RESEARCH HIGHLIGHTS

T- C E L L D E V E LO P M E N T

Location, location, location
During development, thymocytes
follow a particular migratory pathway in the thymus that allows themto interact with distinct populations of stromal cells, but the precise mechanisms that control
intrathymic localization and migration are not well understood. Now,
two studies — one focusing on themigration of early thymic immigrants from the corticomedullary
region to the subcapsular region,and the other focusing on themigration of positively selected thymocytes from the cortex to themedulla — report that signallingthrough CC-chemokine receptor 7(CCR7) is crucial for intrathymic
migration and for the maintenance ofthymic architecture.

In the first study, Misslitz andcolleagues found that CCR7 isexpressed mainly by double-negative(DN) thymocytes and by semimature and mature single-positive(SP) thymocytes. CCR7-deficient
mice and plt/plt mice (which are
deficient in the CCR7 ligands
CCL19 and CCL21) were found tohave abnormal thymic architecture.
By 6–8 weeks of age, CCR7-deficientand plt/plt mice also had reduced
numbers of thymocytes but normal
proportions of SP and double-positive(DP) cells. By contrast, older mice
had reduced numbers of DP cells,
which correlated with a severe blockin T-cell transition at early DN
stages. Both types of mouse produced normal T cells, althoughfewer than wild-type mice, indicating that CCR7 or CCL19/CCL21
deficiency can be compensated in an
unknown way. Next, the authors
examined the anatomical localization of the DN cells and found that

M U C O S A L I M M U N O LO G Y

Recognizing the good guys
The recognition of pathogenic bacteria byToll-like receptors (TLRs) has a crucial rolein eliciting protective innate immuneresponses. However, it is not known how
TLRs can distinguish between pathogenic
and commensal bacteria — which sharepathogen-associated molecular patterns
such as lipopolysaccharide (LPS). In thegut, it is thought that inappropriate responses
to commensal bacteria can result in
inflammatory bowel diseases such as Crohn’sdisease. Now, new research published inCell shows that TLR-mediated recognitionof commensal bacteria, rather than being an
unwanted side-effect, is actually importantfor maintaining epithelial integrity and fortissue repair.
Using oral administration of dextran
sulphate sodium (DSS) as a model ofintestinal injury and inflammation,
Medzhitov and colleagues show that mice
deficient in MyD88 — an essential adaptor
molecule for TLR signalling — have increased
morbidity and mortality after DSS
administration compared with wild-type
mice. Similar results were obtained using
TLR2- or TLR4-deficient mice. Death was

associated with colonic epithelial injury,
severe colonic bleeding and anaemia.
The increased epithelial injury was not due
to uncontrolled growth of commensal bacteria
in the absence of restrictive TLR-dependent
responses, because commensal depletion
using antibiotics did not improve disease.
Neither were there any differences in leukocyte
infiltration of the colonic epithelium between
wild-type and MyD88-deficient mice. Instead,they found that TLR signalling through
MyD88 controls homeostasis of the intestinal
epithelium. MyD88-deficient mice had
increased basal levels of proliferation ofcolonic-crypt cells, making these cells moresusceptible to damage. As a result, intestinal
tissue was unable to undergo compensatory
proliferation in response to epithelial injury
caused by γ-irradiation. These mice also had
decreased production of cytoprotective andrepair factors, such as interleukin-6 and heatshock proteins, both before and after DSS
administration.
These results indicated a pathway by which
TLR signalling through MyD88 can protect
against the colonic epithelial injury caused byDSS. To show that commensal bacteria trigger

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they accumulated at the corticomedullary junction in older mice.
Misslitz and colleagues suggest apossible explanation for the worsening defect in older mice: thymocytes
in young mice are derived from fetal
progenitors, which enter the immature thymus when it is an unstructured organ; however, after a few
weeks, progenitors enter a structured organ in which signals from
CCR7 might be more crucial formigration.
In the second study, Ueno andcolleagues also studied mice deficient in signals from CCR7. In support of their previous studies, theyobserved defective T-cell production in the neonatal period but normal peripheral T-cell numbers in
adult mice. Thymic architecture wasalso found to be abnormal in bothyoung and adult mice, and mature
SP thymocytes accumulated in thecortex. This study shows that thedefect is worse in newborn mice,

this signalling pathway, the authors depleted
wild-type mice of all detectable commensal
bacteria using antibiotics and then treatedthem with DSS. The mice suffered severe
morbidity and mortality, similar to MyD88deficient animals. The administration ofpurified LPS or lipoteichoic acid — TLR
ligands expressed by Gram-negative andGram-positive bacteria, respectively —completely protected animals.
Medzhitov and colleagues therefore propose
a two-step model that involves epithelial
maintenance through the constitutive
detection of commensal bacteria by TLRs
present on intestinal epithelial cells, andepithelial repair through the detection ofcommensals that breach the epithelial barrier
by TLRs present on underlying fibroblasts
and macrophages. Because many clinicaltreatments that are associated with intestinal
damage, such as chemotherapy, areaccompanied by antibiotic treatment toprevent opportunistic infections, thediscovery of this new pathway for thebeneficial effects of commensal bacteria
has important clinical implications.
Kirsty Minton
References and links
ORIGINAL RESEARCH PAPER Rakoff-Nahoun, S.,
Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov, R.
Recognition of commensal microflora by Toll-like receptors isrequired for intestinal homeostasis. Cell 118, 229–241 (2004).
FURTHER READING Macpherson, A. J. & Harris, N. L.
Interactions between commensal intestinal bacteria and theimmune system. Nature Rev. Immunol. 4, 478–485 (2004).

