cd8+ recent thymic emigrants home to and efficiently repopulate the small intestine epithelium
TRANSCRIPT
CD8+ recent thymic emigrants home to and efficientlyrepopulate the small intestine epithelium
Tracy L Staton1, Aida Habtezion2, Monte M Winslow1, Tohru Sato2, Paul E Love4 & Eugene C Butcher1–3
Prevailing knowledge dictates that naive ab T cells require activation in lymphoid tissues before differentiating into effector
or memory T cells capable of trafficking to nonlymphoid tissues. Here we demonstrate that CD8+ recent thymic emigrants
(RTEs) migrated directly into the small intestine. CCR9, CCL25 and a4b7 integrin were required for gut entry of CD8+ RTEs.
After T cell receptor stimulation, intestinal CD8+ RTEs proliferated and acquired a surface phenotype resembling that of
intraepithelial lymphocytes. CD8+ RTEs efficiently populated the gut of lymphotoxin-a-deficient mice, which lack lymphoid
organs. These studies challenge the present understanding of naive ab T cell trafficking and suggest that RTEs may be
involved in maintaining a diverse immune repertoire at mucosal surfaces.
The immune system is compartmentalized into primary, secon-dary and tertiary organs1. Mature lymphocytes are generated inprimary lymphoid organs. After export, these naive lymphocytes arethought to recirculate in the secondary lymphoid organs, where theycan be activated by cognate antigen. Once activated, lymphocytescan enter tertiary nonlymphoid sites, such as the skin and intestine,where they can function in clearing infection. Present knowledgesuggests that naive ab T cell circulation is restricted to secondarylymphoid tissues1,2.
The small intestine tertiary site is important in host defense. Thegut epithelium forms a border between the outside environment andthe body interior and is a potential entry site for antigens3. Of theimmune cells that take up residence in the epithelium (called‘intraepithelial lymphocytes’ (IELs)), more than 90% are CD8+
T cells. Because of the large surface area of the small intestine, IELscomprise a considerable fraction of the body’s T cells, and approxi-mately 1 � 109 IELs can be found in the human small intestine4–6.IELs have an activated or memory phenotype7 and, once associatedwith the epithelium, do not recirculate throughout the body8. CD8+
IELs have high expression of aE integrin, which interacts withE-cadherin on epithelial cells and allows retention of IELs in the gutepithelium9. The CD8+ IEL compartment contains CD8aa+ gd andab T cells as well as conventional CD8ab+ ab T cells10–12. Studiessuggest that CD8ab+ ab T cell receptor (TCR)–positive IELs originatefrom the thymus13,14, specifically via naive T cells that are activated inthe Peyer’s patch that then migrate to the gut epithelium15–17.
Recent thymic emigrants (RTEs), a distinct subpopulation of naiveab T cells, have ‘preferential’ access to survival factors and niches andare ‘preferentially’ incorporated into the peripheral naive ab T cell
pool18. Splenic RTEs are less responsive than other naive ab T cells toTCR stimulation19 and may be especially susceptible to tolerization20.CD8+ RTEs have unique adhesive and chemotactic properties. Unlikeother naive ab T cells, RTEs express aE integrin and CCR9, which isinvolved in recruiting memory T cells to the intestines21,22. CCR9 is achemoattractant receptor expressed on small intestine–specific T cells,and its ligand, CCL25, is expressed by small intestine epithelial cells23.CD8+ RTEs also express a4b7 integrin, which is the receptor for theintestinal vascular addressin MAdCAM-1. Those features support thehypothesis that CD8+ RTEs might home to the mucosal surface ofthe small intestine without prior activation and ‘reprogramming’ ofhoming properties in peripheral lymphoid tissues. Moreover, becauseRTEs establish and contribute to the TCR diversity of the peripheralab T cell pool, we reasoned that RTEs might also maintain the TCRdiversity of the IEL population24.
Here we demonstrate that CD8+ RTEs homed to the gut and showthat this migration required a4b7 integrin, CCR9 and CCL25. RTE guthoming occurred independently of prior antigen recognition inperipheral lymphoid tissues. After entry into the gut wall, CD8+
RTEs proliferated and differentiated, ultimately acquiring the pheno-type of resident IELs. CD8+ RTEs populated the IEL compartment oflymphopenic mice more efficiently than naive non-RTE CD8+ T cells.In the steady state, CD8ab+ TCRab+ RTEs were readily recoveredfrom the resident IEL population and expressed a diverse TCRrepertoire. Our data indicate that trafficking of naive CD8+ abT cells, particularly RTEs, is not limited to secondary lymphoidtissues. Instead, CD8+ RTEs can home directly into the intestinalwall, where they might help to maintain the TCR diversity of themucosal CD8ab+ ab T cell pool.
