REVIEWS
`
`The light and dark sides of intestinal
`intraepithelial lymphocytes
`
`Hilde Cheroutre*, Florence Lambolez*and Daniel Mucida‡
`
`Abstract | The intraepithelial lymphocytes (IELs) that reside within the epithelium of the
`intestine form one of the main branches of the immune system. As IELs are located at this
`critical interface between the core of the body and the outside environment, they must balance
`protective immunity with an ability to safeguard the integrity of the epithelial barrier: failure to
`do so would compromise homeostasis of the organism. In this Review, we address how the
`unique development and functions of intestinal IELs allow them to achieve this balance.
`
`Pathogens
`Opportunistic organisms that
`cause acute or chronic disease
`following host infection.
`Derived from the Greek word
`‘pathos’, which means
`‘suffering’.
`
`Intraepithelial lymphocytes
`(IELs). These lymphocyte
`populations consist mostly of
`T cells and are found within the
`epithelial layer of mammalian
`mucosal linings, such as the
`gastrointestinal tract and
`reproductive tract. However,
`unlike conventional naive
`T cells, IELs are antigen-
`experienced T cells and, on
`encountering antigens, they
`immediately release cytokines
`or mediate killing of infected
`target cells.
`
`*Laboratory of
`Developmental Immunology,
`La Jolla Institute for Allergy
`and Immunology, La Jolla,
`California 92037, USA.
`‡Laboratory of Mucosal
`Immunology, The Rockefeller
`University, New York,
`New York 10065, USA.
`Correspondence to H.C. and
`D.M.
`e-mails: hilde@liai.org;
`mucida@rockefeller.edu
`doi:10.1038/nri3007
`Published online 17 June 2011
`
`The epithelium of the intestine digests and absorbs
`nutrients and fluids, and in adult humans it spans
`an area of about 200–400 m2 (REF. 1). This huge sur-
`face is made up of a single cell layer of epithelial
`cells, which lines the lumen of the intestine to form
`a physical barrier between the core of the body and
`the environment and forms the largest entry port for
`pathogens. Probably as a consequence of this, sophis-
`ticated and complex innate and adaptive immune
`networks intensely communicate and synergize to
`tightly control the integrity of this critical interface.
`Although a major task of the mucosal immune system
`is to provide protection against intestinal pathogens,
`it is important that excessive or unnecessary immune
`responses are avoided.
`In this Review, we focus on the intraepithelial
`lymphocytes (IELs), which, by their direct contact with
`the enterocytes and by their immediate proximity to
`antigens in the gut lumen, form the front line of immune
`defence against invading pathogens. IELs essentially
`comprise antigen-experienced T cells belonging to
`both the T cell receptor-γδ (TCRγδ)+ and TCRαβ+ lin-
`eages. We discuss the thymic (natural) and peripheral
`(induced) differentiation of various IEL subpopulations,
`as well as their beneficial roles (that is, their ‘light side’)
`in preserving the integrity of the mucosal barrier and in
`preventing pathogen entry and spreading. Conversely,
`we also expose the ‘dark side’ of IELs and discuss how
`these cells can contribute to immune pathology and
`inflammatory diseases.
`
`Mucosal IELs are unique and heterogeneous
`IELs are extremely heterogeneous, and the various IEL
`subsets are distributed differently in the epithelium
`of the small and large intestine (TABLE 1). This pattern of
`distribution is probably influenced by the distinct
`
`digestive functions and the physiological conditions
`that allow these two compartments to cope with infec-
`tions while simultaneously maintaining tolerance to
`innocuous antigens from the diet or from resident
`non-invasive commensals. Nevertheless, these IELs
`also share characteristics that distinguish them from
`the conventional T cell pools in the periphery.
`Gut IELs are almost exclusively T cells, and esti-
`mates based on histological sections indicate that there
`are more T cells in the intestinal epithelium than in the
`spleen2. IELs include a significant proportion of TCRγδ+
`cells, which can constitute up to 60% of small intestinal
`IELs3–5. These IELs are antigen-experienced cells that
`typically express activation markers, such as CD44 and
`CD69 (REF. 6). Furthermore, studies using parabiotic
`mice and intestinal grafting indicate that these antigen-
`experienced T cells do not recirculate7,8. The majority of
`IELs contain abundant cytoplasmic granules for cyto-
`toxic activity, and they can express effector cytokines,
`such as interferon-γ (IFNγ), interleukin-2 (IL-2), IL-4
`or IL-17 (REFS 9–16). Furthermore, they characteristi-
`cally express both activating and inhibitory types of
`innate natural killer (NK) cell receptors, which typify
`them as stress-sensing (activated) yet highly regulated
`(resting) immune cells9,11,17–20.
`IELs constitutively express CD103 (also known as the
`αE integrin), which interacts with E-cadherin on intes-
`tinal epithelial cells21,22, and most of them, especially in
`the small intestine, express CD8αα homodimers, which
`is a hallmark of their activated phenotype18,23–25 (BOX 1).
