International Immunology. Vol. 7, No. 11, pp. 1861-1871 © 1995 Oxford University Press

Migration pathways of CD4 T cell subsetsin vivo: the CD45RC" subset enters thethymus via a4 integrin-VCAM-1 interactionEric B. Bell, Sheila M. Sparshott and Ann Ager1

Immunology Research Group, Biological Sciences, University of Manchester, Manchester M13 9PT, UK1Division of Cellular Immunology, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK

Keywords: CD4, CD45RC", 04 integrin, T cell, thymus, VCAM-1

Abstract

The present investigation examines the localization and migration of purified T cell subsets Incomparison with B cells, CD8 T cells and CD4+CD8" single-positive thymocytes. CD4 T cellsubsets in the rat are defined by mAb MRC 0X22 (anti-CD45RC), which distinguishes resting CD4 Tcells (CD45RC+) from those (CD45RC") which have encountered antigen In the recent past—subpopulatlons often referred to as 'naive' and 'memory'. Purified, 51Cr-labelled CD45RC+ CD4 Tcells broadly reflected the migration pattern of CD8 T cells and B cells. Early localization to thespleen was followed by a redistribution to mesenterlc lymph nodes (MLN) and cervical lymphnodes (CLN), B cells migrating at a slightly slower tempo. There was almost no localization ofthese subpopulatlons to the small or large Intestine [Peyer's patches (PP) excluded]. In contrast,CD45RC" CD4 T cells (Indistinguishable In size from the CD45RC+ subset) localized in largenumbers to the intestine; they were present here at the earliest time point (0.5 h), persisted for atleast 48 h but did not accumulate, indicating a rapid exit. Numerically, localization of CD45RC" CD4T cells In the MLN could be accounted for entirely by afferent drainage from the intestine.Unexpectedly, CD45RC CD4 T cells (but not other subsets) localized and accumulated In thethymus. In vivo treatment with mAb HP2/1 against the Integrin 04 subuntt inhibited almost entirelyCD45RC- CD4 T cell migration into the PP (98.1%), Intestine (87.1%), MLN (89.1%) and thymus(93.5%); migration into the CLN was only reduced by half. To distinguish between recognition ofMAdCAM-1 and VCAM-1 by c^-contalning Integrins, recipients were treated with mAb 5F10 againstrat VCAM-1. Except for the thymus and a small reduction in CLN, localization of CD45RC~ CD4 Tcells was unaffected; entry to the thymus was almost completely blocked (92.3%) by anti-VCAM-1.The results Indicated (I) that CD45RC" CD4 T cells alone showed enhanced localization to the gutand PP, probably via (X4py-MAdCAM-1 interaction; (II) that many CD45RC cells entered non-mucosal LN independently of 04 integrin or VCAM-1; and (ill) that entry of mature recirculatingCD45RC" CD4 T cells into the thymus across thymlc endothellum was apparently regulated by 04integrin-VCAM-1 interaction.

Introduction

The effectiveness of the immune system depends on bringingtogether rare specific lymphocytes with minute quantities ofantigen. One arm of the response requires a mechanism fortransporting antigen to a central site—lymph nodes (LN)—the other of ensuring that lymphocytes have an opportunityto engage that antigen. LN have thus evolved high endothelialvenules (HEV) to extract selectively, from amongst a variety ofblood-borne cells, lymphocytes irrespective of their receptorspecificity. Specific cells are physically trapped by antigen

(1), whereas the remainder return via efferent lymphatics tothe blood and continue to recirculate (2-4). Within a few daysdifferentiated progeny of antigen-stimulated lymphocytes alsore-enter the blood (5), but with a different objective, e.g. todestroy an infectious agent. These cells may now need toinfiltrate non-lymphoid tissues at the site of insult.

Our understanding of the mechanisms controlling the migra-tion of lymphocytes has advanced rapidly in recent years.Selective extravasation across endothelial cells into lymphoid

Correspondence to. E. B. Bell

Transmitting editor. M. Mayasaka Received 18 May 1995, accepted 18 August 1995

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1862 Migration pathways of CD4 T cell subsets

and non-lymphoid tissues depends on a co-ordinated multi-step adhesion cascade (6-9). Knowledge of the specificadhesion molecules/ligands involved has largely been derivedfrom in vitro systems and there is a need to test rigorouslythe concepts in vivo.

We were interested in the distribution and migration ofpurified lymphocyte subsets, and in particular in CD4 T cellsbefore and after encounter with antigen. It is commonly heldthat 'naive' and 'memory' T cells recirculate by differentpathways (10), a conclusion derived from the observedpredominance of T cells expressing the lowest molecularweight isoform of CD45 (CD45RO in humans) in afferentlymph and in sites of inflammation (11,12). In the rat mAb0X22 [anti-CD45RC (13)] divides CD4 T cells into twofunctionally distinct subsets (14). The high (CD45RC+) andlow (CD45RC") molecular weight isoforms of CD45 in thisand other species were frequently used as markers of 'naive'and 'memory'T cells respectively (15-18), although the validityof the equation has been challenged (19-21).

Many investigators in the past have used whole lymphocytepopulations, but few studies have monitored the migration ofpurified B and T cells, and none has explored CD45RC"1" andCD45RC" subpopulations directly. The localization pattern ofimmunoblasts, which include CD45RC" CD4 T cells, wasstudied in detail by Ford and colleagues (22) and morerecently by others (23-26). However, a majority of CD45RC"T cells are not blast cells and resemble in size the CD45RC+

subset. Here we show that these antigen-experienced Thy-1- CD45RC" CD4 T cells [immature Thy-1+ CD45RC" CD4recent thymic emigrants (RTE) (27,28) were excluded], incontrast to CD45RC+ CD4 T cells, showed enhanced migra-tion to the gut [presumably lamina propria since Peyer'spatches (PP) were excluded]—a migration pathway that wascompletely inhibited by mAb to the 04 integrin subunit.Furthermore, we were surprised to discover that the CD45RC"subset also entered thymus in substantial numbers and thatmigration from the bloodstream depended on 04 integrin-VCAM-1 interaction.