Received 21 November 2005; accepted 14 February 2006; published online 2 April 2006; corrected after print 2 May 2006 (details online); doi:10.1038/ni1319
1Program in Immunology and 2Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California94305, USA. 3Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304, USA. 4Laboratory of MammalianGenes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. Correspondenceshould be addressed to E.C.B. ([email protected]).
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RESULTS
Homing of CD8+ RTEs to nonlymphoid tissue
To analyze the peripheral homing patterns and tissue distribution ofCD8+ RTEs in adult mice, we labeled emigrating thymocytes byinjection of intrathymic fluorescein isothiocyanate (FITC). At 24 hafter FITC injection, all peripheral FITC+ cells are RTEs25,26. Inagreement with published data, we detected FITC+ CD8+ RTEs inthe spleen, peripheral lymph nodes, mesenteric lymph nodes, Peyer’spatches and blood26 (Fig. 1a). To determine if CD8+ RTEs could alsolocalize to the small intestine, we isolated IELs and lamina proprialymphocytes. FITC+ cells expressing the RTE markers aE integrin andCCR9 were present in these gut T cell populations and displayed anaive CD62LhiCD44lo surface phenotype (Fig. 1a and data notshown). These results suggest that CD8+ RTEs migrate both to thegut and to secondary lymphoid organs.
Naive CD8+ RTEs among IELs
We next sought to determine if naive T cells could be found in thesteady-state resident IEL population. There was a small (comparedwith secondary lymphoid organs) but detectable fraction ofCD44loCD62LhiCD8ab+ ab T cells (2–10% in 4- to 12-week-oldmice) in the IEL population (Fig. 1a, left). A large percentage ofCD62LhiCD44lo IELs also expressed CCR9 and aE integrin, suggestingthat RTEs were enriched for this population (Fig. 1a and data notshown). In addition, there were similar proportions of FITC+ cells inthe CD44loCD62Lhi CD8ab+ ab T cell populations in the secondarylymphoid organs and the small intestine (Fig. 1a, right). These resultsfurther support the idea that RTEs can migrate to the gut.
Thymic origin of gut CD8+ RTEs
To rigorously examine the mechanisms regulating RTE trafficking, wedeveloped a short-term homing assay using enriched CD8+ single-positive (SP) thymocytes as a source of RTEs. CD8+ SP thymocytes arethe immediate precursors of RTEs, and adoptive transfer of CD8+ SPthymocytes results in their forced emigration27,28. We isolated CD8+
SP thymocytes by depleting samples of CD4+CD8+ double-positiveand CD4+CD8– SP thymocytes, followed by staining for CD8 (to
exclude CD4–CD8– double-negative thymocytes). CD8+ SP thymo-cytes had high expression of aE integrin and CD62L and intermediateexpression of a4b7 integrin (in contrast to high expression on memorycells) and CCR9 (Fig. 1b and data not shown). The amounts of aE
integrin, CD62L, CCR9 and a4b7 integrin on CD8+ SP thymocyteswere similar to those on FITC+ CD8+ RTEs in the spleens of micegiven intrathymic injection of FITC (data not shown). FITC+ CD8+
RTEs and adoptively transferred FITC-labeled CD8+ SP thymocyteshad similar tissue distribution (Fig. 1a,c), suggesting that the twopopulations had similar migratory capabilities. Unlike methods usedbefore to identify RTEs (such as TCR rearrangement excision circleanalysis or the use of recombination-activating gene–green fluorescentprotein (GFP) mice), detection of RTEs in mice receiving FITC-labeled CD8+ SP thymocytes is not influenced by cell division29,30.
CD8+ RTE localization in the small intestine
To identify the specific intestinal region to which CD8+ SP thymocytesmigrate, we transferred carboxyfluoroscein succinimidyl ester (CFSE)–labeled Thy-1.2+ CD8+ SP thymocytes into Thy-1.1+ congenic reci-pients. Staining of small intestine tissue sections isolated 1d (Fig. 2a)or 8 d (data not shown) after transfer demonstrated the presence ofThy-1.2+ cells in the lamina propria as well as in the region adjacent tothe epithelium. Counterstaining for the CD31 endothelial markerconfirmed that few if any Thy-1.2+ cells were present in vessels ateither time point (data not shown).