`A ligand for CD8αα, the thymus leukaemia antigen (TLA),
`which is a non-classical MHC class I molecule, is abun-
`dantly expressed on mouse small intestinal epithelial
`cells26,27. Many TCRαβ IELs express CD8αα together
`with CD4 or CD8αβ; however, a large fraction expresses
`CD8αα alone16. Finally, under normal conditions, and
`
`NATURE REVIEWS | IMMUNOLOGY
`
` VOLUME 11 | JULY 2011 | 445
`
`© 2011 Macmillan Publishers Limited. All rights reserved
`
`Genome Ex. 1058
`Page 1 of 12
`
`

`

`R E V I E W S
`
`Table 1 | Characteristics of the different intestinal T cells subsets in mice
`Cell
`Characteristics
`type
`Antigen
`IEL phenotype
`dependency
`
`Kinetics of
`accumulation
`
`Frequency of
`TCRγδ and
`TCRαβ IELs
`
`Development and
`TCR repertoire
`
`Potential
`beneficial
`functions
`
`Potential
`pathogenic
`functions
`
`Small intestinal epithelium
`Natural
`Self and
`CD2–CD5–CD28–;
`IELs
`non-self
`CTLA4–THY1–/low;
`B220+/– CD69hi;
`NK receptors
`(+++++)
`
`TCRγδ+ (+++):
`– CD4–CD8– (++)
`– CD8αα+ (+++)
`TCRαβ+ (+++):
`– CD4–CD8– (+)
`– CD8αα+ (+++)
`
`Present
`at birth;
`decrease with
`age
`
`Induced
`IELs
`
`Non-self
`
`CD2+CD5+CD28+/–;
`CTLA4+/–THY1hi;
`B220–CD69hi;
`NK receptors
`(++++)
`
`TCRαβ+ (+++):
`– CD8αβ+ (+)
`– CD8αβ+CD8αα+
` (+++)
`– CD4+ (+)
`– CD4+CD8αα+ (++)
`
`Absent at
`birth; increase
`with age
`
`Anti-inflammatory
`responses;
`antimicrobial
`responses;
`tolerance to
`intestinal antigens;
`immune regulation;
`homeostasis of the
`epithelium
`Oral tolerance;
`protective
`antimicrobial
`responses
`
`‘Alternative’
`positive selection
`for CD8αα+TCRαβ+
`IELs in the thymus;
`oligoclonal TCR
`repertoire; unknown
`MHC restriction
`
`‘Conventional’
`positive selection
`in the thymus;
`oligoclonal TCR
`repertoire; restricted
`on MHC class I or
`MHC class II
`
`Small intestinal lamina propria
`T cells
`Non-self
`Not applicable
`
`TCRαβ (+++):
`– CD8αβ+ (++)
`– CD8αβ+CD8αα+ (+)
`– CD4+ (+++)
`– CD4+CD8αα+ (+)
`
`Absent at
`birth; increase
`with age
`
`Oral tolerance;
`protective
`antimicrobial
`responses
`
`‘Conventional’
`positive selection
`in the thymus;
`polyclonal TCR
`repertoire; restricted
`on MHC class I or
`MHC class II
`
`Promote
`epithelial
`damage during
`intestinal
`inflammation
`
`Excessive
`inflammation
`and cytotoxicity
`in response
`to luminal
`antigens;
`exacerbate
`coeliac disease
`
`Ileitis;
`inflammation
`
`Large intestinal epithelium
`Natural
`Self and
`CD2–CD5–CD28–;
`IELs
`non-self
`CTLA4–THY1–/low;
`B220+/–CD69hi;
`NK receptors (+++)
`
`Induced
`IELs
`
`Non-self
`
`CD2+CD5+CD28+;
`CTLA4+THY1hi;
`B220–CD69hi;
`NK receptors (++)
`
`TCRγδ+ (+):
`– CD4–CD8– (++)
`– CD8αα+ (+)
`TCRαβ (++):
`– CD4–CD8– (++)
`– CD8αα+ (+)
`TCRαβ+ (++):
`– CD8αβ+ (+)
`– CD8αβ+CD8αα+ (+)
`– CD4+ (++)
`– CD4+CD8αα+ (+)
`
`Present
`at birth;
`decrease with
`age
`
`‘Alternative’ positive
`selection for
`CD8αα+TCRαβ+ IELs
`in the thymus; TCR
`repertoire and MHC
`restriction unknown
`
`Anti-inflammatory
`responses;
`antimicrobial
`responses;
`tolerance to
`intestinal antigens
`
`Promote
`epithelial
`damage during
`intestinal
`inflammation
`
`Absent at
`birth; increase
`with age
`
`‘Conventional’
`positive selection
`in the thymus; TCR
`repertoire not
`defined; restricted
`on MHC class I or
`MHC class II
`
`Protective
`antimicrobial
`responses
`
`Drive excessive
`inflammation
`and
`cytotoxicity
`in response to
`microbiota-
`derived
`antigens;
`exacerbate IBD
`
`Large intestinal lamina propria
`T cells
`Non-self
`Not applicable
`
`TCRαβ+ (+++):
`– CD8αβ+ (++)
`– CD8αβ+CD8αα+ (+)
`– CD4+ (+++)
`– CD4+CD8αα+ (+)
`
`Absent at
`birth; increase
`with age
`
`Drive excessive
`inflammation
`and cytotoxicity
`in response to
`microbiota-
`derived
`antigens;
`exacerbate IBD
`Plus symbols represent frequency of expression, from low (+) to high (+++++). CTLA4, cytotoxic T lymphocyte A4; IBD, inflammatory bowel disease;
`IEL,  intraepithelial lymphocyte; NK, natural killer; TCR, T cell receptor.
