VU Research Portal

VU Research Portal

Innate Lymphoid Cells in Human Peripheral Blood and Secondary Lymphoid Organs

Bar-Ephraim, J.E.

2017

document version

Publisher's PDF, also known as Version of record

Link to publication in VU Research Portal

citation for published version (APA)

Bar-Ephraim, J. E. (2017). Innate Lymphoid Cells in Human Peripheral Blood and Secondary Lymphoid Organs.

General rights

Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain

• You may freely distribute the URL identifying the publication in the public portal ?

Take down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

E-mail address:

Chapter 1

General Introduction

ēęėĔĉĚĈęĎĔē

Ž–Š‘—‰Š–Š‡ϐ‹”•–‡„‡”‘ˆ–Š‡ˆƒ‹Ž›™ƒ•ƒŽ”‡ƒ†›†‡•…”‹„‡†‹–Š‡ͳͻ͹Ͳ•1_,

‹–™ƒ•‘Ž›•‹…‡–Š‡Žƒ–‡ͻͲ•ƒ†ϐ‹”•–†‡…ƒ†‡‘ˆ–Š‡ʹͳst _{century that it became}

clear that the classical Natural Killer (NK) cells can be seen as members of a larger family of tissue resident cytokine producing innate lymphoid cells (ILCs).

As the name of the family suggests, ILCs are cells of lymphoid origin. Like B and T cells ILCs develop from the common lymphoid precursor (CLP). In contrast to adaptive lymphocytes, ILCs lack a rearranged antigen receptor and their development is independent of expression of recombinase activated gene (RAG) 1 and 22_{. ILCs depend on the transcription factors GATA3}3_{, TOX}4_,

inhibitor of DNA binding (Id)25 _{and at least in part on NFIL3}6,7_{. ILCs are}

ˆ—”–Š‡”†‡ϐ‹‡†ƒ•…‡ŽŽ•Žƒ…‹‰ƒŽŽ‘™Ž‹‡ƒ‰‡ƒ”‡”•ǡ™Š‹Ž‡‡š’”‡••‹‰ –Š‡ƒŽ’Šƒ…Šƒ‹‘ˆ–Š‡Ǧ͹”‡…‡’–‘”ȋǦ͹”Ƚǡͳʹ͹Ȍ2,8,9_.

In recent years, ILCs have been shown to reside mainly in epithelial tissues where they contribute to tissue integrity and homeostasis9,10_{. During an}

‹ϐŽƒƒ–‘”› ”‡•’‘•‡ǡ • Šƒ˜‡ „‡‡ •Š‘™ –‘ ”‡•’‘† “—‹…Ž› „› ’”‘†—…‹‰ ’”‘Ǧ‹ϐŽƒƒ–‘”› …›–‘‹‡•ǡ –Š‡”‡„› ƒ••‹•–‹‰ –Š‡ ‹‹–‹ƒ–‹‘ ‘ˆ ƒ ‹—‡ ”‡•’‘•‡ ƒ‰ƒ‹•– ‹˜ƒ†‹‰ ’ƒ–Š‘‰‡•Ǥ ˆ–‡” –Š‡ ‹ϐŽƒƒ–‹‘ resolves, ILCs can then contribute to tissue repair following injury and tissue damage11_{. While ILCs are crucially involved in the formation of secondary}

lymphoid organs (SLOs)12,13 _{and still present within adult SLOs}14-17_{, not much}

1

ĘĚćĘĊęĘĎēĒĎĈĊĆēĉčĚĒĆēĘ

Within the ILC family a subdivision in family members is made in analogy to –Š‡™‡ŽŽǦ‘™…‡ŽŽ…Žƒ••‹ϐ‹…ƒ–‹‘Ǥ……‘”†‹‰–‘–Š‹•…Žƒ••‹ϐ‹…ƒ–‹‘•…Š‡‡ǡ NK cells can be seen as the innate counterpart of cytotoxic CD8+ _{T cells, while}

–Š‡ ‘–Š‡”  •—„•‡–• …ƒ „‡ †‡ϐ‹‡† ƒ• Š‡Ž’‡” Ž‹‡ ‹ƒ–‡ Ž›’Š‘‹† …‡ŽŽ• (helper ILC) harboring three subsets based on expression and dependence of transcription factors as well as the cytokines secreted by the cells8_.

ILC1

Type I ILCs (ILC1) are dependent on the transcription factor T-bet (Tbx21) ƒ†•‡…”‡–‡ƒ‹Ž›‹–‡”ˆ‡”‘ȋ Ȍɀ‹”‡•’‘•‡–‘•–‹—Žƒ–‹‘„›‹–‡”Ž‡—‹ (IL) 12, IL-15 and IL-18, in both mouse and man18,19_{. ILC1 can be found within}

the intestinal lamina propria in both mouse and man, while their numbers ™‡”‡ˆ‘—†–‘„‡‹…”‡ƒ•‡†‹’ƒ–‹‡–••—ˆˆ‡”‹‰ˆ”‘‹ϐŽƒƒ–‘”›„‘™‡Ž disease (IBD)20_.

ILC2

‹‡Šʹ…‡ŽŽ•ǡ–›’‡•ȋʹȌƒ”‡†‡’‡†‡–ˆ‘”•—„•‡–†‡ϐ‹‹–‹‘‘–Š‡ transcription factor GATA321,22 _{and, once activated via cytokine receptors,}

secrete type II cytokines in both mouse and man (mainly IL-5 and IL-13, although IL-4, IL-6 and IL-9 production has also been reported)23-27_{. Apart}

from GATA3, ILC2 also depend on the expression of the transcription factor ‡–‹‘‹… ƒ…‹† ”‡…‡’–‘”Ǧ”‡Žƒ–‡† ”’Šƒ ‡…‡’–‘” ƒŽ’Šƒ ȋȽȌǡ ϐ‹ͳǡ ƒ† TCF-128-30_{. Cytokines that can induce type II cytokine production are mainly}

IL-25 (IL-17E), IL-33 and thymic stromal lymphopoitin (TSLP) in mice26,31,32

and humans23 _{and prostaglandin D2 in humans}33_{. ILC2 can be found in}

homeostasis in skin34_{, mucosal tissues (i.e. lung}32,35-38 _{and gastrointestinal}

tract26,39_{), in adipose tissue}25,40,41 _{and to some extent in lymphoid organs}

(e.g. spleen and mLN)24,42,43_{. ILC2 have been shown to play an important}

role in resistance to parasitic helminths and nematodes. In contrast to this ’”‘–‡…–‹˜‡ ”‘Ž‡ǡ ʹ …ƒ …‘–”‹„—–‡ –‘ ƒ ‹ϐŽƒƒ–‘”› ”‡•’‘•‡ †—”‹‰ allergic diseases23,26,35,37,42,44-46_{Ǥ‡•‹†‡•–Š‡‹”’”‘Ǧ‹ϐŽƒƒ–‘”›ƒ…–‹‘•ǡ–Š‡›}

can also play a role in tissue regeneration after injury, as has been shown ‹‹ˆ‡…–‹‘‘†‡Ž•ˆ‘”‹ϐŽ—‡œƒ–Š”‘—‰Š’”‘†—…–‹‘‘ˆƒ’Š‹”‡‰—Ž‹47_{. In}

addition, ILC2 have been shown to play a role in beiging of white adipose tissue, thereby limiting obesity48_.

ILC3

and the aryl hydrocarbon receptor (AHR)8,9_{. Depending on the stimulus,}

these cells express Th17 associated cytokines (mainly IL-22, IL-17A and granulocyte/macrophage colony stimulating factor (GM-CSF, CSF2)) 14,15,49-59_{ǡƒŽ–Š‘—‰Šƒ’‘’—Žƒ–‹‘‘ˆɀ–}+ _{ILCs also expresses T-bet and produces}

 ɀ60_{. In general, a number of subsets of ILC3 can be distinguished based}

on their expression of the chemokine receptor CCR6 and natural cytotoxicity receptors (NCRs)9 _{and a subset of intestinal CCR6}- _{ILC3 in mice and humans}

‡š’”‡••‡• ’Ͷ͸ —†‡” ‹ϐŽ—‡…‡ ‘ˆ –Š‡ –”ƒ•…”‹’–‹‘ ˆƒ…–‘” Ǧ„‡–60-62_.

Human ILC3 can additionally express the NCRs NKp3017 _{and/or NKp44, of}

which the latter coincides with IL-22 expression17,56_.

Tissue resident CCR6+ _{ILC3 (NCR}+ _{and NCR}-_{) are continuously present in a}

number of epithelial tissues, where they maintain epithelial integrity and regulate mucosal immunity via secretion of IL-2263,64_{. Additionally, ILC3}

produce GM-CSF, which sustains intestinal phagocytes50 _{and marginal}

zone (MZ) B cell function in the spleen indirectly, via the effect of GM-CSF on neutrophil activity52_{. Besides this indirect effect on the function of MZ B}

cells, ILC3 can also directly stimulate MZ B cells via secretion of BAFF, and membrane expression of CD40L and Notch ligand Delta-like 1 (DLL1)52_.

Š”‘—‰Š–Š‡‹”’”‘†—…–‹‘‘ˆ•‘Ž—„Ž‡Ž›’Š‘–‘š‹ȋȌȽ₃ƒ†Ƚ₁Ⱦ₂ ILC3 can additionally regulate IgA production by intestinal B cells65_.

Finally, like ILC2, ILC3 play a role in tissue repair after injury, such as in the case of regeneration of splenic white pulp following infection with murine lymphocytic choriomeningitis virus (MLCV)66_{, restoration of intestinal}

epithelium upon damage (e.g. colitis, chemotherapy or graft versus host disease (GvHD))64,67,68 _{and thymic regeneration in mice following sublethal}

total body irradiation69_.