Secondary lymphoid tissue–independent gut localization
After antigen stimulation in secondary lymphoid tissues, naive abT cells are ‘reprogrammed’ to home to nonlymphoid sites31.
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Figure 1 Homing of CD8+ RTEs to the gut. (a) CD44loCD62Lhi (naive) cells
of the CD8ab+ fraction (left) and FITC+ (RTE) cells of the CD44loCD62Lhi
CD8ab+ fraction (right) in various tissues (left margin). Numbers beside
boxed areas indicate the percentage of cells in each fraction. PLN,
peripheral lymph node; MLN, mesenteric lymph node; PP, Peyer’s patch.
(b) Expression of aE integrin, CD62L, CCR9 and a4b7 on CD8+ SP
thymocytes. Shaded histograms indicate isotype control staining.
(c) Analysis of FITC-labeled CD8+ SP thymocytes 24 h after transfer. Plotsare gated on CD8ab+ CD62Lhi cells; numbers beside boxed areas indicate
the percent of CD44loCD62Lhi CD8ab+ cells that are FITC+. All data are
representative of at least three independent experiments.
Uninjected Thy-1.1 Thy-1.2 RTE into Thy-1.1
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Figure 2 Localization of CD8+ SP thymocytes in the gut. (a) Staining of
small intestine sections from Thy-1.1+ congenic BALB/c recipients 1 d after
transfer of CFSE-labeled Thy-1.2+ CD8+ SP thymocytes (right) or no cells
(left). Sections were stained with an anti-Thy-1.2 and were developed with
NovaRED. Original magnification, �20 (top row) and �40 (bottom row).
(b) Flow cytometry of FITC-labeled CD8+ SP thymocytes after transfer into Lta–/–
recipients. Plots are gated on CD8ab+CD62Lhi cells; numbers beside boxed
areas indicate percent FITC+. All data are representative of three experiments.
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Therefore, although unlikely because of the short 24-hour time frameof our experiment, it was possible that before gut localization,adoptively transferred CD8+ SP thymocytes could have passed throughPeyer’s patches or mesenteric lymph nodes and could have been‘reprogrammed’ to allow gut homing. To rule out a requirement for‘reprogramming’ in lymphoid tissues, we analyzed lymphotoxin-a-deficient (Lta–/–) mice, which lack organized secondary lymphoidtissues and lymphoid structures in the small intestine and splenicwhite pulp32,33. Even in the absence of secondary lymphoid structures,naive ab T cells were present in the CD8ab+ IEL compartment inthe small intestines of Lta–/– mice (data not shown), and adopt-ively transferred FITC-labeled CD8+ SP thymocytes migrated to thegut epithelium of Lta–/– mice (Fig. 2b). These results support the hypo-thesis that CD8+ RTEs can transit directly from the thymus to the gut.
Homing mechanisms regulating gut localization
To investigate the mechanisms regulating gut localization of CD8+ SPthymocytes, we analyzed the involvement of several candidate surfacemolecules. We first focused on CCR9 because it is expressed by CD8+
RTEs but not by other naive cells and is required for intestinallocalization of activated and memory ab T cells21,22,34. We mixedwild-type and Ccr9–/– CD8+ SP thymocytes labeled with the redfluorescent cell linker PKH26 and FITC, respectively, and transferredthe cells into wild-type recipient mice. Although wild-type and CCR9-deficient CD8+ SP thymocytes migrated with equal efficiency tolymph nodes, Peyer’s patches, spleen and blood, CCR9-deficientCD8+ SP thymocytes migrated much less efficiently than wild-typeCD8+ SP thymocytes to the small intestine epithelium (Fig. 3a,b).We obtained similar results in experiments using reciprocal labeling(FITC-labeled wild-type and PKH26-labeled Ccr9–/– CD8+ SP
thymocytes), and FITC- and PKH26-labeled wild-type CD8+ SPthymocytes transferred together migrated with equal efficiency tothe gut, confirming that the dyes did not have differential effects onhoming. In addition, we isolated CD4+ SP thymocytes, which do notexpress CCR9, from wild-type and Ccr9–/– mice, labeled the cells withFITC or PKH26 and transferred them together into wild-type reci-pients. Wild-type and Ccr9–/– CD4+ SP thymocytes had similarhoming patterns (Fig. 3b).