`
`Protective
`antimicrobial
`responses
`
`‘Conventional’
`positive selection
`in the thymus;
`polyclonal TCR
`repertoire; restricted
`on MHC class I or
`MHC class II
`
`in contrast to systemic and lamina propria lymphocytes
`(LPLs), CD4+ cells are greatly under-represented in the
`IEL compartment, especially in the small intestine28,29.
`Although all IELs have an antigen-experienced phe-
`notype, they can be divided into two major subsets based
`on the mechanisms by which they become activated
`and on the cognate antigens that they recognize. The
`
`‘natural’ IELs (which were previously known as ‘type b’
`IELs)5 acquire their activated phenotype during develop-
`ment in the thymus in the presence of self antigens, whereas
`the ‘induced’ IELs (which were previously known as ‘type a’
`IELs)5 are the progeny of conventional T cells that are
`activated post-thymically in response to peripheral
`antigens (FIG. 1; TABLE 1).
`
`446 | JULY 2011 | VOLUME 11
`
` www.nature.com/reviews/immunol
`
`© 2011 Macmillan Publishers Limited. All rights reserved
`
`Genome Ex. 1058
`Page 2 of 12
`
`

`

`Box 1 | CD8αα as a repressor
`CD8αα, which is composed of two CD8α subunits, can be induced on activation
`through the T cell receptor (TCR)–CD3 complex, with the level of its expression
`being proportional with the signal strength. Therefore, CD8αα can be used as an
`activation marker for T cells43. CD8αα has the ability to recruit TCR–CD3 signalling
`components, including LCK (also known as p56LCK) and linker for activation of
`T cells (LAT)165. However, owing to its physical separation from the TCR-activation
`complex166, CD8αα neither functions as a TCR co-receptor nor can it substitute for
`the CD4 or CD8αβ co-receptors (see the figure, part a). Consistent with this, CD8αα
`is unable to support positive selection of MHC class I-restricted thymocytes or
`promote activation of mature MHC class I-restricted T cells167. Instead, co-expression
`of CD8αα on CD8αβ+ (or CD4+) T cells downmodulates, rather than enhances, the
`functional avidity of the MHC–TCR activation complex113. The ability of CD8αα to
`function as a negative regulator is partly due to its exclusion from the TCR activation
`complex (see the figure, part b). Consequently, CD8αα can sequester signalling
`components outside the immunological synapse, thereby interfering with their
`recruitment to the CD8αβ or the CD4 co-receptor–TCR–CD3 activation complex.
`Thus, CD8αα serves as a TCR repressor (see the figure, part b) rather than as a
`TCR co-receptor (see the figure, part a). See REF. 43 for further details.
`
`Ag, antigen; APC, antigen-presenting cell; ITAM, immunoreceptor tyrosine-based
`activation motif.