Methods

Animals

Congenic PVG strain rats, distinguished by the RT7a and RT7b

allotype markers, were used as recipient and donor animalsrespectively. PVG-rnu/mu athymic nude rats were used as asource of B cells. All animals were bred and maintained inthe Animal Unit of Manchester University Medical School.Donor rats were male, recipients were females of -150 gbody wt. PVG-RT1U strain animals, also carrying RT7a andRT7b allotype markers, were used in one set of experiments.Thoracic duct cannulation was performed by a previouslydescribed, established technique (29).

Purification of CD45RC subsets

CD4+ CD45RC+ and CD45RCT subsets were prepared, aspreviously described, from whole thoracic duct lymphocytes(TDL) by two or three rounds of immunomagnetic depletionfollowing staining of the cells with a cocktail of mAb (30). Forpurification of CD45RC"1" cells TDL were incubated with MRC

0X12 (anti-lgic), MRC 0X6 (anti-class II), MRC 0X7 [anti-Thy-1, a marker of RTE in the rat (27,28)] and MRC 0X8 (anti-CD8) prior to separation, while CD45RC" cells were preparedby prior incubation with 0X6, 0X7 and 0X8 with the additionof MRC 0X22 and MRC 0X32 [both anti-CD45RC but non-competing (13)]. mAb, as elsewhere in this study, were eitherprepared from ascites generated within the laboratory or werepurchased as ascites from Serotec (Kidlington, UK). Thepurity of CD45RC" TDL was in all cases >96.5%, as measuredby FITC-anti-mouse IgG staining, with >98.5% the norm.CD45RC"1" TDL purified by negative selection were routinely87.5-93.0% pure (>99.0% CD4+). Stained cell populationswere analysed using Consort 30 software in conjunction witha FACScan cytometer (Becton Dickinson, Cowley, UK).

Preparation of CDS* and B lymphocytes

TDL from donor rats were purified using the standard mAbcocktail incubation/immunomagnetic depletion techniques(30). CD8+ cells were prepared by incubation with 0X12,0X6, 0X35 and W3/25 (the last two mAb being non-competinganti-rat CD4), followed by two or three rounds of depletion asrequired. Cell purities were routinely high (97.5-99.0%). Bcells were obtained from TDL of athymic nude rats and usedwithout further purification.

Preparation of CD4+CD8- 'single-positive' (SP) CD45RCthymocytes '

Thymi were extracted from 4- to 8-week-old PVG-RT7b ratsand the thymocytes released by the application of gentlepressure. The released thymocytes were then incubated withmAb 0X8, 0X22 and 0X32, followed by two or three roundsof immunomagnetic depletion. Most CD4~CD8~ 'double-nega-tive' thymocytes (including thymic precursors) expressCD45RC (27,31) and therefore were removed during purifica-tion. A residual population (-18%) of CD45RC"CD4-CD8-thymocytes, shown to have no regenerative potential (31),could not be eliminated from the CD4+CD8" SP thymocyteslabelled for injection.

51Cr-labelling

Purified subsets were labelled with 51Cr (sodium chromate CJS1P; Amersham, Amersham, UK) using 10 nCi (0.37 mBq) per108 subsetted cells. A detailed protocol appears elsewhere(32).

Treatment with anti-a4 integrin subunit and anti-VCAM-1 mAb

mAb HP2/1, anti-human 04 chain of a ^ (VLA-4) or (X4P7,cross-reactive with rat (33), was the kind gift of Dr Sanchez-Madrid (Madrid, Spain). CD45RCT CD4 T cells were purifiedand labelled with 51Cr as described above, and then incu-bated with either HP2/1 or with W3/25 (anti-rat CD4) asan isotype-matched control non-depleting mAb. Incubationswere for 30 min on ice at a concentration of 100 ng/ml. Thetreated cells were injected i.v. without further treatment (i.e.complete with antibody) and were followed immediately bythe injection of 1 mg of appropriate antibody in order tomaintain the level in vivo. mAb 5F10, anti-rat VCAM-1, wasthe kind gift of Dr R. Lobb (Biogen, Cambridge, MA). Purifica-tion and 51Cr-labelling of the CD45RC" CD4 T cells was asbefore; they were injected i.v. without further in vitro treatment.

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Migration pathways of CD4 T cell subsets 1863

After 30 min recipient rats received 1 mg of either 5F10 orW6/32 (anti-human HLA class II, non-cross-reactive with rat)as the isotype-matched non-depleting control mAb.