Developmental defects intrinsic to Ccr9–/– thymocytes might haveindirectly affected their migration patterns. To confirm that CCR9 wasrequired for trafficking of CD8+ SP thymocytes to the gut, we assessedthe function of the CCR9 ligand CCL25. Gut homing of CD8+ SP
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Figure 3 CCR9 and CCL25 in gut homing of CD8+ SP thymocytes. (a) Flow cytometry of PKH26-labeled wild-type (WT) and FITC-labeled Ccr9–/– (KO) CD8+
SP thymocytes transferred together into wild-type recipients. Plots are gated on CD8a+ cells; numbers in top right corners indicate the Ccr9–/–/wild-type ratio
from the boxed areas. (b) Ratio of transferred SP thymocytes in the peripheral lymph node (PLN) versus small intestine (SI IEL). SP thymocytes isolated from
wild-type or Ccr9–/– mice were differentially labeled and transferred together into wild-type mice (horizontal axis). Each dot represents an individual recipient
mouse. (c,d) Flow cytometry of FITC-labeled CD8+ SP thymocytes transferred into mice treated with isotype control antibody or anti-CCL25. Numbers beside
boxed areas in plots (c) and graphed data (d) indicate the percent of cells in the CD8a+ population that are FITC+. In d, data represent mean ± s.e.m. of
values obtained with various tissues (horizontal axes) normalized to values obtained for peripheral lymph nodes of four individual mice per group. *, P o 0.08.
CD8α
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Isotype NSαE integrin
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Figure 4 The a4 integrin in gut homing of CD8+ SP thymocytes. FITC-
labeled CD8+ SP thymocytes were pretreated with isotype control or
antibodies specific for a4 integrin (a,b; n ¼ 4), CD62L (c,d; n ¼ 3) or aE
integrin (e,f; n ¼ 3) and were adoptively transferred into wild-type recipient
mice, followed by flow cytometry. Representative plots (a,c,e) are gated on
CD8a+ cells; numbers beside or above boxed areas indicate the percent ofCD8a+ cells that are FITC+. Graphed data (b,d,f) represent mean ± s.e.m.
of the percentage of CD8a+ cells that are FITC+ in each tissue, normalized
to that of peripheral lymph nodes (b) or spleen (d,f) *, P o 0.02; **,
P o 0.003; ***, P o 0.001; NS, not significant.
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thymocytes was less efficient in mice treated with a neutralizingantibody specific for CCL25 than in mice treated with an isotypecontrol antibody (Fig. 3c,d). These results suggest that CCR9 isrequired for gut homing of CD8+ SP thymocytes.
Next we characterized the function of adhesion molecules. Wetransferred FITC-labeled CD8+ SP thymocytes into mice treatedwith either an isotype control antibody or a neutralizing antibodyspecific for a4b7 integrin, a4 integrin, CD62L or aE integrin. Wemaintained saturating quantities of blocking antibodies throughoutthe experiments, as confirmed by staining of cells from treated micewith a labeled version of the blocking antibody (SupplementaryFigure 1 online).
Blockade of a4 integrin inhibited homing of CD8+ SP thymocytesto the gut and Peyer’s patches (Fig. 4a,b). The a4 integrin can pairwith the b1 or b7 chain to form a4b1 or a4b7 integrin. Blockade withan antibody specific for the a4b7 integrin also inhibited gut homing ofCD8+ SP thymocytes (data not shown). CD62L is essential for homingof naive cells to the lymph nodes. CD62L blockade inhibited homingof CD8+ SP thymocytes to the peripheral lymph nodes but did notalter homing of CD8+ SP thymocytes to the small intestine (Fig. 4c,d).The aE integrin distinguishes CD8+ RTEs from other naive CD8+
cells34. However, antibody blockade of aE integrin did not inhibittrafficking of CD8+ SP thymocytes to the small intestine (Fig. 4e,f).These results indicate that a4b7 integrin is required for the homing ofCD8+ SP thymocytes to the small intestine.
Gut CD8+ RTE proliferation
To determine whether CD8+ SP thymocytes proliferate in the periph-ery, we analyzed CFSE dilution of adoptively transferred CFSE-labeled
CD8+ SP thymocytes. By 8 d after transfer,CD8+ SP thymocytes in the small intestinehad undergone multiple rounds of cell divi-sion (Fig. 5a). CD8+ SP thymocytes at othersites, including peripheral lymph nodes,mesenteric lymph nodes, Peyer’s patches,spleen and blood, did not undergo suchextensive proliferation (Fig. 5b and data notshown). We used Thy-1-mismatched recipi-ents in similar experiments, which indicatedthat CFSE labeling alone was adequatefor tracking transferred cells accurately (datanot shown).