`
`(cid:67)
`
`(cid:35)(cid:50)(cid:37)
`
`(cid:54)(cid:2)(cid:69)(cid:71)(cid:78)(cid:78)
`
`(cid:68)
`
`(cid:37)(cid:38)(cid:26)β
`(cid:37)(cid:38)(cid:26)α
`
`(cid:47)(cid:42)(cid:37)(cid:2)(cid:69)(cid:78)(cid:67)(cid:85)(cid:85)(cid:2)(cid:43)
`(cid:35)(cid:73)
`(cid:54)(cid:37)(cid:52)αβ
`
`(cid:37)(cid:38)(cid:26)α
`(cid:37)(cid:38)(cid:26)α
`
`(cid:37)(cid:38)(cid:26)β
`(cid:37)(cid:38)(cid:26)α
`
`(cid:46)(cid:35)(cid:54)
`
`ε γ ζζ δδ
`
`(cid:46)(cid:35)(cid:54)
`
`(cid:46)(cid:35)(cid:54)
`
`(cid:46)(cid:35)(cid:54)
`
`ε γ ζζ δδ
`
`(cid:46)(cid:37)(cid:45)
`
`(cid:46)(cid:75)(cid:82)(cid:75)(cid:70)(cid:2)(cid:84)(cid:67)(cid:526)
`
`(cid:43)(cid:54)(cid:35)(cid:47)
`
`(cid:46)(cid:37)(cid:45)
`
`(cid:46)(cid:37)(cid:45)
`
`(cid:46)(cid:37)(cid:45)
`
`(cid:54)(cid:37)(cid:52)(cid:2)(cid:85)(cid:75)(cid:73)(cid:80)(cid:67)(cid:78)
`
`(cid:37)(cid:38)(cid:26)αα(cid:2)(cid:85)(cid:87)(cid:82)(cid:82)(cid:84)(cid:71)(cid:85)(cid:85)(cid:71)(cid:85)
`(cid:54)(cid:37)(cid:52)(cid:2)(cid:85)(cid:75)(cid:73)(cid:80)(cid:67)(cid:78)(cid:78)(cid:75)(cid:80)(cid:73)
`
`(cid:54)(cid:37)(cid:52)(cid:2)(cid:85)(cid:75)(cid:73)(cid:80)(cid:67)(cid:78)(cid:78)(cid:75)(cid:80)(cid:73)
`
`Thymus leukaemia antigen
`(TLA). A non-polymorphic,
`non-classical MHC class I
`molecule (MHC class I-b family)
`with a restricted expression
`pattern. It is constitutively
`expressed on intestinal epithelial
`cells and can be induced on
`antigen-presenting cells. TLA is
`structurally incapable of binding
`or presenting peptide antigens
`and it does not engage with
`T cell receptors. However, the
`α3 extracellular domain of
`TLA interacts with CD8α. TLA
`displays stronger affinity for
`CD8αα homodimers compared
`with CD8αβ heterodimers, and
`CD8αα expression can be
`detected with TLA-specific
`tetramers.
`
`(cid:48)(cid:67)(cid:86)(cid:87)(cid:84)(cid:71)(cid:2)(cid:52)(cid:71)(cid:88)(cid:75)(cid:71)(cid:89)(cid:85)(cid:2)(cid:94)(cid:2)(cid:43)(cid:79)(cid:79)(cid:87)(cid:80)(cid:81)(cid:78)(cid:81)(cid:73)(cid:91)
`
`Natural IELs. Natural IELs are either CD8αα+ or
`CD8αα– T cells that express TCRγδ or TCRαβ but
`do not express either CD4 or CD8αβ. Typically, they
`are also negative for expression of CD2, CD5, CD28,
`lymphocyte function-associated antigen 1 (LFA1; also
`known as αLβ2 integrin) and THY1 (REFS 9,28,30,31).
`In addition, the majority of TCRαβ+ natural IELs
`are B220+CD44low/midCD69+, a phenotype that is
`rarely found among peripheral T cells32,33. Many of
`the natural IELs also share molecules expressed by
`NK cells, such as CD16, CD122, DNAX-activation
`protein 12 (DAP12; also known as TYROBP), lympho-
`cyte antigen 49A (Ly49A), Ly49E, Ly49G2, and NK1.1
`(REFS 9,18,34,35). Lastly, they frequently express a CD3
`complex that is composed of either CD3ζ–FcεRIγ
`heterodimers or FcεRIγ–FcεRIγ homodimers instead
`of the CD3ζ–CD3ζ homodimers that are expressed by
`conventional T cells9,36–38.
`
`R E V I E W S
`
`Induced IELs. Induced IELs arise from conventional
`CD4+ or CD8αβ+ TCRαβ+ T cells, which are MHC
`class II-restricted and MHC class I-restricted, respec-
`tively. In contrast to the natural IELs, induced IELs
`acquire an activated phenotype in response to cog-
`nate antigens encountered in the periphery 33,39–42.
`Consistent with this, they typically express a ‘memory-
`like phenotype’ (CD2+CD5+CD44+LFA1+THY1+)9,28,30,
`together with the activation marker CD8αα43 (BOX 1).
`Contrary to the polyclonal nature of the conventional
`T cells in the periphery, the TCR repertoire of induced
`IELs is oligoclonal and does not significantly overlap
`with the limited TCR repertoire of the natural TCRαβ+
`IEL compartment44.
`
`Thymic development of IEL precursor cells
`Similarly to peripheral T cells, all IEL subsets are prog-
`eny of bone marrow precursor cells that initially develop
`in the thymus45. However, thymocyte maturation is not
`uniform, and multiple pathways exist that ultimately
`determine the diversity of mature T cells, including IELs.
`
`Development of natural IELs. The origin and develop-
`ment of TCRγδ and TCRαβ natural IELs are the subjects
`of long-standing debates24,46–49 (BOX 2). For an overview
`of recent advances on this topic, we refer the reader to
`previously published reviews6,45,50,51. Briefly, the term
`‘natural’ refers to the ontogeny of the precursor cells.
`For the natural IELs, these precursors go through an
`‘alternative’ self-antigen-based thymic maturation
`process that results in the functional differentiation
`of mature CD4 and CD8αβ double-negative, TCRγδ-
`expressing or TCRαβ-expressing T cells that directly
`migrate to the intestinal epithelium23,24,52 (FIG. 1).