Localization studies

Approximately equal numbers (6.1-10X106) of purified, redblood cell free 51Cr-labelled CD8 T cells, B cells, CD45RC+or CD45RC" CD4 T cells were injected i.v. into syngeneicrats. Animals given CD4+CD8~ SP thymocytes received1-2X107 cells. Recipients were killed 0.5, 2.0, 24 or 48 hlater. The migration patterns between experiments were highlyreproducible and therefore the results of three (CD8 T cells),two (CD45RC+ T cells), six (CD45RCT T cells) and two(CD4+CD8~ SP thymocytes) separate experiments werepooled within lymphocyte types. Tissues were cleaned,drained of excess blood and weighed, the associated radio-activity measured in an LS280 gamma counter (LKB-Wallac,Milton Keynes, GB) and calculated (with background valuessubtracted) first as a percentage of the injected dose, thenas the percentage of the injected dose per gram of tissuesampled. Smith and Ford (34) argue the merits of monitoringthe localization as a function of concentration rather than perorgan. For example, when comparing organs such as liverand cervical LN (CLN) which differ in size by some 50-fold, but may contain similar quantities of radioactivity (34),meaningful comparison requires consideration of radioactivityper gram of tissue. In other circumstances, however, it maybe useful to assess the distribution of lymphocytes betweencompartments and in Table 1 the data are expressed aspercentage of injected dose per organ. Localization of theinjected cells was measured in lung, spleen and gut, PP, CLNand mesenteric LN (MLN), bone marrow (BM; both tibias)and in thymus, avoiding parathymic LN. Samples of liver andkidney were taken as indicators of cell damage which mighthave been incurred during the preparation. The rare experi-ments in which liver levels were unacceptably high wereexcluded from this study; kidney levels were always withinnormal limits. A 3-4 ml blood sample was withdrawn fromeach recipient before death and the radioactivity associatedwith 1.0 ml plasma and 2.0 ml blood mononuclear cells (white

blood cells) separated by density gradient centrifugation onHistopaque 1083 (Sigma, Poole, UK) determined. Levels ofsignificance were assessed by Student's Mest.

Results

CD8 T cells and B cells

TDL from the PVG strain comprise B cells (- 40%), CD8 Tcells (-8%), CD45RC+ CD4 (-35%) and CD45RC" CD4 Tcells (-14%). The CD45RC~CD4 subset is further divided intoimmature Thy-1+ RTE (-7%) and mature antigen-experiencedThy-1" CD4 T cells (27). The results of B cell and CD8 T celllocalization are given in Fig. 1 as % injected dose/g and inTable 1 at 24 h as % injected dose/organ. At 0.5 h, bothpopulations showed some retention in the lung capillaries,high levels in the peripheral blood and immediate entry intothe BM and spleen. By 2 h, B cells and CD8 T cells werecleared from both the lung and peripheral blood, and nowlocalized predominantly to the spleen. Apart from the BM,few CD8 T cells were found elsewhere at this time. B cellsshowed an early accumulation in PP and lower numbers inthe spleen. By 24 h, CD8 T cells and to a lesser extent Bcells had undergone a major redistribution into CLN, MLNand PP. The number of B cells found in CLN and MLN at24 h was significantly less (P < 0.001) than the number ofCD8 T cells.

CD45RC+ and CD45RC CD4 T cells

TDL were depleted of slg+ B cells, CD8+ T cells and Thy-1+

CD45RC~ RTE. The remaining lymphocytes were 51Cr-labelledand referred to as CD45RC+ T cells (99% CD4+, 87-93%CD45RC+), although they would also contain mature Thy-1"CD45RC" antigen-experienced T cells (Fig. 2a). We opted totolerate a small contamination of CD45RC" T cells in order toavoid purification by positive selection—a procedure whichwould coat the CD45RC+ subset with mAb. Earlier workshowed that half the CD45RC" CD4 T cell populationexpressed Thy-1 (35). Therefore, the CD45RC" subset wasalways purified by depleting TDL of Thy-1+ RTE in addition

Table 1. Summary of the distribution of purified 51Cr-labelled lymphocyte subsets in lymphoid tissues 24 h after injection,expressed as the mean percentage injected dose per organ (background subtracted) ± SD

Type oflymphocyte

B cellsCD8 T cellsCD45RC+CD45RC-CD4+CD8" SP

Tissue weight

No. of cellsinjected(X106)

10.09.7-10.08.5-9.46.1-9.4

10.0-20.0

factor"

c.p.m. injected(xiO"3)8

16.144.3-^18.933.3-35.217.5-42.331.0-138.7

n

46766

Spleen

23.81 218.72 218.08 213.73 2

• 19.86 2

t 1.19t 1.44t 1.88t 1.04t 1.04

401.6 mg

CLN

3.37 26.70 26.83 22.57 21.84 2

i 0.39i 1.00i 1.26t 0.72i 0.51

83.9 mg

MLN

4.17:t 0.638.00 ± 2.028.33:6.18 :

t 1.59t 1.65

3.02 ± 0.94

122.2 mg

PP

3.62 24.43 24.92 25.22 22.58 2

t 0.53:0.53:0.95i 0.43i 0.65

181.0 mg

Gut

1.75 ±0.822.01 ± 1.221.98 ±0.959.57 ± 1.180.72 ± 0.48

3.46 g

Thymus

0.08 ± 0.030.07 ± 0.060.07 ± 0.020.40 ± 0.120.08 ± 0.03

171.1 mg

"Radioactivity injected per recipient. The combined recovery of radioactivity from the above tissues together with lung, liver, kidney, plasma,WBC and BM (two tibiae) was -60-67% of the injected dose.

"Values given are means derived from the pooled weights of the tissues of the experimental animals in this table (n = 29), except for gut(a mean of 10 non-injected but age-matched control rats) and PP (obtained from ref. 37).