The proliferation of gut CD8+ SP thymo-cytes could be driven by TCR signals or bysignals received specifically in the intestinalenvironment (such as growth factors or cyto-kines). To determine whether proliferation ofCD8+ SP thymocytes was antigen induced,we used OT-I TCR-transgenic recombina-tion-activating gene 1–deficient (Rag1–/–)mice35,36. These mice express a single TCRand therefore cannot respond to as diverse apanel of antigens as wild-type mice can. Welabeled OT-I+Rag1–/– and C57BL/6 CD8+ SPthymocytes with CFSE and transferred thecells into separate C57BL/6 recipient mice. By5 d after transfer, gut OT-I+Rag1–/– CD8+ SPthymocytes had undergone fewer divisionsthan gut C57BL/6 CD8+ SP thymocytes(Fig. 5a). In contrast, there was no differencein the proliferation profiles of OT-I+Rag1–/–
and C57BL/6 CD8+ SP thymocytes at other peripheral sites (Fig. 5band data not shown). These results indicate that CD8+ SP thymocytesproliferated in response to an antigen present exclusively in the gut.OT-I+Rag1–/– and C57BL/6 CD8+ SP thymocytes migrated with equalefficiency to the gut (Supplementary Figure 2 online), suggesting thatTCR specificity does not affect homing.
Intraintestinal differentiation of CD8+ RTEs
Resident IELs have an aE integrin–high, CD62Llo surface phenotype,whereas CD8+ SP thymocytes have an aE integrin–intermediate,CD62Lhi phenotype. At 2 d after adoptive transfer, gut OT-I+Rag1–/–
and C57BL/6 CD8+ SP thymocytes retained their aE integrin–inter-mediate, CD62Lhi surface phenotype. However, by 8 d after transfer,gut OT-I+Rag1–/– and C57BL/6 CD8+ SP thymocytes downregulatedCD62L and upregulated aE integrin to amounts seen on resident IELs(Fig. 5c). CD8+ SP thymocytes at other peripheral sites retained theiroriginal aE integrin–intermediate, CD62Lhi surface phenotype (Fig. 5dand data not shown). These data indicate that proliferation is notmandatory for differentiation from naive CD8+ SP thymocytes intoresident IELs and that differentiation can occur in the absence ofspecific TCR stimulation.
Efficiency of CD8+ RTE gut homing
As RTEs are distinguished from non-RTE naive CD8+ T cells by theirunique pattern of CCR9 and aE integrin expression, we sought todetermine whether CD8+ RTEs home more efficiently than non-RTEnaive CD8+ T cells to the gut. We mixed purified RTE and non-RTEnaive CD8+ T cells labeled with FITC and PKH26, respectively, andinjected the cells into wild-type mice. CD8+ SP thymocytes consistently
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Figure 5 Intraintestinal proliferation and differentiation of CD8+ SP thymocytes. (a,b) CFSE dilution
analysis of cell division. C57BL/6 (B6) or OT-I+Rag1–/– (OT-I) CD8+ SP thymocytes were isolated,
labeled with CFSE and transferred into C57BL/6 recipient mice, which were killed on day 2, 5 or
8 after cell transfer. Based on CFSE dilution, cells were classified as having undergone zero to threecell divisions (horizontal axes). Data represent the mean ± s.e.m. of three individual recipients in each
group. *, P o 0.001. (c,d) Representative CD62L and aE integrin profiles of CD8ab+CFSE+ cells (from
a,b) on day 2, 5 or 8 after transfer (n ¼ 3).
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homed to the gut more efficiently than non-RTE naive CD8+ T cells(Fig. 6a). However, both populations homed equally efficiently toother peripheral tissues. In contrast, CD8+ SP thymocytes and per-ipheral naive CD8+ RTEs (aE integrin–positive, CD62L+) homed withequal efficiency to all peripheral tissues (Fig. 6b). These data furtherconfirm the validity of our CD8+ SP thymocyte adoptive transfermodel, as CD8+ SP thymocytes homed comparably to splenic andperipheral CD8+ T cells with an RTE surface phenotype.