‹…‡ŽŽ•

The CCR6+_{…‡ŽŽ•ƒŽ•‘‹…Ž—†‡–Š‡ϐ‹”•–ɀ–†‡’‡†‡–’‘’—Žƒ–‹‘–‘„‡}

described, the subset of lymphoid tissue inducer (LTi) cells16,70_{. In mice this}

subset can be further divided into two populations based on the expression of CD471_{. As the name already suggests, LTi cells are key players in the formation}

of lymph nodes (LN) and Peyer’s Patches (PP), although development of other secondary lymphoid tissue (e.g. white pulp of the spleen) can occur in an LTi independent manner (see below,12,13,72,73_{). Apart from their lymphoid}

1

ėĊĈĚėĘĔėĘęĔ

All ILCs develop from a common lymphoid precursor (CLP) in the bone marrow. For mice it was shown that all helper ILC subsets are derived from the common helper-like ILC precursor (CHILP), characterized as Lin

-Id2+_CD127+_CD25-_ȽͶȾ͹+ 75 _{(see Fig. 1). Although thought to belong to the}

subset of type I ILCs8_{, classical NK cells are only partially dependent on}

Id276 _{and T-bet}77,78 _{for their development, yet completely dependent on the}

transcription factor eomesodermin (EOMES)78_{. Accordingly, NK cells seem}

to stem from an early Cxcr6+ _{ILC precursor (ILC/NK precursor), which}

can further develop into all types of helper like ILCs, including LTi cells79_.

Downstream from this NK/ILC precursor, the differentiation of NK cells seems to split off from the rest of the ILC family75,80_{. Although ILC1 cluster}

closer to classical NK cells than to ILC2 or ILC3 based on transcriptome analysis81_{ǡ…‡ŽŽ•ƒ†ͳ†‘‘–•Šƒ”‡ƒ—‹“—‡…‘‘’”‡…—”•‘”}75,80_{. It}

–Š—•ƒ›„‡“—‡•–‹‘‡†™Š‡–Š‡”–Š‡•‡…‡ŽŽ–›’‡•„‡Ž‘‰–‘–Š‡•ƒ‡•—„•‡–Ǥ Downstream of the CHILP precursor, both ILC1 and ILC3 have been shown –‘”‡Ž›‘–Š‡–”ƒ•…”‹’–‹‘ˆƒ…–‘”—š͵ƒ†‹–•†‘™•–”‡ƒ–ƒ”‰‡– ǦȾ for differentiation and survival, while ILC2 are not82_{. This may lead to}

hypothesize that ILC1 and ILC3 derive from the same precursor, downstream ‘ˆǡ™Š‹Ž‡ʹǡ™Š‹…Šƒ”‡’”‡•‡–‹„‘–Š—š͵ƒ† ǦȾ‘…‘—– mice, may have a distinct precursor downstream of CHILP (Fig. 1).

†‡‡†ǡƒ†‡ϐ‹‡†’”‡…—”•‘”™ƒ•ˆ‘—†ˆ‘”ʹȋʹ’”‘‰‡‹–‘”ǡʹȌǡ„—– not for ILC1 and ILC322_{. Furthermore, ILC2 depend for their development on}

Bcl11b, as in Bcl11b-/- _{mice ILC2 lost their ILC2 phenotype and upregulated}

ILC3 related genes83_{. Reversely, retinoic acid (RA)-mediated signaling was}

shown to block ILC2 differentiation while allowing ILC3 formation84_{. As ILC2}

have been shown to have different migration and transcription patterns than ILC1 and ILC381,85_{ǡʹ…ƒ„‡†‡ϐ‹‡†ƒ•ƒ•—„•‡–†‹•–‹…–ˆ”‘ͳ}

and ILC3 cells. However, under certain circumstances plasticity between the different types of ILCs is feasible, similar as what has been shown for CD4+ _{T cells}86_{. In line with this, it has been demonstrated that a subset of}

ͳ†‡˜‡Ž‘’•ˆ”‘‘”ɀ–+_{͵—’‘Ž‘••‘ˆ‘”ɀ–‡š’”‡••‹‘™Š‹Ž‡Ǧ„‡–}

expression is being induced. This upregulation of T-bet coincided with ‹…”‡ƒ•‡† ‡š’”‡••‹‘ ‘ˆ ’Ͷ͸ ƒ†  ɀ •‡…”‡–‹‘ ‹ ‹…‡ǡ ™Š‹Ž‡ ‹ –Š‡ absence of T-bet ILC3 mainly produce IL-2260-62_{. Also in humans, ILC3 were}

•Š‘™ –‘ †‹ˆˆ‡”‡–‹ƒ–‡ ‹–‘ ͳ —†‡” ‹ϐŽ—‡…‡ ‘ˆ Ǧͳʹ ™Š‹Ž‡ ”‡˜‡”•‡Ž›ǡ ͳ …ƒ †‡˜‡Ž‘’ ‹–‘ ͵ —†‡” ‹ϐŽ—‡…‡ ‘ˆ ǦͳȾ ƒ† Ǧʹ͵ ‹ …‘…‡”– with RA87_.

1

PLZF+ _{ILC precursor, which gives rise to ILC1, -2, and -3 (see Fig. 1)}71,80_{. In}

agreement, LTi cells have been found to have their own gene transcription signature, including higher expression of ”’ͷ and CD25, in comparison with other ILC381_.

Collectively, helper ILCs are a heterogeneous, possibly plastic subset of cells that participate in maintaining the integrity of barrier tissues during Š‘‡‘•–ƒ•‹•ǡ™Š‹Ž‡”‡•’‘†‹‰“—‹…Ž›†—”‹‰ƒ‹ˆ‡…–‹‘‹„‘–Š„ƒ””‹‡” tissues and lymphoid tissues.

ĘĆėĊĎēĘęėĚĒĊēęĆđęĔęčĊĉĊěĊđĔĕĒĊēęĔċĘĔĒĊǡćĚęēĔę

ĆđđĘ

One of the hallmarks of the mammalian immune system is the presence of specialized locations throughout the body at which immune activation can readily take place. These SLOs are organized in a way in which activation of –Š‡ƒ†ƒ’–‹˜‡„”ƒ…Š‘ˆ–Š‡‹—‡•›•–‡‹•‘•–‡ˆϐ‹…‹‡–12_{. In steady state}

in mice, LNs have additionally been shown to be an important site where the induction of immune tolerance takes place via the presentation of peripheral tissue antigens (PTA) to both CD4+ _{and CD8}+ _{T cells in the absence of}

co-stimulation88-94_{. The group of SLOs encompasses the spleen, peripheral and}

mesenteric LNs and a variety of mucosal associated lymphoid tissues (MALT, i.e. Peyer’s patches (PP) in the small intestine, colonic patches in the colon, cryptopatches and isolated lymphoid follicles (ILFs) in both the colon and the small intestine, nasal associated lymphoid tissue (NALT) in the nasal passages, tonsils and adenoids in the nasopharynx and tear duct associated lymphoid tissue (TALT)). In addition, fat-associated lymphoid clusters (FALC) in the mesentery and the milky spots in the omentum are examples of fat-associated lymphoid tissue. Although these different tissues are captured under the same name of SLOs and have comparable architecture with separated B

Figure 1. Development of innate lymphoid cells. All ILCs develop from the common

Ž›’Š‘‹† ’”‡…—”•‘” ȋȌ ‹ –Š‡ „‘‡ ƒ””‘™Ǥ š’”‡••‹‘ ‘ˆ ϐ‹Ž͵ ƒ† ‘š Ž‡ƒ† –‘ –Š‡ †‡˜‡Ž‘’‡– ‘ˆ –Š‡ Ȁ …‡ŽŽ ’”‡…—”•‘” ƒ† Ž‘•• ‘ˆ  ƒ†  …‡ŽŽ ’‘–‡–‹ƒŽǤ —„•‡“—‡– Dx5 and Eomes expression lead to development of cNK cells, while Id2 and Gata3 lead to †‹ˆˆ‡”‡–‹ƒ–‹‘‘ˆ–Š‡ȽͶȾ͹+ _{CHILP. Helper like ILCs develop further upon expression of PLZF,}

while LTi cells branch off from the rest of the hILC lineages at this point in their development. Further increase in Gata3 expression leads to the development of the ILC2 precursor (ILC2P) ƒ†•—„•‡“—‡–Ž›ʹǡ™Š‹Ž‡ͳƒ†͵†‡˜‡Ž‘’†‹”‡…–Ž›ˆ”‘–Š‡—’‘‡š’”‡••‹‘ ‘ˆǦ„‡–ƒ†‘”ɀ–”‡•’‡…–‹˜‡Ž›Ǥ•ͳƒ†͵ƒ”‡„‘–Š†‡’‡†‡–‘–Š‡‡š’”‡••‹‘‘ˆ Runx3 it can be hypothesized that there is a separate Runx3+ _{ILC1/3 precursor, although no}

and T cell areas, their development is not necessarily directed by the same cellular signaling pathways. For instance, the splenic white pulp, LNs and PP develop during embryogenesis95_{, while TALT and NALT develop after birth}96,97_.

Additionally, cryptopatches also develop after birth98_{, while their maturation}

into ILFs within the small intestines in addition depends on colonization by (commensal) microbes99-101_{. In the colon, development of colonic patches starts}

during embryonic development and is completed after birth, while cryptopatch maturation is entirely post-natal and independent of colonization102_.

As there is a variation in the timeframe in which the various lymphoid organs develop, the hematopoietic cells that can be marked as inducer cells are not ƒŽ™ƒ›•‘”ɀ–+ _{LTi cells. LTi cells are crucial for the development of LNs (both}

mesenteric and peripheral), PP, colonic patches and cryptopatches70,73,103_.