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1864 Migration pathways of CD4 T cell subsets

CD8TcellsB cells

hours after injection

Fig. 1. In vivo migration and localization of purified populations of B cells and CD8 T cells in the rat. Lymphocytes were purified from TDL,labelled with 51Cr and injected i.v. Recipients were killed at 0.5, 2, 24 or 48 h later, tissues removed, weighed and the accumulation ofradioactivity determined Results were expressed as percentage of injected dose per gram of tissue or per 2 ml blood (WBC) or per tibia(BM) Each bar represents the mean ± SE (CD8 0.5, 2, 24 h: n = 6, 48 h: n = 2; B cells all times: n = 4)

to B cells, CD8 T cells and CD45RC+ T cells (Fig. 2c-f). Theresulting population was >98% CD4+ (Fig. 2e) and had thecharacteristics (defined by forward and side scatter analysis)of small lymphocytes (cf. Fig. 2b and d). Blast cells, alsoCD45RC" (36), were lost during the purification procedure.

The localization of the CD45RC+ T cells broadly resembledthat of B cells and CD8 T cells, although there was lessretention in the lungs at 0.5 h, with a compensating accumula-tion in the spleen (Fig. 3). At 2 h, most of the CD45RC+ Tcells were in the spleen, with relatively few in LN, PP, BM,intestine or blood. By 24 h and later (48 h), the CD45RC"1"subset had redistributed to both MLN and CLN (Table 1).Approximately 40% fewer CD45RC+ T cells were found inBM at 24 h compared with CD8 T cells or B cells.

The migration pattern of the CD45RC" (Thy-T) subset stoodin marked contrast to the above three subpopulations. Twiceas many CD45RC" as CD45RC+ T cells were found in thelung at 0.5 h, but by 2 h these cells had been released andwere found along with the CD45RC+ T cells in the spleen(Fig. 3). By 24 h, CD45RCT T cells were redistributed fromthe spleen to LN and PP (Table 1), although there weresignificantly fewer (compared with CD45RC+ T cells) in the

MLN (P < 0.05) and CLN (P < 0.01). There was a furtherloss of CD45RC- T cells from MLN, CLN, PP and bloodbetween 24 and 48 h.

The most striking deviation from the norm was the earlyand sustained migration of CD45RC" T cells to PP-freesmall intestine (Fig. 3, Table 1)—presumably lamina propria.Approximately 7% of the injected dose localized rapidly(0.5 h) to the intestine, more than five times the number ofCD45RC+ T cells. However, there was no evidence thatCD45RC" T cells were accumulating in the gut between0.5 and 48 h (Fig. 3), suggesting that CD45RC" T cells werenot static in this site but continuing to migrate via afferentlymphatics back to the blood. Recirculation studies inmesenteric lymphadenectomized rats have confirmed a rapidexit of CD45RC" T cells from the intestine via afferent lymph(S. M. Sparshott and E. B. Bell, in preparation).

The second major change observed was an unexpectedentry of CD45RC~ T cells into the thymus. In agreementwith earlier work using unseparated lymphocyte populations(37,38), B cells, CD8 T cells and CD45RC+ CD4 T cells allfailed to localize to the thymus in significant numbers (Table1). In contrast, a modest accumulation of CD45RC" T cells at

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CD45RC FSC

no.

1h 1!\ f

CD4 a4 Intagrln

Fig. 2. A representative analysis of CD45RC+ and CD45RC" CD4 Tcells purified from TDL and used to study lymphocyte migration,(a) CD45RC"1" subset: TDL stained with mAb 0X22 (anti-CD45RC)followed by FITC-F(ab)'2 anti-mouse IgG before (dotted line) andafter (solid line) depletion. Around 99.9% of B cells, CD8 T cells andRTE were removed by three rounds of immunomagnetic depletion;the resulting population was 99.7% CD4+, 87.5% CD45RC+. (c)CD45RC" subset: TDL stained with FITC-OX22 before (dotted line)and after (solid line) depletion. Around 99.5% of B cells, CD8 Tcells, CD45RC"1" cells and RTE were removed by three rounds ofimmunomagnetic depletion; the remaining cells were 96.2% CD45RC"by FITC-OX22 staining. The forward versus side scatter profile of thepurified CD45RC" subset (d) displayed a pattern of size andgranularity characteristic of small lymphocytes; compare withunseparated TDL (b) which show a typical blast cell population(arrow) (e) The purified CD45RC" subset (solid line) was 98 1%CD4+ (mAb W3/25 followed by FITC-F(ab)'2 anti-mouse IgG) and iscompared with TDL before separation (dotted line), (f) UnseparatedTDL (dotted line) and the CD45RC" subset (solid line) show identicalstaining for the a4 integrin subunit [biotinylated HP2/1 followedby streptavidm-phycoerythrin (SAPE)]; control staining (biotinylatedcontrol mAb followed by SAPE) is shown by the dashed line.

2 h anticipated the relatively high levels (2.9% injected dose/g tissue) present at 24 h (Fig. 3 and Table 1). Numbers fellat 48 h suggesting a subsequent exit of donor cells fromthe thymus.

CD45RCT CD4+CD8~ SP thymocytes

Since the recirculation patterns of CD45RC+ and CD45RC"subsets of CD4 T lymphocytes were so clearly different it was

Migration pathways of CD4 T cell subsets 1865

of interest to determine to what extent this could be attributedto the presence or absence of the CD45RC isoform per se.The migration patterns of CD4+CD8~ SP thymocytes, a Thy-1 + CD45RC" population destined to join the peripheral pool asRTE (27), were therefore examined using identical techniques.