We compared the ability of CD8+ SP thymocytes and non-RTEnaive CD8+ cells to reconstitute the lymphopenic intestine of micewith severe combined immunodeficiency (SCID). The intestinalepithelium of SCID mice is devoid of CD8ab+ ab T cells12. However,we noted the presence of a small population of CD8aa+ gd T cells ingut of SCID mice. As CD8+ RTEs express CD8ab+ and TCRab, theseCD8aa+ gd T cells were ‘gated out’ of our analyses. We sorted CD8+
SP thymocytes from ‘actin-GFP’ mice and non-RTE naive CD8+
T cells from wild-type mice, mixed the cells and transferred themtogether into SCID recipient mice (Fig. 6c). We analyzed reconstitu-tion at various time points from 2 d to 4 weeks after transfer. By 7 dafter transfer, the RTE/non-RTE ratio was higher in the gut than inthe blood of each mouse (Fig. 6d). These results indicate that RTEshome more efficiently to the gut than do their non-RTE naive CD8+
T cell counterparts (Supplementary Fig. 3 online).
DISCUSSION
Our results have demonstrated that CD8+ RTEs have a unique surfacephenotype that allows them to migrate directly from the blood tothe small intestine. Using gene-targeted mice and in vivo antibodyblockade, we have shown that this gut entry required a4b7
integrin, CCR9 and CCL25. CD8+ SP thy-mocytes were more efficient than non-RTECD8+ T cells in entering and repopulatingthe small intestine. After entry into the gut,CD8+ SP thymocytes underwent post-thymicdifferentiation involving TCR-dependentproliferation and TCR-independent, micro-environment-dependent acquisition of aresident IEL surface phenotype.
It is generally believed that naive T cellhoming is restricted to organized secondarylymphoid tissues, particularly the spleen,lymph nodes and Peyer’s patches. Therefore,our substantial recovery of naive CD8+ T cellsfrom the small intestine epithelium was unex-pected. We did several rigorous experimentsto show that naive CD8+ T cells, particularlyRTEs, were present in the small intestine andwere not due to contamination from Peyer’spatches or blood. There were naive CD8+
T cells and RTEs in IEL populations fromwild-type and from Lta–/– mice, suggestingthat the presence of these cells in the gut wasnot due to contamination from Peyer’spatches or small lymphoid aggregates in thesmall intestine. Histological localization ofadoptively transferred CD8+ SP thymocytesthat could be identified by fluorescence andgenetics demonstrated that these cells werelocalized specifically in the lamina propriaand IEL compartments. Recruitment ofRTEs to the gut was specifically blocked
by inhibition of CCR9-CCL25 interactions or by blockade of a4
integrin, ruling out the possibility of blood contamination.The finding that naive CD8+ T cells comprise a substantial
percentage of small intestine IELs suggests that the trafficking ofnaive ab T cells is not limited to secondary lymphoid organs. It will benecessary to evaluate the extent of homing and the functionalrelevance of naive ab T cells in other nonlymphoid peripheral tissues.In particular, our results raise the possibility that ab T cell populationsresident in the skin, brain, liver and lungs may also be derived, at leastin part, from naive ab T cells recruited directly from the blood.
After entry into the small intestine (but not after entry intosecondary lymphoid tissues), CD8+ SP thymocytes underwent multi-ple rounds of cell division and adopted a resident IEL phenotype.Specifically, aE integrin, which tethers IELs to neighboring epithelialcells, was induced9. Our studies with OT-I+Rag1–/– RTEs suggestedthat specific TCR stimulation was necessary for maximal gut prolif-eration of CD8+ SP thymocytes. Those results do not rule out thepossibility that growth factors cooperate with TCR signals in regulat-ing proliferation, but they indicate that the local population expansionof RTEs is driven by their response to antigens in the small intestine.In contrast, acquisition of a resident IEL phenotype (aE integrin–high,CD62Llo) occurred independently of TCR signals. That change in cellsurface phenotype may have been due to transforming growth factor-b receptor signaling, as there is high expression of transforminggrowth factor-b in the intestinal environment37 and it is involved inaE integrin induction in vitro38,39.
The mechanisms regulating antigen presentation in the small intes-tine differ from those that regulate antigen presentation in lymphnodes. Specifically, epithelial cells are the main antigen-presenting cells
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Time after SCID reconstitution
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Figure 6 ‘Preferential’ homing of CD8+ RTEs to the gut. (a,b) FITC-labeled CD8+ SP thymocytes were
transferred together with equal numbers of PKH26-labeled CD8+, aE integrin–negative, CD62L+ cells (a)
or CD8+, aE integrin–positive, CD62L+ cells (b) into wild-type recipient mice. At 24 h after transfer, the
FITC/PKH26 ratios were calculated for various tissues (horizontal axes) and were normalized to those for
peripheral lymph nodes. A ratio of more than 1 indicates a competitive homing advantage for RTEs.