Accordingly, mice which lack LTi cells, such as Id2-/-5_{ƒ†‘”ɀ–}-/- _mice73_{, are}

devoid of these SLOs. Inversely, mice which have increased LTi numbers (e.g. mice over expressing IL-7) also have more SLOs104,105_{. However, the}

splenic white pulp, NALT, TALT, milky spots and FALC develop normally in ‘”ɀ–-/- _{mice, which lack LTi cells}73,96,97,106_{. Although independent of LTi cells}

for their formation, NALT and FALC do not develop in Id2-/- _mice25,97_{, which}

implies a role for other types of ILCs, possibly ILC2, in the formation of these tissues. Altogether, with the exception of the splenic white pulp, all SLOs which develop before birth are dependent on the presence of LTi cells. SLOs which develop after birth, with the exception of cryptopatches, seem to be independent of the presence of LTi cells, although other ILC subsets may play a role in this process.

ĘĆėĊĘęĎđđĕėĊĘĊēęĎēĆĉĚđęĘ

As ILCs are located not only in mucosal tissues and developing SLOs, but also ‹ƒ–—”‡•‹„‘–Š‹…‡ƒ†‹Š—ƒ•ȋ•‡‡ƒ„Ž‡ͳȌǡ–Š‡“—‡•–‹‘…ƒ „‡”ƒ‹•‡†™Šƒ–”‘Ž‡–Š‡•‡…‡ŽŽ•ˆ—Žϐ‹ŽŽƒ––Š‡•‡Ž‘…ƒ–‹‘•Ǥ•–Š‡‹”’”‡•‡…‡‹ developing SLOs is necessary for the development of these organs, ILCs and ‘”‡•’‡…‹ϐ‹…ƒŽŽ›‹…‡ŽŽ•…‘—Ž†„‡…‘•‹†‡”‡†‘„•‘Ž‡–‡‹ƒˆ—ŽŽ›ˆ‘”‡† unless they play an additional role. Moreover, the presence of ILCs in spleen, which can develop independent of ILCs, further supports a role for ILCs in the immune response in adult SLOs. Importantly, as for adaptive lymphocytes, the conditions within a LN are favorable for ILC residence. As such, there are a number of indications that show that SLO stroma is important for proper ILC function. For instance, podoplanin+ _{splenic stroma was shown to}

1

ƒ „Ž‡ͳǣ ˆ—…–‹‘‹Š—ƒƒ†‘—•‡ • A bbri viations: pLN- peripher al lymphnode, mLN- m esent eric ly mph node, PP - Pa ye r’s pat ches, IL C- innat e lymphoid cell, IFN- int erf er on, IL- int er leukin, GM-C SF - gr anulocyt e macr ophage colon y stimulat ing fa ct or , B A FF - , APRIL- , L T- lymphot ox in, TNF - t umor necr os is fact or, M Z- mar

ginal zone, CD- clust

er of diff

er

entiation, MR

C- mar

ginal r

eticular cell, SCS- subcapsular sinus, IgA

ILC numbers are increased, suggesting that competition for the favorable LN niche restricts ILC numbers in WT animals due to the presence of adaptive lymphocytes109_{. Similar as for T cells}110_{, IL-7 was shown to enhance ILC}

survival and/or proliferation104,105_{. Moreover, IL-7 was shown to support LTi}

ˆ—…–‹‘‹‘–‘‰‡›˜‹ƒ–Š‡‹†—…–‹‘‘ˆȽ₁Ⱦ₂ expression111,112_{. The}

IL-7-‡†‹ƒ–‡†‹†—…–‹‘‘ˆȽ₁Ⱦ₂ might also be relevant in the adult organism when regeneration of lymphoid tissue is needed, such as seen during the recovery period after an LCMV infection66_{. Upon resolution of the infection,}

stromal cells enhance production of IL-7113_{. This can then lead to induced}

expression of LT on tissue resident ILCs, which in turn can further activate stroma. This may then result, in analogy to SLO formation during ontogeny, in restoration of lost lymphoid tissue. Altogether, these observations imply that IL-7 is not only involved in ILC survival and proliferation, but also in ILC †‹ˆˆ‡”‡–‹ƒ–‹‘ƒ†•—„•‡“—‡–Ž›‹ϐŽ—‡…‡•–Š‡‹””‘Ž‡‹ƒ†—Ž–Ž›’Š‘‹†–‹••—‡Ǥ ‹…‡†ʹƒ†‘”ɀ–†‡ϐ‹…‹‡–ƒ‹ƒŽ•†‘‘–†‡˜‡Ž‘’•ǡ‹–‹•…ŠƒŽŽ‡‰‹‰ to study the role of ILCs (or at least ILC3) in matured SLOs. However, as the development and organization of the white pulp of the spleen is independent of the presence of LTi cells114_{, data obtained using this organ can lead to more}

insights in the function of ILCs in adult SLOs.

đĔĈĆđĎğĆęĎĔēĜĎęčĎēĘ

During murine SLO development, a change in the localization of LTi cells can be observed. In newborn murine pLN, LTi cells are present throughout –Š‡Ǥ‹–Š‹–Š‡ϐ‹”•–™‡‡ƒˆ–‡”„‹”–Š†‹•–‹…–Ǧƒ†…‡ŽŽœ‘‡•ˆ‘” and LTi cells start to localize to the cortical area of the LN, in the vicinity of the newly forming B cell follicles16,70_{. Finally, in adult mice, LTi cells are}

present in close association with stromal marginal reticular cells (MRCs), in between the B cell follicles108,115_{. This change in the anatomic positioning}

of ILCs in the highly organized environment of the SLO during development •—‰‰‡•–•–Šƒ––Š‡›ƒŽ•‘ƒ›ˆ—Žϐ‹ŽŽƒ†‹ˆˆ‡”‡–ˆ—…–‹‘‹ƒ†—Ž–•Ǥ†‡‡† ILC1, present within the interfollicular region of LNs, were shown to assist subcapsular macrophages in killing of bacteria that enter via the lymphatics „›’”‘†—…–‹‘‘ˆ ɀ116_{. Additionally, localization of ILC3s in the splenic}

marginal zone (MZ) in both mice and humans was shown to be important for sustaining IgA production by MZ B cells. As mentioned before, this occurs both directly, via BAFF/APRIL, CD40 and/or DLL signaling to the MZ B cells, or indirectly via GM-CSF dependent maintenance of MZ B cell helper neutrophils which in their turn sustain the MZ B cells52_.

1

change in expression pattern of a number of markers, e.g. NKp44, NKp46,

͵ͲǡͶͲƒ†Ƚ₁Ⱦ₂17,114,117_{. Interestingly, LTi cells derived from spleens}

of adult Rag-/- _{mice were shown to induce Vcam}+ _{clusters in cxcr5}-/- _mice,

which may represent ILF anlagen105_{. This implies that adult SLO-derived}

ILC3 can still act as „‘ƒǦϔ‹†‡ LTi cells. In a different setting, LTi cells derived from mLN of neonatal but not adult mice were found to induce ectopic SLOs in newborn and adult mice118_{. This seeming discrepancy can be explained by}

the fact that in the latter experiments whole mLN cell suspensions, containing relatively high numbers of LTi cells, were injected in the recipients, while LTi form a minor population in adult LN. It is thus possible that the difference „‡–™‡‡–Š‡ƒ†—Ž–ƒ†‡‘ƒ–ƒŽ†‘‘”•‹•ƒ”‡ϐŽ‡…–‹‘‘ˆ–Š‡Ž‘™‡”—„‡”• of LTi cells injected when adult mLNs were used, although a difference in activation state of LTi cells could have additionally contributed to these results. Strikingly, only SLOs induced in neonatal, but not in adult mice were properly organized, although organization was restored upon immune activation in the adult mice118_{. These data imply that not only the LTi cells,}

but also the environment in which they act differ between adult and neonatal mice resulting in differences in cellular behavior.

ĘċĆĈĎđĎęĆęĊĆĉĆĕęĎěĊĎĒĒĚēĊėĊĘĕĔēĘĊĘ

Across the literature, various roles for ILCs in adult SLOs have been suggested, mainly restoring lymphoid tissue integrity after damage and regulating the adaptive immune response in a number of ways. Importantly, ILC1, 2 and 3 are all present in most adult LN and in the spleen, albeit in different proportions14_.

ILCs, mostly ILC3, have been implicated in initiation and support of both T cell dependent and T cell independent antibody production by B cells in the intestine, spleen and LNs via LT, CD40, BAFF/APRIL and/or DLL signaling65,119_{. In agreement, adult (splenic) ILC3 have been shown to sustain}

CD4+ _{T cell memory, which also affects germinal center reactions and}

•—„•‡“—‡– ƒ–‹„‘†› ’”‘†—…–‹‘ǡ ˜‹ƒ ͶͲ ƒ† ͵Ͳ117,120,121_{. It can be}

“—‡•–‹‘‡† ™Š‡–Š‡” –Š‡ •—‰‰‡•–‡† Ž‹ –‘ ‡‹–Š‡” ͶͲ ‘” ͵Ͳ ‹• –Š‡ only mechanism by which LTi cells mediate this, since CD8+ _{T cell memory}

is not supported in vivo121_{, while OX40 and CD30 are both expressed by}

activated CD8+ _{T cells. It thus seems that additional factors are involved in}

ILC-mediated support of CD4+ _{T lymphocytes in SLOs.}

The distinct effect that ILCs have on CD8+ _{and CD4}+ _{T cells might point to}

a role for class II MHC (MHC-II). A number of groups have already shown that ILCs in neonatal LNs and adult spleens, LNs and intestines express this molecule and present antigen to CD4+ _{T cells, although to a lesser extent}

ILC2 was reported to be crucial for a proper Th2 response against a worm challenge and in case of allergies43,124_{, mLN-derived MHC-II expressing ILC3s}

were shown to be important in inducing tolerance to commensal bacteria in steady state123_{ǤŠ‹•‹•†‘‡„›•‡“—‡•–‡”‹‰Š‹‰Šƒ‘—–•‘ˆǦʹǡ–Š—•}

depriving activated T cells of essential growth factors in combination with lack of co-stimulation via CD28123,125_{Ǥ ‘˜‡”•‡Ž›ǡ —†‡” ‹ϐŽƒƒ–‘”›}

conditions (i.e.•–‹—Žƒ–‹‘™‹–ŠǦͳȾȌǡ•’Ž‡‹…ǡ„—–‘–Žƒ‹ƒ’”‘’”‹ƒȋȌ ILC3 were shown to up-regulate MHC-II and co-stimulatory molecules and support T cell activation and proliferation122_{. This implies that ILCs from}

different tissues may react differently under similar conditions. This is in agreement with recent microarray data, in which it was shown that ILC1 ˆ”‘ †‹ˆˆ‡”‡– –‹••—‡• Šƒ† ƒ –‹••—‡ •’‡…‹ϐ‹… ‰‡‡ –”ƒ•…”‹’–‹‘ •‹‰ƒ–—”‡81_.