The migration of CD45RC" CD4+ SP thymocytes wascharacterized by their exceptionally slow passage throughthe lung (Fig. 3). Three times as many CD45RC" CD4+ SPthymocytes as mature CD45RC" peripheral T cells remainedin the lung at 30 mm after injection. Compared with CD45RC+

T cells, there was a 5-fold excess. Even 2 h after injection,>30% of injected CD45RC" CD4+ SP thymocytes per gramwere still entrapped within the lung, although by 24 h theirnumbers were no different from other lymphocyte populations.Sequestration in the lungs coloured the other elements of theearly localization, with very low levels in the spleen, BM, PPand blood. Most notably, there was almost no localization atall of CD45RCT CD4+ SP thymocytes into gut or thymus, twotissues into which CD45RC" T cells had shown enhancedmigration (Table 1). By 24 h, there was evidence that migrationinto secondary lymphoid tissues had begun; in CLN, CD45RC~CD4+ SP thymocytes were in slight excess compared withCD45RC" T cells, although levels in MLN and PP remainedlower. By 48 h, migration into CLN, MLN and PP had not quiteachieved parity with mature CD45RC" and CD45RC+ T cells,perhaps reflecting the relative immaturity of the injectedthymocytes. In summary, CD45RC" CD4+ SP thymocyteswere programmed to migrate as CD45RC"1" T cells. The factthat they expressed the low molecular weight isoform of CD45had no bearing on their migration potential.

Adhesion molecules controlling localization of CD45RC~ Tcells

Considering the relatively high localization of CD45RC" T cellsto the small intestine we asked whether migration to this sitewas regulated by adhesion molecules expressing the <x4

integrin subunit. When paired with (3-|, cells expressing theresulting a$-\ (VLA-4) molecule bind to endothelial cellsexpressing VCAM-1 (39,40). Alternatively, when paired withp7, the a4p7 integrin is known to bind to the mucosal addressinMAdCAM-1 (41,42). Weak binding between a4p7 and VCAM-1 was also observed, although this was restricted tolymphomas and required phorbol ester activation (39,41,43).

The CD45RC" subset, when incubated with mAb HP2/1(anti-a4 integrin), showed a single population of stained cells,a profile that was indistinguishable from that of unseparatedTDL (Fig. 2f) and similar to published profiles for peripheralblood lymphocytes (44). Levels of a4 integrin on CD45RC+

and CD45RC" T cells were identical (data not shown). Puri-fied 51Cr-labelled CD45RC" T cells were incubated with mAbHP2/1 and injected without washing. Recipients received anadditional 1 mg of HP2/1 i.v. to maintain effective levels in vivo.Controls included the same 51Cr-labelled cells incubated andinjected with isotype-matched mAb W3/25 (anti-rat CD4), amAb which would also coat the surface of the injectedcells. Additional recipients were injected with untreated 51Cr-labelled cells.

CD45RC" T cells treated with control mAb W3/25 showedonly slight variations from untreated cells (Fig. 4) indicatingthat the presence of mAb on the surface per se was not

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1866 Migration pathways of CD4 T cell subsets

SPLEEN

D CD45RC- CD4 T cells• C045RC- CD4T cells0 CD4*8- SPthymocytes

2 4 4 1

O.5 2 2 4 4 8

hours after iniertinn

Fig. 3. CD45RC" CD4 T cells, unlike CD45RC+ T cells or CD4+ SP thymocytes, show enhanced localization to the gut and thymus. Purifiedcell populations were 51Cr-labelled and injected i.v. Recipients were killed 0.5, 2, 24 or 48 h later, tissues removed, weighed and theaccumulation of radioactivity determined. Results were expressed as the percentage of injected dose per gram of tissue or per 2 ml blood(WBC) or per tibia (BM). Each bar represents the mean ± SE of four to 11 recipients per group.

sufficient to interfere with the ability of lymphocytes to re-circulate. Treatment with anti-a4 mAb had profound effectson the localization of CD45RC" T cells. Migration to the smallintestine, PP, MLN and thymus was blocked almost entirely,while localization to CLN was halved (Fig. 4). In contrast, thenumber of labelled cells in the spleen doubled. That this wasnot due to sequestration of antibody-coated cells is indicatedby the fact that more than twice the usual number of cells(control recipients) were circulating in the blood. CD45RC" Tcells unable to localize to their traditional sites remained inthe circulation.

To determine whether the a4 integrin-dependent entry ofCD45RC- T cells was due to VCAM-1 or MAdCAM-1 recogni-tion, a similar experiment was done using mAb 5F10 whichrecognises rat VCAM-1. mAb W6/32 (anti-human, non-cross-reacting with rat) was used as a control. Anti-VCAM-1did not inhibit migration into the small intestine, PP, MLN,brachial LN, BM or spleen (Fig. 5); localization to the CLN wasslightly reduced (28.5%, P < 0.05). Unexpectedly, however,entrance into the thymus was almost completely (92.3%)inhibited.

The combined blocking studies using anti-a4 and anti-VCAM-1 suggested that CD45RC" T cell migration into gut,PP and MLN was primarily mediated by a4p7-MAdCAM-1interaction. Entrance of CD45RC" T cells into CLN wasonly partially dependent on <x4 integrin expression, whereasmigration into the thymus depended entirely on cc4 integrinexpression and recognition of VCAM-1 on the thymic vas-culature.

Discussion

The kinetics of migration of unseparated lymphocytes follow-ing i.v. injection in rats have been intensively studied andfollow defined patterns (34,45). In the early stages (30 min)there is an accumulation of the injected cells in the lungand spleen, followed at 2-2.5 h by a major concentration inthe spleen. Distribution to secondary lymphoid organs suchas CLN and MLN follows more slowly and progresses to astate of equilibrium, from which there is little subsequentvariation. A majority of the injected cells are therefore foundin peripheral LN at 24-48 h as they redistribute from the

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Migration pathways of CD4 T cell subsets 1867

spleen. In the present experiments, significant variations fromthis generalized overview were found when the migrationpatterns of individual subsets were examined.