www.nature.com/reviews/immunol©2004 Nature Publishing GroupRESEARCH HIGHLIGHTS
and adult mice can export T cells tothe periphery in the absence of signals from CCR7. The authors suggestthat CCR7-dependent migration to
the medulla is more important for
the export of T cells from the thymus in newborns than in adultmice, and they highlight the factthat REL-B-deficient mice, whichlack thymic medullas, can exportfunctional T cells.
These two studies show thatsignalling through CCR7 is important for intrathymic migration ofT cells, but there is clearly more tobe learned.
Elaine Bell
References and links
ORIGINAL RESEARCH PAPERS Misslitz, A.

et al. Thymic T cell development and progenitor
localization depend on CCR7. J. Exp. Med. 200,481–491 (2004) | Ueno, T. et al. CCR7 signals are
essential for cortex–medulla migration ofdeveloping thymocytes. J. Exp. Med. 200,493–505 (2004).
FURTHER READING Petrie, H. T. Cell migrationand the control of post-natal T-cell lymphopoiesis
in the thymus. Nature Rev. Immunol. 3, 859–866(2003).

B CELLS

AID provided by RPA
Antibody diversification through somatic
hypermutation (SHM) — the process by which
non-templated point mutations are introduced
into the variable region of immunoglobulin heavy
and light chains, thereby enabling the selection ofB cells that generate higher-affinity antibodies —
requires the single-stranded DNA (ssDNA)
deaminase known as activation-induced cytidine
deaminase (AID). However, the way in which AID
is targeted to sites of mutation was unclear until
a paper published in Nature identified replication
protein A (RPA) as crucial for this process.
More than half of all SHMs occur around a
DNA motif known as the RGYW motif (whereR denotes A or G, Y denotes C or T, and W denotes
A or T), leading to the suggestion that unknown
factors might target AID to these sites. To identify
these putative cofactors Chaudhuri et al. used anin vitro assay for AID activity. This assay relied on
the observation that although AID purified from
B cells can by itself deaminate ssDNA templates,
it cannot by itself deaminate an actively transcribeddouble-stranded DNA SHM substrate (that is,a substrate containing multiple RGYW motifs
that cannot form R loops — structures generated
during transcription that displace the nontemplate strand as ssDNA). The ability of protein
fractions isolated from B cells to facilitate AID
deamination of the SHM substrate led to theidentification of a heat-labile complementing
factor (CF) of 120–170 kDa.
In further experiments, a 30–35 kDa proteinthat was highly enriched in fractions containing
CF activity was shown to associate with AID.
Subsequent analysis identified this as the 32 kDa
subunit of RPA (RPA32). RPA is heterotrimeric— consisting of a 70 kDa (RPA70) and a 14 kDa(RPA14) subunit, in addition to RPA32 — andNATURE REVIEWS | IMMUNOLOGY

has previously been implicated in DNA
replication, recombination and repair.
Confirmation of RPA as the CF was provided
by the observation that recombinant human
RPA containing all three subunits could replace
CF in facilitating AID deamination of the activelytranscribed SHM substrate. As well as complexes
that contain AID, RPA and RGYW-containing
DNA substrates, smaller complexes that contain
only RPA and the RGYW-containing substrate
were observed. Because these smaller complexes
were not detected when the deamination reaction
was inhibited, the authors suggest that AIDmediated deamination leads to the release ofAID from the AID–RPA–DNA complex andthat the DNA-bound RPA might enable therecruitment of DNA-repair proteins.
Interestingly, AID that was purified fromthe 293 non-lymphoid cell line ectopically
expressing AID was unable to mediate
deamination of the actively transcribed SHM
substrate in the presence of RPA, and RPA andAID did not associate in this cell line. Because
AID was found to be more highly
phosphorylated in B cells than in the 293 cellline, these results indicate that the AID–RPAinteraction is B-cell specific and that thisspecificity probably depends, at least in part,on the post-translational modification of AID.
This study identified RPA as the factor thattargets AID to RGYW DNA motifs — that is,
the sites of SHM — and it defined a mechanism
by which SHM is a B-cell specific event. Further
studies will focus on whether RPA has a similarfunction in class-switch recombination (whichalso requires AID), an outcome that the authors’
initial experiments support.
Karen Honey
References and links
ORIGINAL RESEARCH PAPER Chaudhuri, J., Khuong, C. &Alt, F. W. Replication protein A interacts with AID to promotedeamination of somatic hypermutation targets. Nature 25 July 2004
(doi:10.1038/nature02821).
FURTHER READING Chaudhuri, J. & Alt, F. W. Class-switch
recombination: interplay of transcription, DNA deamination
and DNA repair. Nature Rev. Immunol. 4, 541–552 (2004).

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