Importantly, at least for ILC2 – T cell interaction, a mutual relationship has been shown to exist between the two cell types, as T cell-derived IL-2 ‡Šƒ…‡•  ˆ—…–‹‘ —’‘ ‹ϐŽƒƒ–‹‘43_{. This again accentuates the}

complex relationship between the different cells of the immune system.

ĎĈėĔǦĊēěĎėĔēĒĊēęĆđĈĔēęėĔđĔċǦĈĊđđĎēęĊėĆĈęĎĔē

The discrepancy in outcome of T cell stimulation between ILC3s derived from the intestines123 _{or from the spleen}122 _{points to a potential}

microenvironmental control of ILC function. While intestinal and mLN-†‡”‹˜‡†͵•‡‡–‘„‡ƒ–‹Ǧ‹ϐŽƒƒ–‘”›ƒ–•–‡ƒ†›•–ƒ–‡123,125_{, splenic ILC3}

…ƒ„‡…‘‡’”‘Ǧ‹ϐŽƒƒ–‘”›‘…‡–”‹‰‰‡”‡†„›ͳE from APC122_{. As the}

immune system should not be triggered by the commensal microbiota in the intestine while bacterial products in the spleen should induce a strong immune response, these functional differences at the various anatomical locations are understandable for proper immune defense. However, the presence of helminth or worm products should in turn trigger a strong type II immune response. In line with this, at steady state mLN and LP resident ILC3 are immunosuppressive123_{, while ILC2 are immuno-stimulatory once}

triggered by either IL-25 or IL-33, which are secreted by the epithelium once an infection with parasites is detected43_{. It is however not clear what factors}

1

ĘĆĘėĊČĚđĆęĔėĘĔċċĚēĈęĎĔē

Indeed, DCs have been shown to be important in regulating cytokine production „›•ǤǦʹ͵ƒ†ǦͳȾ†‡”‹˜‡†ˆ”‘•™ƒ••Š‘™–‘‹†—…‡–Š‡’”‘†—…–‹‘ of IL-22 by ILCs in the intestine in case of an intestinal barrier breach, which occurs during infection with ‹–”‘„ƒ…–‡” ”‘†‡–‹—129,130_{. Furthermore,}

epithelium-derived IL-33 and IL-25 suppress the production of IL-22 and lead to the production of type II cytokines by ILC2s67_{. Likewise, DC-derived IL-23}

blocks IL-33 signaling in Tregs in the murine intestine131 _{and this might very}

well be the case for ILCs. Also in humans, it was shown that IL-12 can drive ͵–‘ƒͳ’Š‡‘–›’‡™Š‹Ž‡Ǧʹ͵ƒ†ǦͳȾ‡†‹ƒ–‡–Š‡‘’’‘•‹–‡‡ˆˆ‡…–87

(Fig. 2A). Thus, microenvironmental factors will mediate the differentiation of different ILC populations, either towards suppressive (i.e. presenting antigen without co-stimulation123_{Ȍ‘”’”‘Ǧ‹ϐŽƒƒ–‘”›•—„•‡–•}43_{, thereby driving}

ILC plasticity (Fig. 2B). In line with this hypothesis, it was recently shown that the ratios between the ILC subtypes (ILC1, 2 and 3) differ between mLN and pLN in mice, again pointing to the microenvironmental factors (mucosal draining versus non-mucosal draining) as instrumental in ILC differentiation and plasticity. It will be interesting to see whether ILCs derived from different LN (mLN, cLN or pLN) interact differently with T cells upon stimulation, as splenic ILCs do seem to differ from mLN or LP ILCs122,123,125_.

An interesting factor that can play a role here, especially in the MALT, is RA. RA is produced by a subset of intestinal DCs, stromal cells, and by the intestinal ‡’‹–Š‡Ž‹—‹–•‡Žˆƒ†‹•—„‹“—‹–‘—•Ž›’”‡•‡–‹–Š‡Ǥ’ƒ”–ˆ”‘„‡‹‰ important in the initiation of lymphoid organ formation132,133_{, RA signaling has}

been shown to play a role in shaping the balance between differentiation of ILCs towards either a type I, II or III phenotype84,87,133,134_{. By directly binding to the}

‘”ɀ–’”‘‘–‘”133,135 _{RA induces differentiation towards a type III phenotype}

in both mice and humans87,134_{, while its absence is hallmarked by the presence}

of more ILC2 in mice84_{. In agreement, ILC3 are enriched for transcripts of RA}

target genes in comparison to ILC1 and ILC281_{. The increase in ILC2 in absence}

‘ˆ‹‰Š–„‡‹†‹”‡…–Ž›…ƒ—•‡†„›•ǡƒ•˜‹–ƒ‹†‡ϐ‹…‹‡…›‹‹…‡Šƒ• been shown to lead to an increase in Th2 skewing of naïve T cells by mLN resident DCs136_{. Considering the similarities between T cell and ILC skewing}

…›–‘‹‡•‹–‹•–‡’–‹‰–‘Š›’‘–Š‡•‹œ‡–Šƒ––Š‡‡ˆˆ‡…–‘ˆ†‡ϐ‹…‹‡…›‘ †‹ˆˆ‡”‡–‹ƒ–‹‘‹•‡†‹ƒ–‡†„›•ƒ†ƒ›ƒŽ•‘‹ϐŽ—‡…‡’Š‡‘–›’‡‹–Š‡ draining mLNs.

It seems thus that DCs in the intestine induce ILCs to differentiate to an ILC3 phenotype in steady state, by the production of RA and IL-23. The ILCs then migrate back to the mLNs14 _{present antigen to the resident T cells in}

ILC3 RorȖt ILC1 T-bet IL-12 IL-18 IL-1ȕ IL-23 RA A B T RA IL-1β IL-23 GM-CSF mLN T IL-2 Helminth/parasite infection RA deficiency IL-25 IL-33 RA Commensal bacteria Food derived RA ILC3 ILC3 ILC3 ILC2 ILC2 ILC2 ILC2 ILC3

Figure 2. Environmental control of ILC function. (A) Dendritic cells producing skewing

1

triggers the production of IL-25 and IL-33 by the epithelium and tissue

resident macrophages while RA production by DCs is reduced, ILCs will then differentiate towards the ILC2 phenotype. In addition, they will express MHC-II in the presence of co-stimulation, thereby supporting a Th2 response31,43

(Fig. 2B). As DCs will also migrate from the intestinal lamina propria to the draining mLNs it can be assumed that also the microenvironment of the mLNs will change, further stimulating differentiation towards the ILC2 subset. Interestingly, apart from its effect on ILCs, DC-derived RA has long been shown to induce immunological tolerance via the induction of Tregs137,138_{. Induction}

of tolerogenic MHC-II expressing ILC3 is in line with the reputation RA has won for itself as an immunosuppressive rather than an immunostimulatory molecule within the intestine.

ĚęĚĆđėĊđĆęĎĔēĘčĎĕćĊęĜĊĊēĘĆēĉĘĎēĘ

It is clear that DCs play an important role in skewing and sustaining cytokine production by ILCs in the periphery50,130,136_{ƒ†–Š‡“—‡•–‹‘…ƒ„‡ƒ•‡†}

whether this relationship is mutual. In the intestine, ILC-derived GM-CSF maintains the CD103+ _{intestinal DC population}50 _{and further stimulates}

its capacity to produce RA139_{. The gut microbiota supports this cross-talk}

„‡–™‡‡‹–‡•–‹ƒŽ’Šƒ‰‘…›–‡•ƒ†‘”ɀ–+ _{ILCs, through the induction of}

IL-ͳȾƒ†’”‘†—…–‹‘„›’Šƒ‰‘…›–‡•™Š‹…Š‹–—”‹‹–‹ƒ–‡•–Š‡’”‘†—…–‹‘ of GM-CSF by the ILCs50_{, thus driving a positive feedback loop.}

Although true for the intestines, it is not clear whether this mutual relationship holds true for SLOs. DC populations in the spleen are not altered in csf2-/- _{mice, while the DC subsets in LNs are affected by the lack of}

GM-CSF140,141_{. As in the intestines, ILCs and DCs reside in close proximity of each}

other, within the inter-follicular- and T cell areas of the LNs115_{. ILCs have}

been shown to support DC populations in the intestine50 _{and it would be}

interesting to see whether the same holds true for LNs. This may then in turn contribute to LN homeostasis as CD11c+ _{LN resident DCs were shown}