In vitro handling of lymphocytes has been shown to influ-ence the kinetics of cell migration (34,45), although the effectsare transient. Migratory behaviour is restored to normal within4-6 h (34). While it is theoretically possible that the purificationand 51Cr-labelling procedures used could themselves havebiased the results, all subsets (with the exception of the nude-derived B cells) were treated comparably. It is unlikely,

Gut Thymus CLN Spleen

mAb

Controlanti-«4

Gut

113.210.8

% of control valueThymus WBC PP

100.46.5

67.1255.1

98.31.9

MLN

133.110.4

CLN

81.254.2

Spleen

128.7195.2

Fig. 4. mAb against the a4 integrin subunit blocks migration ofCD45RC" CD4 T cells into the gut, PP, MLN, CLN and thymus. 51Cr-labelled CD45RC" CD4 T cells were untreated (open bars, n = 1) orincubated in vitro with control mAb W3/25 (hatched bars, n = 4)or anti-a4 mAb HP2/1 (solid bars, n = 4) and injected i.v. Ratsreceiving mAb treated cells were given an additional i.v. injection ofthe same mAb (1 mg) at the time of cell transfer. All recipients werekilled 24 h later and the accumulation of radioactivity in tissuesdetermined. Results (means ± SE) were expressed as percentageof injected dose per gram of tissue or per 2 ml blood (WBC).

therefore, that the differences observed in early localization(0.5 and 2 h) are non-specific and arise from our preparativetechniques, while the major differences in localizationobserved at 24 and 48 h occurred long after cells hadresumed normal migration.

B cells localized early (0.5-2 h) to PP, ahead of CD8 Tcells, but by 24 h no differences were observed. In the spleensignificantly fewer B cells (P < 0.01-0.001) than CD45RC"1"CD4 or CD8 T cells had entered at 2 h after injection, butagain this difference had disappeared by 24 h.

In the case of T cells, these departed from the spleen morequickly and were redistributed to LN. These observations areconsistent with the fact that compared with B cells, T cells werefound to migrate through tissues with accelerated kinetics (46-48). The localization of B cells to LN at 24 h never matchedthat of CD4 and CD8 T cells (30-42% less, P < 0.01-0.001).CD45RC+ CD4 T cells, CD8 T cells and B cells all failed tolocalize in large numbers to the gut (excluding PP) nor didthey enter the thymus

The subset which displayed the greatest deviation fromthe generalized pattern was the CD45RC" CD4 T cell subset,a Thy-1" mature T cell population that has encounteredantigen in the recent past (15,18,20). Although antigen-induced proliferation initially generates CD45RC" blast cells(36), the population investigated here was at a post-activationstage and composed of small lymphocytes—blast cells didnot co-purify with the CD45RC" subset to any great extent asjudged by forward and side scatter profiles on cytofluoro-graphic analysis (Fig. 2d). When injected, CD45RC" T cellslocalized initially to the spleen but by 24 h had becomeredistributed to LN and PP. The numbers of CD45RC" T cellsin the PP by 24 h was normal, in MLN slightly reduced(P < 0.05) and in CLN substantially less (P < 0.01). By 48 h,the numbers in both MLN and CLN had declined further( P < 0.001) and could not be accounted for by a compensat-ing accumulation elsewhere. The overall loss of CD45RC" as

Gut Thymus BM MLN CLN BrLN Spleen

mAb

ControlVCAM-1

Gut

78.685.6

Thymus

76.17.8

%

BM

87.889.8

of control valueWBC PP

109.5204.8

91.7107.8

MLN

93.392.1

CLN

92.271.5

BrLN

82.673.3

Spl««n

97.5103.4

Fig. 5. Migration of CD45RC~ CD4 T cells into the thymus is blocked by anti-rat VCAM-1. 51Cr-labelled CD45RC" CD4 T cells were injectedinto untreated recipients (open bars, n = 2) or recipients given control mAb W6/32 (hatched bars, n = 3) or mAb 5F10 (anti-VCAM-1) (solidbars, n = 4). All recipients were killed 24 h later and the accumulation of radioactivity in tissues determined. Results (means ± SE) wereexpressed as the percentage of injected dose per gram of tissue or per 2 ml blood (WBC) or per tibia (BM).

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Migration pathways of CD4 T cell subsets 1867

spleen. In the present experiments, significant variations fromthis generalized overview were found when the migrationpatterns of individual subsets were examined.

In vitro handling of lymphocytes has been shown to influ-ence the kinetics of cell migration (34,45), although the effectsare transient. Migratory behaviour is restored to normal within4-6 h (34). While it is theoretically possible that the purificationand 51Cr-labelling procedures used could themselves havebiased the results, all subsets (with the exception of the nude-derived B cells) were treated comparably. It is unlikely,

Out Thymua WBC CLN SplMn

mAb

Controlanti-a4

Out

113.210 J

% of control valueTKynu WBC PP

100.48.5

•7.1255.1 1.9

MLN

1S3.110.4

CLN

81.254 2

SplMn

128.7195.2

Fig. 4. mAb against the 04 integrin subunit blocks migration ofCD45RC" CD4 T cells into the gut, PP, MLN, CLN and thymus. 51Cr-labelled CD45RC~ CD4 T cells were untreated (open bars, n- 1) orincubated in vitro with control mAb W3/25 (hatched bars, n = 4)or anti-O4 mAb HP2/1 (solid bars, n = 4) and injected i.v. Ratsreceiving mAb treated cells were given an additional i.v. injection ofthe same mAb (1 mg) at the time of cell transfer. All recipients werekilled 24 h later and the accumulation of radioactivity in tissuesdetermined. Results (means ± SE) were expressed as percentageof injected dose per gram of tissue or per 2 ml blood (WBC).

therefore, that the differences observed in early localization(0.5 and 2 h) are non-specific and arise from our preparativetechniques, while the major differences in localizationobserved at 24 and 48 h occurred long after cells hadresumed normal migration.