Data represent mean ± s.e.m. of four individual mice per group. *, P o 0.01; NS, not significant.
(c) Flow cytometry of CD8+ SP thymocytes from actin-GFP mice and CD8+, aE integrin–negative,
CD62L+ cells from wild-type mice, mixed and transferred together into SCID recipient mice. In the
first three plots, boxed areas indicate sort gates; dot plots show the purity of sorted populations afterreanalysis. Numbers beside boxed areas (far right plot) indicate percent GFP– cells (left) or GFP+
cells (right). Plots are representative of three independent experiments. (d) GFP+/GFP– ratio among
CD8ab+TCRb+TCRgd– cells in the blood and small intestines of SCID mice analyzed at various times
after transfer (horizontal axis). Data from six mice are presented individually (1–6, below bars).
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in the gut40. In the uninflamed state, intestinal epithelial cells arethought to present antigen in a suppressive way because they havelower expression of costimulatory molecules than do ‘professional’antigen-presenting cells41. In addition, transforming growth factor-bpresent in the gut epithelium contributes to the generation of animmunosuppressive microenvironment37. That may explain why RTEsand other IEL subsets proliferate and have an effector or memoryphenotype but do not normally cause overt inflammatory symptoms.
Published work suggests a thymic origin for CD8ab+TCRab+
IELs13. IELs were thought to originate from naive ab T cells activatedin Peyer’s patches, which subsequently migrated into the smallintestinal epithelium15,16. Here we have shown that at least someresident IELs trafficked in a naive state directly from the thymusthrough the blood into the gut. Moreover, although the lymphopenicgut can be reconstituted by total lymph node cells42, our resultshave shown that the gut could be reconstituted by a naive CD8+ SPthymocyte population ‘preprogrammed’ in the thymus to migrate tothis site.
What can RTEs contribute to the IEL population? In contrast to thepreviously activated cells ‘programmed’ in the Peyer’s patches to enterthe gut, RTEs have a diverse repertoire of TCRs. The TCRb repertoireof naive CD8+ IELs is similar in diversity to that of splenic and lymphnode RTEs and CD8+ SP thymocytes but is less diverse than that ofmemory CD8+ ab T cells (data not shown)43,44. It is expected that theTCR repertoire of IELs becomes more restricted over time because ofgut-specific antigen recognition and clonal amplification. Thus, con-tinuous RTE recruitment may be an important mechanism formaintaining TCR diversity at mucosal surfaces.
METHODSMice. BALB/c, C57BL/6, SCID and Lta–/– mice were purchased from Jackson
Laboratories. OT-I+Rag1–/– and C57BL/6 control mice were purchased from
Taconic Farms35,36. Thy-1.1+ congenic BALB/c mice were a gift from
I. Weissman (Stanford University School of Medicine, Stanford, California).
Ccr9–/– mice45 and actin-GFP mice (transgenic mice expressing enhanced GFP
under the control of the promoter of the gene encoding chicken b-actin)46 have
been described. Unless stated otherwise, mice used were between 4 and 12
weeks of age. Animals were housed in a specific pathogen–free facility. All
animal protocols were in accordance with institutional, federal and state
guidelines and were approved by the Veterans Affairs Palo Alto Health Care
System Institutional Animal Care and Use Committee (Palo Alto, California).
Flow cytometry. Antibodies to CD4, CD8a, CD44, CD49d, CD62L, CD103–aE
integrin, a4b7, TCRb and TCRgd and rat IgG2ak and IgG2bk isotype controls
were from BD Pharmingen. Antibody to CD8b (anti-CD8b) was from
eBioscience. Cells were stained in Hank’s balanced-salt solution containing
1% bovine serum and were analyzed on an LSRII (BD Biosciences) or
were sorted with a FACSVantage (BD Biosciences). CCR9 staining (clone
242503) was done according to the manufacturer’s protocol (R&D Systems).
Data were analyzed with CellQuest software (BD Biosciences) and FlowJo
software (Treestar).
IEL preparation. IELs were isolated as described47 with some modifications.