–‘„‡…”—…‹ƒŽˆ‘”ϐ‹–‡••–Š”‘—‰Š‡†‹ƒ–‡†•‹‰ƒŽ‹‰142_{. In fact, in the}

•‘ˆ‹…‡‹™Š‹…Š͹™ƒ••’‡…‹ϐ‹…ƒŽŽ›†‡Ž‡–‡†‘Ž›‹•ǡ‹…‘—–• ™‡”‡Ž‘™‡”™Š‡…‘’ƒ”‡†–‘–Š‡•‹–—ƒ–‹‘™‹–Š͹•—ˆϐ‹…‹‡–•ƒ† cells143_{. There may thus be an interaction of DCs and ILCs within lymphoid}

ĎĒĆēĉĔĚęđĎēĊĔċęčĎĘęčĊĘĎĘ

As discussed above, ILCs have been found to affect adaptive immunity and lymphoid tissue repair in a number of ways, but also to be of importance for homeostasis within SLOs while on the other hand SLO stroma creates a favorable niche for ILC survival. In this thesis, the complex relationship between ILCs and adult SLOs was further studied. As SLO stroma plays ‹’‘”–ƒ–”‘Ž‡•‹–Š‡‹—‡•›•–‡ƒ†‹ϐŽ—‡…‡•„‡Šƒ˜‹‘”ǡ™‡•‡–‘—– to set up a method for isolation and in vitro culture of human SLO stromal cells from tonsils, which is described in chapter 2. In this chapter we also show that human SLO stromal cells are functional and support ILC survival in culture. Following the observation that ILC3 from human adult PB do not respond –‘ •–‹—Žƒ–‹‘ ™‹–Š ǦͳȾ ƒ† Ǧʹ͵ „› ’”‘†—…‹‰ Ǧʹʹ ƒ• †‡•…”‹„‡† ˆ‘” SLO ILC3, we investigated the differences between human PB and human SLO (i.e. spleen, LN and tonsil) ILCs on a transcriptional level in chapter 3. The analysis provided evidence that PB ILCs are an immature population, •Š‘™‹‰ƒ–”ƒ•…”‹’–‹‘ƒŽ’”‘ϐ‹Ž‡•‹‹Žƒ”–‘–Šƒ–‘ˆƒÃ˜‡ǦŽ‹‡͸ʹ+ _{ILC3 as}

described in human tonsils144_{. Further comparison of PB ILC3 with NKp44}

-ILC3 from human LN, tonsils and spleens revealed that the transcriptional •‹‰ƒ–—”‡ ‘ˆ –Š‡ ϐ‹”•– ‹• ‡”‹…Š‡† ˆ‘” ‰‡‡• ‹˜‘Ž˜‡† ‹ ƒ–—”ƒ–‹‘ ƒ† differentiation of leukocytes.

—„•‡“—‡–Ž›ǡ™‡•Š‘™‹chapter 4, that like naïve T cells, ILCs in human adult PB expressing CD62L, a homing receptor for SLOs, lack activation ƒ”‡”•Ǥ  ‹…‡ǡ –Š‹• ͸ʹ ‡š’”‡••‹‰ ’‘’—Žƒ–‹‘ ™ƒ• ƒŽ•‘ ‹†‡–‹ϐ‹‡† ‹ „‘–Š • ƒ†  ƒ† ™ƒ• ˆ‘—† –‘ ƒ……‘”†‹‰Ž› Žƒ… Ž‹‡ƒ‰‡ †‡ϐ‹‹‰ markers. Like naïve T cells, CD62L+ _{ILCs were found to migrate into pLNs in}

a CD62L dependent fashion, and leave the pLN via S1PR. Finally, we show that in Crohn’s disease patients, this population is diminished in PB, further showing that CD62L marks immature, re-circulating ILCs.

Although ILCs are known to be present in adult SLOs, their function in human SLOs is not fully understood. In chapter 5, we looked into one of the potential roles of human ILCs in adult SLOs, being regulation of the T cell responses. We show that ILCs can present antigen to CD4+ _{T cells in the}

context of MHC-II yet lack expression of co-stimulatory molecules. As was shown for mouse ILCs123,125_{, T cell suppression by human ILCs was at least}

partially dependent on scavenging of IL-2.

–Š‡’ƒ•–‹–Šƒ•„‡‡•Š‘™–Šƒ–ˆ—…–‹‘‹•‹ϐŽ—‡…‡†„›–Š‡’”‡•‡…‡ of RA84,87,133-135_{. For this reason, in chapter 6, we investigated the changes in}

1

in the course of treatment. Also, less ILC2s and more ILC3s are found in the

PB of patients, which can be caused both by altered differentiation or by altered homing of the cells.

Finally, in chapter 7™‡“—‡•–‹‘‡†™Š‡–Š‡”Š—ƒ•ƒ”‡‹ϐŽ—‡…‡† by a fully developed commensal microbiota, as has been shown in ‡š’‡”‹‡–•™‹–Š‰‡”ˆ”‡‡‹…‡Ǥ‘ƒ††”‡••–Š‹•“—‡•–‹‘ǡ™‡‹˜‡•–‹‰ƒ–‡† ILC populations in adult PB and umbilical cord blood (UCB) for differences in expression of membrane markers and transcription factors. Here, we found that the ILC population in UCB was larger than in adult blood, with a relative enrichment for CRTH2+ _{ILC2 at the expense of ILC1.and that the markers}

ĊċĊėĊēĈĊĘ

1 Kiessling, R., Klein, E., Pross, H. & Wigzell, H. “Natural” killer cells in the mouse. II. Cytotoxic cells ™‹–Š•’‡…‹ϐ‹…‹–›ˆ‘”‘—•‡‘Ž‘‡›Ž‡—‡‹ƒ…‡ŽŽ•ǤŠƒ”ƒ…–‡”‹•–‹…•‘ˆ–Š‡‹ŽŽ‡”…‡ŽŽǤ—”Ǥ Ǥ—‘Ž 5, 117-121, doi:10.1002/eji.1830050209 [doi] (1975).

2 Spits, H. & Cupedo, T. Innate lymphoid cells: emerging insights in development, lineage relationships, and function. —Ǥ ‡˜Ǥ —‘Ž 30, 647-675, doi:10.1146/annurev-immunol-020711-075053 [doi] (2012).

͵ ‡”ƒϐ‹‹ǡǤ et al. Gata3 drives development of RORgammat+ group 3 innate lymphoid cells. Ǥš’Ǥ

Med 211, 199-208, doi:jem.20131038 [pii];10.1084/jem.20131038 [doi] (2014).

4 Aliahmad, P., de la Torre, B. & Kaye, J. Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages. ƒ–Ǥ —‘Ž 11, 945-952, doi:ni.1930 [pii];10.1038/ni.1930 [doi] (2010).

5 Yokota, Y. et al. Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. ƒ–—”‡ 397, 702-706, doi:10.1038/17812 [doi] (1999).

6 Seillet, C. et al.ϐ‹Ž͵‹•”‡“—‹”‡†ˆ‘”–Š‡†‡˜‡Ž‘’‡–‘ˆƒŽŽ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•—„•‡–•Ǥ Ǥš’Ǥ‡† 211, 1733-1740, doi:jem.20140145 [pii];10.1084/jem.20140145 [doi] (2014).

7 Geiger, T. L. et al.ϐ‹Ž͵‹•…”—…‹ƒŽˆ‘”†‡˜‡Ž‘’‡–‘ˆ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•ƒ†Š‘•–’”‘–‡…–‹‘ƒ‰ƒ‹•– intestinal pathogens. Ǥš’Ǥ‡† 211, 1723-1731, doi:jem.20140212 [pii];10.1084/jem.20140212 [doi] (2014).

8 Spits, H. et al. Innate lymphoid cells--a proposal for uniform nomenclature. ƒ–Ǥ‡˜Ǥ—‘Ž 13, 145-149, doi:nri3365 [pii];10.1038/nri3365 [doi] (2013).

9 Artis, D. & Spits, H. The biology of innate lymphoid cells. ƒ–—”‡ 517, 293-301, doi:nature14189 [pii];10.1038/nature14189 [doi] (2015).

10 Gasteiger, G., Fan, X., Dikiy, S., Lee, S. Y. & Rudensky, A. Y. Tissue residency of innate lymphoid cells in lymphoid and non-lymphoid organs. …‹‡…‡, doi:science.aac9593 [pii];10.1126/science.aac9593 [doi] (2015).

11 Romera-Hernandez, M., Aparicio-Domingo, P. & Cupedo, T. Damage control: Rorgammat+ innate lymphoid cells in tissue regeneration. —””Ǥ’‹Ǥ—‘Ž 25, 156-160, doi:S0952-7915(13)00008-3 [pii];10.1016/j.coi.2013.01.007 [doi] (2013).

12 Mebius, R. E. Organogenesis of lymphoid tissues. ƒ–Ǥ ‡˜Ǥ —‘Ž 3, 292-303, doi:10.1038/ nri1054 [doi];nri1054 [pii] (2003).

13 van de Pavert, S. A. & Mebius, R. E. New insights into the development of lymphoid tissues. ƒ–Ǥ‡˜Ǥ

—‘Ž 10, 664-674, doi:nri2832 [pii];10.1038/nri2832 [doi] (2010).

14 Mackley, E. C. et al. ͹Ǧ†‡’‡†‡– –”ƒˆϐ‹…‹‰ ‘ˆ ‰ƒƒȋΪȌ • …”‡ƒ–‡• ƒ —‹“—‡ microenvironment within mucosal draining lymph nodes. ƒ–Ǥ‘— 6, 5862, doi:ncomms6862 [pii];10.1038/ncomms6862 [doi] (2015).

15 Cupedo, T. et al. Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells. ƒ–Ǥ —‘Ž 10, 66-74, doi:ni.1668 [pii];10.1038/ ni.1668 [doi] (2009).

16 Mebius, R. E., Rennert, P. & Weissman, I. L. Developing lymph nodes collect CD4+CD3- LTbeta+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells. —‹–› 7, 493-504, doi:S1074-7613(00)80371-4 [pii] (1997).