B cells localized early (0.5-2 h) to PP, ahead of CD8 Tcells, but by 24 h no differences were observed. In the spleensignificantly fewer B cells (P < 0.01-0.001) than CD45RC+CD4 or CD8 T cells had entered at 2 h after injection, butagain this difference had disappeared by 24 h.

In the case of T cells, these departed from the spleen morequickly and were redistributed to LN. These observations areconsistent with the fact that compared with B cells, T cells werefound to migrate through tissues with accelerated kinetics (46-48). The localization of B cells to LN at 24 h never matchedthat of CD4 and CD8 T cells (30-42% less, P < 0.01-0.001).CD45RC"1" CD4 T cells, CD8 T cells and B cells all failed tolocalize in large numbers to the gut (excluding PP) nor didthey enter the thymus.

The subset which displayed the greatest deviation fromthe generalized pattern was the CD45RC" CD4 T cell subset,a Thy-1" mature T cell population that has encounteredantigen in the recent past (15,18,20). Although antigen-induced proliferation initially generates CD45RC" blast cells(36), the population investigated here was at a post-activationstage and composed of small lymphocytes—blast cells didnot co-purify with the CD45RC" subset to any great extent asjudged by forward and side scatter profiles on cytofluoro-graphic analysis (Fig. 2d). When injected, CD45RC" T cellslocalized initially to the spleen but by 24 h had becomeredistributed to LN and PP. The numbers of CD45RC" T cellsin the PP by 24 h was normal, in MLN slightly reduced( P < 0.05) and in CLN substantially less ( P < 0.01). By 48 h,the numbers in both MLN and CLN had declined further(P< 0.001) and could not be accounted for by a compensat-ing accumulation elsewhere. The overall loss of CD45RC~ as

Out Thymui BM WBC MLN CLN BrLN Spltcn

mAb

ControlVCAM-1

Gut

76.685.6

Thymus

76.17.8

%ofBM

87.889.8

control valueWBC PP

109.5204.8

91.7107.8

MLN

92.1

CLN

92.271.5

BrLN

82.673.3

Spleen

973103.4

Fig. 5. Migration of CD45RC" CD4 T cells into the thymus is blocked by anti-rat VCAM-1. s1Cr-labelled CD45RC CD4 T cells were injectedinto untreated recipients (open bars, n = 2) or recipients given control mAb W6/32 (hatched bars, n = 3) or mAb 5F10 (anti-VCAM-1) (solidbars, n = 4). All recipients were killed 24 h later and the accumulation of radioactivity in tissues determined. Results (means ± SE) wereexpressed as the percentage of injected dose per gram of tissue or per 2 ml blood (WBC) or per tibia (BM).

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1868 Migration pathways of CD4 T cell subsets

compared with CD45RC+ T cells was consistent with theshorter half-life of this subset reported earlier (21).

The enhanced localization of CD45RC" T cells to the smallintestine was most notable, even at the earliest time point.The peak level in the gut was attained with great rapidity anddid not depend, as do PP and LN, on an accumulation over24 h. Exit of CD45RC" T cells was equally brisk (withinhours) as shown in a separate study (Sparshott and Bell, inpreparation). The MLN, the termination point of intestinallymphatics, would be the immediate beneficiary of this rapiddrainage. An analysis of the kinetic changes suggests thatthe MLN may acquire practically all CD45RC" T cells fromafferent lymph rather than from the blood across HEV (seelater discussion). The net result was that the number ofCD45RC" T cells in the MLN at 24 h was only slightly reducedfrom that of other subsets, despite the fact that low levels inthe blood would deliver fewer CD45RCT T cells to HEV. CLNon the other hand, deprived of the large intestinal component,already had significantly fewer (P < 0.01) CD45RC" T cellsat 24 h.

The experiments showed unexpectedly that CD45RC" Tcells also entered the thymus in significant numbers. Thisappears to differ from earlier studies which failed to findperipheral lymphocytes entering adult thymus (37,38),although during the first 2 days of neonatal life the thymuswas receptive to both CD4 and CD8 T cells (49). There areat least two reasons which could account for the presentobservations, (i) Firstly, that CD45RC" T cells represent arelatively rare subset whose localization would be masked bythe other populations. It was only when CD45RC" T cells wereisolated from the whole that selective migration into the thymus(and gut) was apparent, (ii) Furthermore, it may be that evenwithin the CD45RC~ subset not all cells have the ability toenter the thymus. For example, the CD45RC" lymphocyteswhich migrate to the gut could be non-overlapping with thosewhich enter the thymus (see later discussion in relation toadhesion molecule expression), in which case the lattersubpopulation would be very small indeed.

The migration pattern of CD45RC" T cells differed from thatseen in the gut in that thymic values increased steadily up to24 h. In a separate investigation we could clearly identify,by immunohistochemistry, adoptively transferred CD45RC"T cells within the thymic medulla (J. Westermann ef a/.,unpublished observations). Others have also reported theappearance of rare allotype-marked donor CD4 and CD8 Tcells in the thymus following adoptive transfer, again localizedprimarily to the medulla (50). However, the re-entry of peri-pheral cells was thought to be restricted to activated T cells(51). The thymic migration observed in the present studywas of mature, recirculating cells with the flow cytometriccharacteristics of small lymphocytes (Fig. 2d).