After removal of Peyer’s patches, small intestines were rinsed twice with Hank’s
balanced-salt solution containing 2% bovine calf serum, were cut into pieces
5 mm in length and were incubated at 37 1C while being stirred for two
10-minute sessions, with a change of media between sessions. IELs were
collected from the interface of a 40–70% Percoll gradient.
CD8+ RTE enrichment and labeling. Total thymocyte suspensions from 4- to
8-week-old mice were depleted of CD4+ thymocytes with anti–mouse CD4
MACS beads and LD columns according to the manufacturer’s protocol
(Miltenyi Biotech). After depletion, 50–70% of the population (CD8+ SP
thymocytes) was CD8+CD4–TCRhi and the remaining cells were CD4–CD8–.
Cells were labeled with FITC (Sigma), CFSE (Molecular Probes) or PKH26
(Sigma) before transfer, as described25,48.
In vivo antibody blockade. FITC-labeled CD8+ SP thymocytes were preincu-
bated with blocking antibody in vitro for 20 min at 25 1C, after which both cells
and antibody were transferred intravenously into recipient mice. Recipient mice
were analyzed 24 h after transfer. For confirmation of complete blockade of
these cell surface receptors, cells from treated mice were stained with a labeled
antibody of the same clone as the blocking antibody and were analyzed by flow
cytometry. Antibodies specific for a4 integrin (clone PS/2; 200 mg), aE integrin
(clone M290; 250 mg), CD62L (clone MEL14; 100 mg), a4b7 integrin (clone
DATK32; 200 mg) and the isotype control anti–human CD44 (clone 9B5) were
generated ‘in house’ (Supplementary Fig. 1). For CCL25 blockade, mice were
pretreated 2 h before transfer of CD8+ SP thymocytes with 100 mg of antibody
specific for CCL25 (clone 89818) or an isotype control antibody (clone
141945). Both antibodies were purchased from R&D Systems.
Intrathymic FITC injection. These injections were done as described49. A
ventral midline incision was made one third down the sternum to expose the
thymus. A Hamilton syringe was used to inject 10 ml of a 1-mg/ml solution of
FITC (Sigma). The skin incision was closed with Vetbond (3M) and mice were
allowed to recover under a heat source.
Adoptive transfer. Each wild-type or Lta–/– recipient was injected intravenously
with approximately 1 � 107 cells after depletion CD8+ SP thymocytes. SCID
mice were reconstituted with a 1:1 mix of approximately 5 � 105 sorted RTEs
and naive non-RTEs.
Histology. Tissues were embedded in optimum cutting temperature com-
pound (Tissue Tek; Sakura) and were frozen at –80 1C. Frozen tissue sections
6 mm in thickness were fixed in acetone, were blocked with PBS containing 2%
BSA and 2% goat serum and were stained with anti-Thy-1.2 or isotype control
antibody. Slides were washed with PBS containing 2% BSA before and after the
incubation steps, and images were visualized with a confocal microscope
(Nikon Eclipse TE300) equipped with Lasersharp software (Bio-Rad Labora-
tories). Immunoperoxidase staining was done on similarly sectioned tissues
with biotin–anti-Thy-1.2 and a streptavidin-peroxidase kit (Zymed Labora-
tories) and the substrate NovaRED, which was used according to the manu-
facturer’s guidelines.
Statistics. All data are presented as mean ± s.e.m. For antibody blockade
experiments in Figures 3 and 4, Student’s t-test was used for comparisons of
two groups. In Figure 5, the data set was collapsed and the w2 test was used. In
Figure 6, a paired Student’s t-test was used for comparison of IELs and blood.
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTSWe thank E. Resurrecion for histology, and L. Rott and C. Crumpton for cellsorting. Supported by the National Institutes of Health (AI47822 and GM37734to E.C.B.; DK07056 to A.H.; and DK060000 to T.S.), the Department ofVeterans Affairs (E.C.B.), the Howard Hughes Medical Institute (M.M.W.)and the Stanford Digestive Disease Center (DK56339).
COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.
Published online at http://www.nature.com/natureimmunology/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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CD8+ recent thymic emigrants home to and efficiently repopulate the small intestine epitheliumTracy L Staton, Aida Habtezion, Monte M Winslow, Tohru Sato, Paul E Love & Eugene C ButcherNature Immunology 7Nature Immunology 7Nature Immunology , 482–488 (2005); published online 2 April 2005; corrected after print 2 May 2006
In the version of this article initially published, the vertical axis label ‘FITC’ is missing from the right column in Figure 1a. The correct figure is presented here.The error has been corrected in the PDF version of the article.
a b
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