17 Hoorweg, K. et al. Functional Differences between Human NKp44(-) and NKp44(+) RORC(+) Innate Lymphoid Cells. ”‘–—‘Ž 3ǡ͹ʹǡ†‘‹ǣͳͲǤ͵͵ͺͻȀϐ‹—ǤʹͲͳʹǤͲͲͲ͹ʹȏ†‘‹ȐȋʹͲͳʹȌǤ

18 Bernink, J. H. et al.—ƒ–›’‡ͳ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•ƒ……——Žƒ–‡‹‹ϐŽƒ‡†—…‘•ƒŽ–‹••—‡•Ǥ

ƒ–Ǥ—‘Ž 14, 221-229, doi:ni.2534 [pii];10.1038/ni.2534 [doi] (2013).

19 Powell, N. et al. Š‡ –”ƒ•…”‹’–‹‘ ˆƒ…–‘” Ǧ„‡– ”‡‰—Žƒ–‡• ‹–‡•–‹ƒŽ ‹ϐŽƒƒ–‹‘ ‡†‹ƒ–‡† „› interleukin-7 receptor+ innate lymphoid cells. —‹–› 37, 674-684, doi:S1074-7613(12)00421-9 [pii];10.1016/j.immuni.2012.09.008 [doi] (2012).

20 Fuchs, A. et al.–”ƒ‡’‹–Š‡Ž‹ƒŽ–›’‡ͳ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•ƒ”‡ƒ—‹“—‡•—„•‡–‘ˆǦͳʹǦƒ†Ǧ 15-responsive IFN-gamma-producing cells. —‹–› 38, 769-781, doi:S1074-7613(13)00091-5 [pii];10.1016/j.immuni.2013.02.010 [doi] (2013).

21 Mjosberg, J. et al. The transcription factor GATA3 is essential for the function of human type 2 innate lymphoid cells. —‹–› 37, 649-659, doi:S1074-7613(12)00420-7 [pii];10.1016/j. immuni.2012.08.015 [doi] (2012).

1

innate lymphoid cells. —‹–› 37, 634-648, doi:S1074-7613(12)00422-0 [pii];10.1016/j.

immuni.2012.06.020 [doi] (2012).

23 Mjosberg, J. M. et al.—ƒǦʹͷǦƒ†Ǧ͵͵Ǧ”‡•’‘•‹˜‡–›’‡ʹ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•ƒ”‡†‡ϐ‹‡†„› expression of CRTH2 and CD161. ƒ–Ǥ—‘Ž 12, 1055-1062, doi:ni.2104 [pii];10.1038/ni.2104 [doi] (2011).

24 Price, A. E. et al. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. ”‘…Ǥ

ƒ–ŽǤ…ƒ†Ǥ…‹ǤǤǤ 107, 11489-11494, doi:1003988107 [pii];10.1073/pnas.1003988107 [doi]

(2010).

25 Moro, K. et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells. ƒ–—”‡ 463, 540-544, doi:nature08636 [pii];10.1038/nature08636 [doi] (2010). 26 Neill, D. R. et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity.

ƒ–—”‡ 464, 1367-1370, doi:nature08900 [pii];10.1038/nature08900 [doi] (2010).

27 Liang, H. E. et al.‹˜‡”‰‡–‡š’”‡••‹‘’ƒ––‡”•‘ˆǦͶƒ†Ǧͳ͵†‡ϐ‹‡—‹“—‡ˆ—…–‹‘•‹ƒŽŽ‡”‰‹… immunity. ƒ–Ǥ—‘Ž 13, 58-66, doi:ni.2182 [pii];10.1038/ni.2182 [doi] (2012).

28 Halim, T. Y. et al. ‡–‹‘‹…Ǧƒ…‹†Ǧ”‡…‡’–‘”Ǧ”‡Žƒ–‡† ‘”’Šƒ —…Ž‡ƒ” ”‡…‡’–‘” ƒŽ’Šƒ ‹• ”‡“—‹”‡† ˆ‘” ƒ–—”ƒŽ Š‡Ž’‡” …‡ŽŽ †‡˜‡Ž‘’‡– ƒ† ƒŽŽ‡”‰‹… ‹ϐŽƒƒ–‹‘Ǥ —‹–› 37, 463-474, doi:S1074-7613(12)00371-8 [pii];10.1016/j.immuni.2012.06.012 [doi] (2012).

29 Yang, Q. et al.…‡ŽŽˆƒ…–‘”ͳ‹•”‡“—‹”‡†ˆ‘”‰”‘—’ʹ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ‰‡‡”ƒ–‹‘Ǥ—‹–› 38, 694-704, doi:S1074-7613(13)00140-4 [pii];10.1016/j.immuni.2012.12.003 [doi] (2013). 30 Wong, S. H. et al. Transcription factor RORalpha is critical for nuocyte development. ƒ–Ǥ—‘Ž

13, 229-236, doi:ni.2208 [pii];10.1038/ni.2208 [doi] (2012).

31 Halim, T. Y., Krauss, R. H., Sun, A. C. & Takei, F. Lung natural helper cells are a critical source of Šʹ…‡ŽŽǦ–›’‡…›–‘‹‡•‹’”‘–‡ƒ•‡ƒŽŽ‡”‰‡Ǧ‹†—…‡†ƒ‹”™ƒ›‹ϐŽƒƒ–‹‘Ǥ—‹–› 36, 451-463, doi:S1074-7613(12)00085-4 [pii];10.1016/j.immuni.2011.12.020 [doi] (2012).

32 Saenz, S. A. et al. IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses. ƒ–—”‡ 464, 1362-1366, doi:nature08901 [pii];10.1038/nature08901 [doi] (2010). 33 Xue, L. et al. Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant

receptor-homologous molecule expressed on TH2 cells. ǤŽŽ‡”‰›Ž‹Ǥ—‘Ž 133, 1184-1194, doi:S0091-6749(13)01771-5 [pii];10.1016/j.jaci.2013.10.056 [doi] (2014).

34 Roediger, B. et al.—–ƒ‡‘—•‹—‘•—”˜‡‹ŽŽƒ…‡ƒ†”‡‰—Žƒ–‹‘‘ˆ‹ϐŽƒƒ–‹‘„›‰”‘—’ʹ‹ƒ–‡ lymphoid cells. ƒ–Ǥ—‘Ž 14, 564-573, doi:ni.2584 [pii];10.1038/ni.2584 [doi] (2013). 35 Wilhelm, C. et al. An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung

‹ϐŽƒƒ–‹‘Ǥƒ–Ǥ—‘Ž 12, 1071-1077, doi:ni.2133 [pii];10.1038/ni.2133 [doi] (2011). 36 Mirchandani, A. S. et al. Type 2 innate lymphoid cells drive CD4+ Th2 cell responses. Ǥ—‘Ž 192,

2442-2448, doi:jimmunol.1300974 [pii];10.4049/jimmunol.1300974 [doi] (2014).

37 Gold, M. J. et al. Group 2 innate lymphoid cells facilitate sensitization to local, but not systemic, TH2-inducing allergen exposures. ǤŽŽ‡”‰›Ž‹Ǥ—‘Ž 133, 1142-1148, doi:S0091-6749(14)00298-X [pii];10.1016/j.jaci.2014.02.033 [doi] (2014).

38 Drake, L. Y., Iijima, K. & Kita, H. Group 2 innate lymphoid cells and CD4+ T cells cooperate to mediate type 2 immune response in mice. ŽŽ‡”‰› 69, 1300-1307, doi:10.1111/all.12446 [doi] (2014). 39 Camelo, A. et al.Ž‘…‹‰Ǧʹͷ•‹‰ƒŽŽ‹‰’”‘–‡…–•ƒ‰ƒ‹•–‰—–‹ϐŽƒƒ–‹‘‹ƒ–›’‡Ǧʹ‘†‡Ž‘ˆ

colitis by suppressing nuocyte and NKT derived IL-13. Ǥƒ•–”‘‡–‡”‘Ž 47, 1198-1211, doi:10.1007/ s00535-012-0591-2 [doi] (2012).

40 Molofsky, A. B. et al. Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. Ǥš’Ǥ‡† 210, 535-549, doi:jem.20121964 [pii];10.1084/ jem.20121964 [doi] (2013).

41 Hams, E., Locksley, R. M., McKenzie, A. N. & Fallon, P. G. Cutting edge: IL-25 elicits innate lymphoid type 2 and type II NKT cells that regulate obesity in mice. Ǥ—‘Ž 191, 5349-5353, doi:jimmunol.1301176 [pii];10.4049/jimmunol.1301176 [doi] (2013).

42 Fallon, P. G. et al.†‡–‹ϐ‹…ƒ–‹‘‘ˆƒ‹–‡”Ž‡—‹ȋȌǦʹͷǦ†‡’‡†‡–…‡ŽŽ’‘’—Žƒ–‹‘–Šƒ–’”‘˜‹†‡•Ǧ 4, IL-5, and IL-13 at the onset of helminth expulsion. Ǥš’Ǥ‡† 203, 1105-1116, doi:jem.20051615 [pii];10.1084/jem.20051615 [doi] (2006).

43 Oliphant, C. J. et al. MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion. —‹–› 41, 283-295, doi:S1074-7613(14)00243-X [pii];10.1016/j.immuni.2014.06.016 [doi] (2014).