`
`Development of induced IELs. Induced IELs are the prog-
`eny of conventional CD4+ or CD8αβ+ TCRαβ+ T cells
`that are selected in the thymus. The development of
`conventional TCRαβ+ thymocytes, including selection
`and lineage commitment has been extensively reviewed
`elsewhere53–55. Following positive selection, mature
`thymocytes exit the thymus and reach the periphery as
`conventional naive CD4+ or CD8αβ+ TCRαβ+ T cells. In
`response to cognate antigens, they can further mature
`into antigen-experienced cells, including induced
`IELs (FIG. 1).
`
`Local differentiation of IELs
`Because of the antigen-experienced phenotype that
`they acquire in the thymus, one can assume that the
`repertoire of the natural IELs is predominantly tuned to
`self antigens, whereas induced IELs are mainly shaped
`by non-self antigens encountered in the periphery.
`Consequently, induced IELs are sparse early in life, but
`the population steadily increases with age in response
`to exposure to exogenous antigens56–59 (FIG. 2; TABLE 1).
`The gradual accumulation of induced IELs allows the
`mucosal immune system to adapt and develop an almost
`‘personalized’ mucosal immune repertoire that is directed
`against those environmental antigens that are most
`likely to be re-encountered by a particular individual.
`
`NATURE REVIEWS | IMMUNOLOGY
`
` VOLUME 11 | JULY 2011 | 447
`
`© 2011 Macmillan Publishers Limited. All rights reserved
`
`Genome Ex. 1058
`Page 3 of 12
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`

`R E V I E W S
`
`(cid:54)(cid:74)(cid:91)(cid:79)(cid:87)(cid:85)
`
`(cid:54)(cid:84)(cid:75)(cid:82)(cid:78)(cid:71)(cid:15)
`(cid:80)(cid:71)(cid:73)(cid:67)(cid:86)(cid:75)(cid:88)(cid:71)
`(cid:86)(cid:74)(cid:91)(cid:79)(cid:81)(cid:69)(cid:91)(cid:86)(cid:71)
`
`(cid:38)(cid:81)(cid:87)(cid:68)(cid:78)(cid:71)(cid:15)
`(cid:82)(cid:81)(cid:85)(cid:75)(cid:86)(cid:75)(cid:88)(cid:71)
`(cid:86)(cid:74)(cid:91)(cid:79)(cid:81)(cid:69)(cid:91)(cid:86)(cid:71)
`
`(cid:47)(cid:46)(cid:48)(cid:85)(cid:2)(cid:81)(cid:84)(cid:2)(cid:50)(cid:71)(cid:91)(cid:71)(cid:84)(cid:111)(cid:85)(cid:2)(cid:82)(cid:67)(cid:86)(cid:69)(cid:74)(cid:71)(cid:85)
`(cid:50)(cid:71)(cid:82)(cid:86)(cid:75)(cid:70)(cid:71)(cid:115)(cid:47)(cid:42)(cid:37)
`(cid:54)(cid:37)(cid:52)
`
`(cid:43)(cid:80)(cid:86)(cid:71)(cid:85)(cid:86)(cid:75)(cid:80)(cid:71)
`
`(cid:110)(cid:37)(cid:81)(cid:80)(cid:88)(cid:71)(cid:80)(cid:86)(cid:75)(cid:81)(cid:80)(cid:67)(cid:78)(cid:111)
`(cid:82)(cid:81)(cid:85)(cid:75)(cid:86)(cid:75)(cid:88)(cid:71)
`(cid:85)(cid:71)(cid:78)(cid:71)(cid:69)(cid:86)(cid:75)(cid:81)(cid:80)
`
`(cid:37)(cid:38)(cid:22)(cid:13)(cid:2)
`(cid:54)(cid:37)(cid:52)αβ(cid:13)
`
`(cid:35)(cid:50)(cid:37)
`
`(cid:48)(cid:67)(cid:75)(cid:88)(cid:71)(cid:2)(cid:37)(cid:38)(cid:22)(cid:13)(cid:2)
`(cid:54)(cid:37)(cid:52)αβ(cid:13)(cid:2)(cid:54)(cid:2)(cid:69)(cid:71)(cid:78)(cid:78)
`
`(cid:37)(cid:38)(cid:26)αβ(cid:13)
`(cid:54)(cid:37)(cid:52)αβ(cid:13)
`
`(cid:35)(cid:50)(cid:37)
`
`(cid:48)(cid:67)(cid:75)(cid:88)(cid:71)(cid:2)(cid:37)(cid:38)(cid:26)αβ(cid:13)
