lung TRM that are sustained by recruitment of TEM and their conversion to TRM51. Additionally, the lung TRM population wane over time⁵¹, a decline that can be delayed by repetitive antigen stimulations, which prolonged circulating TEM survival and their conversion to lung TRM52.

2.1.3 TRM differentiation signals

Signals driving TRM differentiation are just starting to emerge. Transforming growth factor-β (TGF-β) signaling is involved in the upregulation of CD103 (Figure 2), since cells lacking the TGF-β-receptor II failed to express CD103 and to be retained within the skin and the gut epithelium^37,49. In vitro, T

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cell treatment with TNF-α and IL-33 synergized with TGF-β to induce CD103 expression³⁴. CD4 T cells have been shown to help CD8⁺ T cells to localize to the lung epithelium in order to receive TGF-β signals⁵⁴. Additionally, T-bet and Eomes downregulation is a prerequisite for CD103 expression since they both repress TGFbRII expression and, moreover, T-bet also directly represses gene transcription at the CD103 promoter^54,55 (Figure 2). While TGF-β signaling is implicated in the differentiation of CD103⁺ TRM cells, the factors involved in the differentiation of CD103^- TRM are not known.

It was recently shown that the presence of CD4⁺ T cells in the brain during TRM differentiation is required to induce a long-term tissue retention without replenishment from the periphery during persistent CNS infection⁵⁶, suggesting that CD4⁺ T cells produce key signals involved in TRM

acquisition of homeostatic program in this infection context. However, the requirement of CD4⁺ T cell help for TRM acquisition of a self-renewal program has never been studied following acute brain infection.

The signals involved in TRM differentiation vary depending on the tissues in which these cells develop, revealing TRM adaptation to their local microenvironment. The most striking example is the dependence on local antigen presentation. Local TCR stimulation is necessary to induce CD103 expression within the brain and the dorsal root ganglia^25,32,34, suggesting that local antigen stimulation is required for the formation of CD103⁺ TRM cells within neural tissues. By contrast, CD103⁺ TRM from epithelial tissues can be generated without the prerequisite for local antigen encounter^34,39.

Another divergent signal for TRM differentiation and maintenance among different tissues is the requirement for IL-15. TRM expression of CD122 (the IL-15 receptor) varies among different tissues^37,38, indicating that the dependency of IL-15 as a surviving signal also varies between different TRM subsets. Additionally, cell-intrinsic mTOR signaling modulates TRM generation in the gut⁵⁷, revealing a potential implication of mTOR TF in TRM differentiation.

Altogether, this indicates that CD127⁺KLRG1- memory precursor cells receive organ-specific signals directly in the infected-tissue, affecting their differentiation towards TRM cells. Moreover, TRM

maintenance programs similarly depend on specific tissue microenvironment signals. Since mediators of TRM regulation diverge among tissues, the description of a common pathway for TRM

generation and retention is difficult to draw. A scheme with key upregulated and downregulated factors involved in TRM regulation in various NLT is represented in Figure 2.