ͶͶ ‹ǡ Ǥ Ǥǡ ‘Œ‘ǡ Ǥ Ǥ Ƭ ”–‹•ǡ Ǥ ƒ–‡ Ž›’Š‘‹† …‡ŽŽ• ƒ† ƒŽŽ‡”‰‹… ‹ϐŽƒƒ–‹‘Ǥ —””Ǥ ’‹Ǥ

45 Boyd, A., Ribeiro, J. M. & Nutman, T. B. Human CD117 (cKit)+ innate lymphoid cells have a discrete –”ƒ•…”‹’–‹‘ƒŽ ’”‘ϐ‹Ž‡ ƒ– Š‘‡‘•–ƒ•‹• ƒ† ƒ”‡ ‡š’ƒ†‡† †—”‹‰ ϐ‹Žƒ”‹ƒŽ ‹ˆ‡…–‹‘Ǥ ‘Ǥ ‡ 9, e108649, doi:10.1371/journal.pone.0108649 [doi];PONE-D-14-29816 [pii] (2014).

46 Chang, Y. J. et al. ƒ–‡ Ž›’Š‘‹† …‡ŽŽ• ‡†‹ƒ–‡ ‹ϐŽ—‡œƒǦ‹†—…‡† ƒ‹”™ƒ› Š›’‡”Ǧ”‡ƒ…–‹˜‹–› independently of adaptive immunity. ƒ–Ǥ—‘Ž 12, 631-638, doi:ni.2045 [pii];10.1038/ni.2045 [doi] (2011).

47 Monticelli, L. A. et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with ‹ϐŽ—‡œƒ˜‹”—•Ǥƒ–Ǥ—‘Ž 12, 1045-1054, doi:ni.2131 [pii];10.1031/ni.2131 [doi] (2011). 48 Brestoff, J. R. et al. Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit

obesity. ƒ–—”‡, doi:nature14115 [pii];10.1038/nature14115 [doi] (2014).

49 Cella, M. et al. A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. ƒ–—”‡ 457, 722-725, doi:nature07537 [pii];10.1038/nature07537 [doi] (2009). 50 Mortha, A. et al. Microbiota-dependent crosstalk between macrophages and ILC3 promotes

intestinal homeostasis. …‹‡…‡ 343, 1249288, doi:science.1249288 [pii];10.1126/science.1249288 [doi] (2014).

51 Buonocore, S. et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. ƒ–—”‡ 464, 1371-1375, doi:nature08949 [pii];10.1038/nature08949 [doi] (2010). 52 Magri, G. et al. Innate lymphoid cells integrate stromal and immunological signals to enhance

antibody production by splenic marginal zone B cells. ƒ–Ǥ —‘Ž 15, 354-364, doi:ni.2830 [pii];10.1038/ni.2830 [doi] (2014).

53 Geremia, A. et al. Ǧʹ͵Ǧ”‡•’‘•‹˜‡ ‹ƒ–‡ Ž›’Š‘‹† …‡ŽŽ• ƒ”‡ ‹…”‡ƒ•‡† ‹ ‹ϐŽƒƒ–‘”› „‘™‡Ž disease. Ǥš’Ǥ‡† 208, 1127-1133, doi:jem.20101712 [pii];10.1084/jem.20101712 [doi] (2011). 54 Tumanov, A. V. et al. Lymphotoxin controls the IL-22 protection pathway in gut innate lymphoid

cells during mucosal pathogen challenge. ‡ŽŽ‘•–Ǥ‹…”‘„‡ 10, 44-53, doi:S1931-3128(11)00192-2 [pii];10.1016/j.chom.2011.06.002 [doi] (2011).

55 Glatzer, T. et al.‰ƒƒ–ȋΪȌ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•ƒ…“—‹”‡ƒ’”‘‹ϐŽƒƒ–‘”›’”‘‰”ƒ—’‘ engagement of the activating receptor NKp44. —‹–› 38, 1223-1235, doi:S1074-7613(13)00239-2 [pii];10.1016/j.immuni.2013.05.013 [doi] (2013).

56 Crellin, N. K., Trifari, S., Kaplan, C. D., Cupedo, T. & Spits, H. Human NKp44+IL-22+ cells and LTi-like cells constitute a stable RORC+ lineage distinct from conventional natural killer cells. Ǥš’Ǥ‡† 207, 281-290, doi:jem.20091509 [pii];10.1084/jem.20091509 [doi] (2010).

57 Crellin, N. K. et al. Regulation of cytokine secretion in human CD127(+) LTi-like innate lymphoid cells by Toll-like receptor 2. —‹–› 33, 752-764, doi:S1074-7613(10)00372-9 [pii];10.1016/j. immuni.2010.10.012 [doi] (2010).

58 Satoh-Takayama, N. et al.‹…”‘„‹ƒŽϐŽ‘”ƒ†”‹˜‡•‹–‡”Ž‡—‹ʹʹ’”‘†—…–‹‘‹‹–‡•–‹ƒŽ’Ͷ͸Ϊ…‡ŽŽ• that provide innate mucosal immune defense. —‹–› 29, 958-970, doi:S1074-7613(08)00504-9 [pii];10.1016/j.immuni.2008.11.001 [doi] (2008).

59 Sanos, S. L. et al. ‰ƒƒ– ƒ† …‘‡•ƒŽ ‹…”‘ϐŽ‘”ƒ ƒ”‡ ”‡“—‹”‡† ˆ‘” –Š‡ †‹ˆˆ‡”‡–‹ƒ–‹‘ ‘ˆ mucosal interleukin 22-producing NKp46+ cells. ƒ–Ǥ—‘Ž 10, 83-91, doi:ni.1684 [pii];10.1038/ ni.1684 [doi] (2009).

60 Klose, C. S. et al. A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells. ƒ–—”‡ 494, 261-265, doi:nature11813 [pii];10.1038/nature11813 [doi] (2013). 61 Sciume, G. et al.‹•–‹…–”‡“—‹”‡‡–•ˆ‘”Ǧ„‡–‹‰—–‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•Ǥ Ǥš’Ǥ‡† 209,

2331-2338, doi:jem.20122097 [pii];10.1084/jem.20122097 [doi] (2012).

62 Vonarbourg, C. et al. Regulated expression of nuclear receptor RORgammat confers distinct functional fates to NK cell receptor-expressing RORgammat(+) innate lymphocytes. —‹–› 33, 736-751, doi:S1074-7613(10)00403-6 [pii];10.1016/j.immuni.2010.10.017 [doi] (2010). 63 Sonnenberg, G. F. et al. Innate lymphoid cells promote anatomical containment of lymphoid-resident

commensal bacteria. …‹‡…‡ 336, 1321-1325, doi:science.1222551 [pii];10.1126/science.1222551 [doi] (2012).

64 Hanash, A. M. et al. Interleukin-22 protects intestinal stem cells from immune-mediated tissue damage and regulates sensitivity to graft versus host disease. —‹–› 37, 339-350, doi:S1074-7613(12)00331-7 [pii];10.1016/j.immuni.2012.05.028 [doi] (2012).

65 Kruglov, A. A. et al. Nonredundant function of soluble LTalpha3 produced by innate lymphoid cells in intestinal homeostasis. …‹‡…‡ 342, 1243-1246, doi:342/6163/1243 [pii];10.1126/ science.1243364 [doi] (2013).

1

tissue-inducer cells with stroma of the T cell zone. ƒ–Ǥ —‘Ž 9, 667-675, doi:ni.1605

[pii];10.1038/ni.1605 [doi] (2008).

67 Sawa, S. et al. RORgammat+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota. ƒ–Ǥ —‘Ž 12, 320-326, doi:ni.2002 [pii];10.1038/ni.2002 [doi] (2011).

68 Aparicio-Domingo, P. et al. Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage. Ǥš’Ǥ‡†, doi:jem.20150318 [pii];10.1084/jem.20150318 [doi] (2015). 69 Dudakov, J. A. et al. Interleukin-22 drives endogenous thymic regeneration in mice. …‹‡…‡ 336,

91-95, doi:science.1218004 [pii];10.1126/science.1218004 [doi] (2012).

70 Eberl, G. et al. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. ƒ–Ǥ—‘Ž 5, 64-73, doi:10.1038/ni1022 [doi];ni1022 [pii] (2004).

71 Sawa, S. et al. Lineage relationship analysis of RORgammat+ innate lymphoid cells. …‹‡…‡ 330, 665-669, doi:science.1194597 [pii];10.1126/science.1194597 [doi] (2010).

72 Randall, T. D. & Mebius, R. E. The development and function of mucosal lymphoid tissues: a balancing act with micro-organisms. —…‘•ƒŽǤ—‘Ž 7, 455-466, doi:mi201411 [pii];10.1038/mi.2014.11 [doi] (2014).

73 Sun, Z. et al.‡“—‹”‡‡–ˆ‘”‰ƒƒ‹–Š›‘…›–‡•—”˜‹˜ƒŽƒ†Ž›’Š‘‹†‘”‰ƒ†‡˜‡Ž‘’‡–Ǥ

…‹‡…‡ 288, 2369-2373, doi:8641 [pii] (2000).

74 Takatori, H. et al. Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22. Ǥš’Ǥ

Med 206, 35-41, doi:jem.20072713 [pii];10.1084/jem.20072713 [doi] (2009).

75 Klose, C. S. et al. Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 157, 340-356, doi:S0092-8674(14)00405-X [pii];10.1016/j. cell.2014.03.030 [doi] (2014).

76 Boos, M. D., Yokota, Y., Eberl, G. & Kee, B. L. Mature natural killer cell and lymphoid tissue-inducing …‡ŽŽ†‡˜‡Ž‘’‡–”‡“—‹”‡•†ʹǦ‡†‹ƒ–‡†•—’’”‡••‹‘‘ˆ’”‘–‡‹ƒ…–‹˜‹–›Ǥ Ǥš’Ǥ‡† 204, 1119-1130, doi:jem.20061959 [pii];10.1084/jem.20061959 [doi] (2007).

77 Townsend, M. J. et al. T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells. —‹–› 20, 477-494, doi:S1074761304000767 [pii] (2004).