`(cid:54)(cid:37)(cid:52)αβ(cid:13)(cid:2)(cid:54)(cid:2)(cid:69)(cid:71)(cid:78)(cid:78)
`
`(cid:39)(cid:82)(cid:75)(cid:86)(cid:74)(cid:71)(cid:78)(cid:75)(cid:67)(cid:78)
`(cid:69)(cid:71)(cid:78)(cid:78)
`
`(cid:54)(cid:37)(cid:52)γδ(cid:13)(cid:2)
`(cid:80)(cid:67)(cid:86)(cid:87)(cid:84)(cid:67)(cid:78)(cid:2)(cid:43)(cid:39)(cid:46)(cid:2)
`(cid:10)(cid:37)(cid:38)(cid:26)αα(cid:13)(cid:17)(cid:115)(cid:11)(cid:2)
`
`(cid:37)(cid:38)(cid:22)(cid:13)
`(cid:75)(cid:80)(cid:70)(cid:87)(cid:69)(cid:71)(cid:70)(cid:2)(cid:43)(cid:39)(cid:46)
`(cid:10)(cid:37)(cid:38)(cid:26)αα(cid:13)(cid:17)(cid:115)(cid:11)
`
`(cid:37)(cid:38)(cid:26)αβ(cid:13)
`(cid:75)(cid:80)(cid:70)(cid:87)(cid:69)(cid:71)(cid:70)(cid:2)(cid:43)(cid:39)(cid:46)
`(cid:10)(cid:37)(cid:38)(cid:26)αα(cid:13)(cid:17)(cid:115)(cid:11)
`
`(cid:54)(cid:84)(cid:75)(cid:82)(cid:78)(cid:71)(cid:15)
`(cid:82)(cid:81)(cid:85)(cid:75)(cid:86)(cid:75)(cid:88)(cid:71)
`(cid:86)(cid:74)(cid:91)(cid:79)(cid:81)(cid:69)(cid:91)(cid:86)(cid:71)
`
`(cid:110)(cid:35)(cid:78)(cid:86)(cid:71)(cid:84)(cid:80)(cid:67)(cid:86)(cid:75)(cid:88)(cid:71)(cid:111)
`(cid:82)(cid:81)(cid:85)(cid:75)(cid:86)(cid:75)(cid:88)(cid:71)
`(cid:85)(cid:71)(cid:78)(cid:71)(cid:69)(cid:86)(cid:75)(cid:81)(cid:80)
`
`(cid:38)(cid:81)(cid:87)(cid:68)(cid:78)(cid:71)(cid:15)
`(cid:80)(cid:71)(cid:73)(cid:67)(cid:86)(cid:75)(cid:88)(cid:71)(cid:2)
`(cid:54)(cid:37)(cid:52)αβ(cid:13)
`
`(cid:54)(cid:37)(cid:52)αβ(cid:13)(cid:2)(cid:80)(cid:67)(cid:86)(cid:87)(cid:84)(cid:67)(cid:78)(cid:2)(cid:43)(cid:39)(cid:46)
`(cid:10)(cid:37)(cid:38)(cid:26)αα(cid:13)(cid:17)(cid:115)(cid:11)(cid:2)
`
`(cid:38)(cid:81)(cid:87)(cid:68)(cid:78)(cid:71)(cid:15)
`(cid:80)(cid:71)(cid:73)(cid:67)(cid:86)(cid:75)(cid:88)(cid:71)(cid:2)
`(cid:54)(cid:37)(cid:52)γδ(cid:13)
`
`(cid:48)(cid:67)(cid:86)(cid:87)(cid:84)(cid:71)(cid:2)(cid:52)(cid:71)(cid:88)(cid:75)(cid:71)(cid:89)(cid:85)(cid:2)(cid:94)(cid:2)(cid:43)(cid:79)(cid:79)(cid:87)(cid:80)(cid:81)(cid:78)(cid:81)(cid:73)(cid:91)
`Figure 1 | Thymic and peripheral differentiation of natural and induced IELs. In the thymus, immature
`CD4+CD8αβ+CD8αα+ (triple positive) thymocytes undergo agonist (‘alternative’) selection and differentiate into
`double-negative T cell receptor-αβ (TCRαβ)+ cells that are the precursors of natural TCRαβ+ intraepithelial
`lymphocytes (IELs). The TCRαβ+ natural IEL precursor cells partly acquire their antigen-experienced phenotype
`‘naturally’ during selection with self antigens. In addition, the precursor cells for TCRαβ+ and TCRγδ+ natural IELs
`upregulate intestinal homing receptors during their maturation in the thymus, which allows these cells to directly
`seed the intestinal epithelium after they leave the thymus. CD4+CD8αβ+ (double positive) thymocytes undergo
`‘conventional’ thymic selection and differentiate into naive CD4+ and CD8αβ+ TCRαβ+ T cells that migrate to the
`periphery. These naive T cells can differentiate into effector T cells in response to peripheral antigens and subsequently
`migrate to the gut and become incorporated into the induced IEL compartment. APC, antigen-presenting cell;
`MLNs, mesenteric lymph nodes.