78 Gordon, S. M. et al. The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation. —‹–› 36, 55-67, doi:S1074-7613(12)00005-2 [pii];10.1016/j. immuni.2011.11.016 [doi] (2012).

79 Possot, C. et al. Notch signaling is necessary for adult, but not fetal, development of RORgammat(+) innate lymphoid cells. ƒ–Ǥ—‘Ž 12, 949-958, doi:ni.2105 [pii];10.1038/ni.2105 [doi] (2011). 80 Constantinides, M. G., McDonald, B. D., Verhoef, P. A. & Bendelac, A. A committed precursor to innate

lymphoid cells. ƒ–—”‡, doi:nature13047 [pii];10.1038/nature13047 [doi] (2014).

81 Robinette, M. L. et al.”ƒ•…”‹’–‹‘ƒŽ’”‘‰”ƒ•†‡ϐ‹‡‘Ž‡…—Žƒ”…Šƒ”ƒ…–‡”‹•–‹…•‘ˆ‹ƒ–‡Ž›’Š‘‹† cell classes and subsets. ƒ–Ǥ—‘Ž 16, 306-317, doi:ni.3094 [pii];10.1038/ni.3094 [doi] (2015). 82 Ebihara, T. et al.—š͵•’‡…‹ϐ‹‡•Ž‹‡ƒ‰‡…‘‹–‡–‘ˆ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•Ǥƒ–Ǥ—‘Ž 16,

1124-1133, doi:ni.3272 [pii];10.1038/ni.3272 [doi] (2015).

83 Califano, D. et al. Transcription Factor Bcl11b Controls Identity and Function of Mature Type 2 Innate Lymphoid Cells. —‹–› 43, 354-368, doi:S1074-7613(15)00272-1 [pii];10.1016/j. immuni.2015.07.005 [doi] (2015).

84 Spencer, S. P. et al.†ƒ’–ƒ–‹‘‘ˆ‹ƒ–‡Ž›’Š‘‹†…‡ŽŽ•–‘ƒ‹…”‘—–”‹‡–†‡ϐ‹…‹‡…›’”‘‘–‡•–›’‡ 2 barrier immunity. …‹‡…‡ 343, 432-437, doi:343/6169/432 [pii];10.1126/science.1247606 [doi] (2014).

85 Kim, M. H., Taparowsky, E. J. & Kim, C. H. Retinoic Acid Differentially Regulates the Migration of Innate Lymphoid Cell Subsets to the Gut. —‹–›, doi:S1074-7613(15)00231-9 [pii];10.1016/j. immuni.2015.06.009 [doi] (2015).

86 Zhou, L., Chong, M. M. & Littman, D. R. Plasticity of CD4+ T cell lineage differentiation. —‹–› 30, 646-655, doi:S1074-7613(09)00198-8 [pii];10.1016/j.immuni.2009.05.001 [doi] (2009). 87 Bernink, J. H. et al. Interleukin-12 and -23 Control Plasticity of CD127(+) Group 1 and

Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria. —‹–› 43, 146-160, doi:S1074-7613(15)00263-0 [pii];10.1016/j.immuni.2015.06.019 [doi] (2015).

88 Baptista, A. P. et al. Lymph node stromal cells constrain immunity via MHC class II self-antigen presentation. Elife 3, doi:10.7554/eLife.04433 [doi] (2014).

via Aire-independent direct antigen presentation. Ǥ š’Ǥ ‡† 207, 681-688, doi:jem.20092465 [pii];10.1084/jem.20092465 [doi] (2010).

90 Nichols, L. A. et al. Deletional self-tolerance to a melanocyte/melanoma antigen derived from tyrosinase is mediated by a radio-resistant cell in peripheral and mesenteric lymph nodes. Ǥ

—‘Ž 179, 993-1003, doi:179/2/993 [pii] (2007).

91 Yip, L. et al. Deaf1 isoforms control the expression of genes encoding peripheral tissue antigens in the pancreatic lymph nodes during type 1 diabetes. ƒ–Ǥ—‘Ž 10, 1026-1033, doi:ni.1773 [pii];10.1038/ni.1773 [doi] (2009).

92 Lee, J. W. et al. Peripheral antigen display by lymph node stroma promotes T cell tolerance to intestinal self. ƒ–Ǥ—‘Ž 8, 181-190, doi:ni1427 [pii];10.1038/ni1427 [doi] (2007).

93 Magnusson, F. C. et al. Direct presentation of antigen by lymph node stromal cells protects against CD8 T-cell-mediated intestinal autoimmunity. ƒ•–”‘‡–‡”‘Ž‘‰› 134, 1028-1037, doi:S0016-5085(08)00176-5 [pii];10.1053/j.gastro.2008.01.070 [doi] (2008).

94 Fletcher, A. L. et al.›’Š‘†‡ϐ‹„”‘„Žƒ•–‹…”‡–‹…—Žƒ”…‡ŽŽ•†‹”‡…–Ž›’”‡•‡–’‡”‹’Š‡”ƒŽ–‹••—‡ƒ–‹‰‡ —†‡” •–‡ƒ†›Ǧ•–ƒ–‡ ƒ† ‹ϐŽƒƒ–‘”› …‘†‹–‹‘•Ǥ Ǥ š’Ǥ ‡† 207, 689-697, doi:jem.20092642 [pii];10.1084/jem.20092642 [doi] (2010).

95 Rennert, P. D., Browning, J. L., Mebius, R., Mackay, F. & Hochman, P. S. Surface lymphotoxin alpha/ „‡–ƒ…‘’Ž‡š‹•”‡“—‹”‡†ˆ‘”–Š‡†‡˜‡Ž‘’‡–‘ˆ’‡”‹’Š‡”ƒŽŽ›’Š‘‹†‘”‰ƒ•Ǥ Ǥš’Ǥ‡† 184, 1999-2006 (1996).

96 Nagatake, T. et al. Id2-, RORgammat-, and LTbetaR-independent initiation of lymphoid organogenesis in ocular immunity. Ǥš’Ǥ‡† 206, 2351-2364, doi:jem.20091436 [pii];10.1084/jem.20091436 [doi] (2009).

97 Fukuyama, S. et al. Initiation of NALT organogenesis is independent of the IL-7R, LTbetaR, and NIK •‹‰ƒŽ‹‰’ƒ–Š™ƒ›•„—–”‡“—‹”‡•–Š‡†ʹ‰‡‡ƒ†͵ȋǦȌͶȋΪȌͶͷȋΪȌ…‡ŽŽ•Ǥ—‹–› 17, 31-40, doi:S1074761302003394 [pii] (2002).

98 Kanamori, Y. et al. †‡–‹ϐ‹…ƒ–‹‘ ‘ˆ ‘˜‡Ž Ž›’Š‘‹† –‹••—‡• ‹ —”‹‡ ‹–‡•–‹ƒŽ —…‘•ƒ ™Š‡”‡ clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop. Ǥš’Ǥ‡† 184, 1449-1459 (1996).

99 Hamada, H. et al.†‡–‹ϐ‹…ƒ–‹‘‘ˆ—Ž–‹’Ž‡‹•‘Žƒ–‡†Ž›’Š‘‹†ˆ‘ŽŽ‹…Ž‡•‘–Š‡ƒ–‹‡•‡–‡”‹…™ƒŽŽ‘ˆ the mouse small intestine. Ǥ—‘Ž 168, 57-64 (2002).

100 Bouskra, D. et al. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. ƒ–—”‡ 456, 507-510, doi:nature07450 [pii];10.1038/nature07450 [doi] (2008).

101 Pabst, O. et al. Adaptation of solitary intestinal lymphoid tissue in response to microbiota and chemokine receptor CCR7 signaling. Ǥ—‘Ž 177, 6824-6832, doi:177/10/6824 [pii] (2006). 102 Baptista, A. P. et al. Colonic patch and colonic SILT development are independent and differentially

regulated events. —…‘•ƒŽǤ —‘Ž 6, 511-521, doi:mi201290 [pii];10.1038/mi.2012.90 [doi] (2013).

103 Tsuji, M. et al. ‡“—‹”‡‡– ˆ‘” Ž›’Š‘‹† –‹••—‡Ǧ‹†—…‡” …‡ŽŽ• ‹ ‹•‘Žƒ–‡† ˆ‘ŽŽ‹…Ž‡ ˆ‘”ƒ–‹‘ and T cell-independent immunoglobulin A generation in the gut. —‹–› 29, 261-271, doi:S1074-7613(08)00303-8 [pii];10.1016/j.immuni.2008.05.014 [doi] (2008).

104 Meier, D. et al. Ectopic lymphoid-organ development occurs through interleukin 7-mediated enhanced survival of lymphoid-tissue-inducer cells. —‹–› 26, 643-654, doi:S1074-7613(07)00255-5 [pii];10.1016/j.immuni.2007.04.009 [doi] (2007).

105 Schmutz, S. et al. Cutting edge: IL-7 regulates the peripheral pool of adult ROR gamma+ lymphoid tissue inducer cells. Ǥ—‘Ž 183, 2217-2221, doi:jimmunol.0802911 [pii];10.4049/ jimmunol.0802911 [doi] (2009).

106 Rangel-Moreno, J. et al. Omental milky spots develop in the absence of lymphoid tissue-inducer cells and support B and T cell responses to peritoneal antigens. —‹–› 30, 731-743, doi:S1074-7613(09)00186-1 [pii];10.1016/j.immuni.2009.03.014 [doi] (2009).

107 Hou, T. Z. et al. Splenic stromal cells mediate IL-7 independent adult lymphoid tissue inducer cell survival. —”Ǥ Ǥ—‘Ž 40, 359-365, doi:10.1002/eji.200939776 [doi] (2010).

108 Hoorweg, K. et al. A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells. Ǥ

—‘Ž, doi:jimmunol.1402584 [pii];10.4049/jimmunol.1402584 [doi] (2015).