`
`The development of these antigen-specific immune cells
`not only provides focused protective immunity at this
`mucosal interface but, at the same time, also reduces the
`risk of unwanted immune responses directed against
`innocuous antigens. By contrast, natural IELs do not
`depend on exogenous antigen-driven differentiation and
`so they are the first type of antigen-experienced T cells
`to populate the gut, even before birth60 (FIG. 2). Although
`direct evidence is still lacking, it is likely that the early
`accumulation of natural IELs provides a self-antigen-
`based, stress-sensing surveillance mechanism that is
`tolerant to dietary antigens and colonizing microbiota
`but provides protective immunity against stress-induc-
`ing invasive pathogens. With time, the population of
`induced IELs gradually becomes larger than the natural
`IEL population, which remains steady in actual numbers
`but represents a minor IEL population at later stages in
`life (FIG. 2).
`Nevertheless, regardless of the nature of the cog-
`nate antigens or the location of the initial differentia-
`tion, all IELs are directly influenced by the intestinal
`environment61. This was demonstrated with elegant
`studies in ‘germ-free’ and ‘antigen-free’ mice (the germ-
`free mice were fed an elementary diet), which showed
`that both the microbiota and dietary proteins have
`a crucial role in the establishment of a normal IEL
`
`repertoire, as virtually all IEL populations, with the
`exception of TCRγδ-expressing T cells, were mark-
`edly reduced in such an antigen-deprived environ-
`ment62–64. Interestingly, although more than 95% of
`the commensal bacteria in the body normally reside in the
`large intestine, the small intestine contains at least ten
`times more IELs than the colon6. Furthermore, mice
`fed an amino-acid-based, protein-free diet, displayed
`a poorly developed intestinal immune system, simi-
`lar to that of germ-free mice, with a strong decrease
`in most IEL populations65. These effects could be due
`to direct effects of the diet on the immune system or
`could result from diet-induced shaping of the intesti-
`nal microbiota. This highlights the importance of the
`diet, in addition to the microbiota, as a major driv-
`ing factor in establishing and shaping this mucosal
`immune branch.
`
`Migration of natural IELs. In addition to their anti-
`gen-experienced phenotype, natural IEL precursors
`may also acquire expression of gut-homing receptors
`(including αEβ7 integrin and CC-chemokine recep-
`tor 9 (CCR9)) in the thymus21,22,66–68. The expression of
`the ligands for αEβ7 integrin and CCR9, E-cadherin
`and CC-chemokine ligand 25 (CCL25), respectively,
`by small intestinal epithelial cells results in the direct
`
`Lamina propria
`Connective tissue that
`underlies the epithelium of the
`mucosa and contains various
`myeloid and lymphoid cells,
`including macrophages,
`dendritic cells, T cells and
`B cells.
`
`Microbiota
`The microorganisms present
`in normal, healthy individuals.
`These microorganisms live
`mostly in the digestive tract
`but are also found in some
`other tissues.
`
`Germ-free mice
`Mice born and raised in sterile
`isolators. They are devoid of
`colonizing microorganisms,
`but after they have been
`experimentally colonized by
`known bacteria, they are said
`to be gnotobiotic.
`
`448 | JULY 2011 | VOLUME 11
`
` www.nature.com/reviews/immunol
`
`© 2011 Macmillan Publishers Limited. All rights reserved
`
`Genome Ex. 1058
`Page 4 of 12
`
`

`

`Box 2 | The distinct pathways of CD8αα+TCRαβ+ IEL differentiation
`This Review does not aim to discuss the debates surrounding the importance of the
`thymic versus the extrathymic differentiation pathway for CD8αα+ T cell receptor-αβ
`(TCRαβ+) intestinal epithelial lymphocytes (IELs), as this has been extensively reviewed
`elsewhere47,48,168. Here, we give a brief overview of the data that have challenged or
`supported the thymic differentiation pathway. CD8αα+ IELs were originally thought to
`differentiate locally in the gut. This attractive idea was fuelled, in part, by the presence
`of self-reactive TCRs in their TCR repertoire, by supporting data derived from studies
`using athymic mouse models, and by the presence of haematopoietic immature cells
`in the small intestine that display characteristics of T cell precursor cells169–171.
`Nevertheless, ample data indicate that the absolute numbers of CD8αα+TCRαβ+ IELs
`in athymic mice are extremely reduced compared with euthymic mice16,172–174. These
`data therefore support the notion that the vast majority of CD8αα+TCRαβ+ IELs are
`the progeny of cells with a thymic origin. Other data suggest that, under certain
`experimental conditions, immature T cell-committed precursors may leave the
`thymus prematurely, before TCR rearrangements have occurred, and complete their
`maturation locally in the gut175. In conclusion, if indeed the gut contains precursors of
`bone marrow and thymic origin that have the potential under physiological conditions
`to further differentiate locally, multiple crucial questions remain unanswered. For
`example, under what circumstances is this local pathway functional? And how can
`the gut environment support selection? However, there is currently no evidence
`available to answer these questions.
`
`Gut-associated lymphoid
`tissues
`Lymphoid structures and
`aggregates associated with the
`intestinal mucosa, specifically
`the tonsils, Peyer’s patches,
`lymphoid follicles, appendix
`and caecal patch. Enriched in
`lymphocytes and specialized
`dendritic cell and macrophage
`subsets.
`
`Peyer’s patches
`Groups of lymphoid nodules
`present in the small intestine
`(usually the ileum). They occur
`in the intestinal wall, opposite
`the line of attachment of the
`mesentery. They consist of a
`dome area, B cell follicles and
`interfollicular T cell areas. High
`endothelial venules are present
`mainly in the interfollicular
`areas.
`
`Mesenteric lymph nodes
`Lymph nodes, located at the
`base of the mesentery, that
`collect lymph (including cells
`and antigens) draining from the
`intestinal mucosa.
`
`Microfold cells
`(M cells). Specialized
`antigen-sampling cells that are
`located in the follicle-associated
`epithelium of the organized
`mucosa-associated lymphoid
`tissues. M cells deliver antigens
`by transepithelial vesicular
`transport from the
`aero-digestive lumen directly to
`subepithelial lymphoid tissues
`of nasopharynx-associated
`lymphoid tissue and Peyer’s
`patches.
`
`recruitment of mature natural IEL precursors to the
`small intestinal epithelium24,66. Consistent with this,
`the seeding of mucosal tissues with these cells is inde-
`pendent of sphingosine-1-phosphate receptor 1 (S1P1,
`also known as S1PR1), which is required for the migra-
`tion of conventionally-selected T cells69. The direct
`and early population of the mucosal barrier with these
`stress-sensing self-reactive effector cells provides a
`layer of pre-existing immunity before the development
`of antigen-specific immune cells in response to exog-
`enous antigens (FIG. 2). Additional local endogenous
`and exogenous factors, such as the cytokine IL-15 and
`vitamins A and D, further expand and adapt the natu-
`ral IEL compartment. In light of this, a recent study
`showed that vitamin D receptor (VDR)-deficient mice
`had reduced numbers of CD8αα+ IELs and that this
`co incided with low levels of IL-10 in the small intestine
`and increased inflammation in the steady state70.
`
`Migration of induced IELs. One study showed that,
`similarly to natural IELs, CD8αβ+ recent thymic emi-
`grants (RTEs) can directly migrate to the small intesti-
`nal compartment66. However, in general, conventionally
`selected T cells do not express mucosal homing recep-
`tors and naive T cells are normally not detected within
`the intestinal epithelium. Instead, naive T cells acquire
`gut-homing capacity following priming in gut-associated
`lymphoid tissues (GALTs), such as Peyer’s patches, and in
`mesenteric lymph nodes (MLNs)71. Specialized intesti-
`nal epithelial cells or microfold cells (M cells), resident
`CX3C-chemokine receptor 1 (CX3CR1)+ macrophages
`and migratory CD103+ dendritic cells (DCs) all have
`the capacity to sample antigens in the gut and pro-
`mote appropriate T cell responses42,72–75. On priming in
`mucosal sites, naive T cells upregulate homing receptors
`that allow them to enter intestinal tissues in response
`to specific ligands expressed by the tissues76. These
`receptor–ligand pairs include: LFA1 and intercellular
`
`R E V I E W S
`
`adhesion molecule 1 (ICAM1); very late antigen 1 (VLA1;
`also known as α1β1 integrin) and collagen; and α4β7
`integrin and mucosal vascular addressin cell adhe-
`sion molecule 1 (MADCAM1). α4β7 integrin can also
`be induced on T cells primed in other sites, allowing
`peripherally activated cells to migrate to the intestine71.
`Upregulation of CCR9 expression further directs
`T cells to the small intestine compartment in response
`to its ligand, CCL25, which is constitutively expressed
`by intestinal epithelial cells.
`Induction of the receptors that promote migration
`of antigen-experienced T cells to the intestine can be
`promoted by environmental factors that are unique
`to the gut. It was shown that a diet-derived factor,
`the vitamin A metabolite retinoic acid, is an impor-
`tant inducer of gut-homing molecules. Retinoic acid
`promotes upregulation of α4β7 integrin and CCR9,
`thereby directing activated T cells mainly to the small
`intestine77. Although migration to the colon is also
`dependent on α4β7 integrin78, retinoic acid seems to
`be neither necessary nor sufficient to induce migra-
`tion to this site78,79. The ability to produce retinoic acid
`and to imprint gut-homing receptors during T cell
`priming is characteristic of the CCR7+CD103+ migra-
`tory DC subset77, which have been programmed previ-
`ously by retinoic acid from intestinal epithelial cells and
`stromal cells80,81. It is important to note that mucosal
`imprinting is not absolutely required for entering the
`intestinal tissues, and T cells primed in the periph-
`ery also readily migrate to the gut as effector cells.
`However, in response to gut-derived antigens, the
`imprinting process might greatly focus the immune
`response to the mucosal effector site.
`The main adhesion molecule that is involved in the
`specific localization of IELs in the ep