In the periphery CD4 T cells expressing the low molecularweight isoform (CD45RC~) include both mature T cells (Thy-1") and immature Thy-1+ RTE (27). We found that CD4+ SPthymocytes migrated to peripheral LN but failed to enterthe small intestine or the thymus despite their CD45RC"phenotype, indicating that they resembled the CD45RC+

subset in migratory behaviour. This is consistent with the factthat RTE differentiate within a week of leaving the thymus intoThy- r CD45RC+ T cells (27,28).

Recent investigations in mice showed that early localization(1 h) of whole LN populations to intestine and PP was partiallyblocked by mAb specific for the 04 integrin subunit or bymAb specific for a combinatorial epitope on the 0C4P7 integrin(52). In rats mAb TA-2 to the rat 04 subunit was shown toinhibit the migration of unseparated lymphocytes into PP andMLN (by 80 and 95% respectively) (53), whereas lymphocytemigration into peripheral LN was not inhibited by the samemAb. Blast cells, which preferentially migrate to the gut andPP (22), were also blocked by this same anti-a* mAb (25).

In the present study we asked which adhesion moleculescontrolled the movement of CD45RC" T cells across theendothelium of the gut and thymus. Treating CD45RC" T cellswith anti-a4 mAb profoundly inhibited migration into gut, PP,MLN and thymus. Migration into the CLN by comparison wasreduced by only 46%. Localization to the spleen followinganti-O4 treatment was almost doubled. It could be arguedthat the failure of CD45RC" T cells to enter tissues wasprimarily the consequence of retention in the spleen, i.e. theclearance from the circulation of lymphocytes coated withmAb. This was unlikely for two reasons, (i) CD45RC" T cellsbearing a control mAb on their surface (the anti-rat CD4 mAbW3/25) displayed a normal migration pattern with nosequestration in the spleen. Surface antibody per se doesnot interfere with lymphocyte recirculation. (ii) In HP2/1 -treatedrats, the number of labelled T cells in the blood was doubled,indicating that the injected cells were viable and circulat-ing, not sequestrated. Having blocked their exit from theblood with antibody, the increased numbers of injected cellswould inevitably raise the levels of radioactivity in the highlyvascularized spleen.

Although the number of mAb recognizing adhesion mole-cules in the rat is limited, by combining the results of anti-04 blocking with those of anti-VCAM-1, it was possible todistinguish between migration based on a ^ from that basedon CI4P7 expression. The fact that migration into the thymuswas abolished by anti-VCAM-1 gives confidence that mAbtreatment was effective in vivo and suggests that CD45RCTT cells entering the thymus expressed Otfiy By deductionthe failure of anti-VCAM-1 to block entry of CD45RC" T cellsto the gut, PP or MLN indicates that migration to these siteswas in some way governed by C^PJ expression mediatedvia recognition of the mucosal addressin MAdCAM-1. It isinteresting to speculate on whether T cells co-express c ^and (X4P7 or, alternatively, whether integrin expression furthersubdivides the CD45RC" subset.

The migration of CD45RC" T cells into PP and gut laminapropria apparently via (X4P7 expression is consistent with thework of others (25,52,53). Entrance of these T cells into PPmay be mediated entirely by expression of MAdCAM-1 onPP HEV. However, entrance into MLN is more complex.Theoretically, CD45RC" T cells could migrate directly acrossMLN HEV via MAdCAM-1 or alternatively gain entrance entirelyfrom intestinal afferent lymph. In the present study we calculatethat at any one instant the gut lamina propria as a wholecontained between 5.4x105(0.5 h) and 7.1x105(24 h) 51Cr-labelled CD45RC" CD4 T cells (mean values). At 24 h, thetime of maximum accumulation, radioactivity in the MLN(mean value) corresponded to 5.1X105 CD45RC" CD4 Tcells. It was clear that the population in the MLN could be

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accounted for entirely by lymphatic drainage alone. Whetherthe CD45RC" subset also migrates across HEV into MLN willrequire additional, more direct evidence.

The migration into CLN was substantially different. FewerCD45RC" than CD45RC+ T cells entered CLN under normalcircumstances. Using unseparated whole I_N cells, otherswere unable to detect any inhibition of migration to CLN byanti-O4 treatment (52,53). Here we found that localization ofpurified CD45RC" T cells following anti-a4 treatment wasreduced by 50%. It is again necessary to entertain thepossibility that the component blocked by anti-O4 representsthose cells originating in afferent lymph. Nevertheless, halfthe CD45RC~~ T cells apparently entered CLN independentlyof the 04 integrin, presumably across HEV. This suggests thatCD45RC" T cells may enter CLN by more than one route(via afferent lymph and HEV) and that the CD45RC" T cellpopulation may be heterogeneous with respect to integrinexpression, although this was not apparent following stainingfor 04 integrin. In previous studies we have reported theexpression of VCAM-1 by HEV in CLN of rats by immunohisto-chemistry (54). In addition we showed that cultured highendothelial cells expressed VCAM-1 and that lymphocytebinding was partially dependent on 04 integrin-VCAM-1recognition. In vivo, anti-VCAM-1 mAb significantly (P< 0.05)reduced localization to CLN but by only 28%. The biologicalimportance of the change is uncertain but is consistent withthe possibility that a minority of CD45RCT T cells express0 ^ — a majority would express o^pV and therefore not beblocked by anti-VCAM-1.

T cell development depends on the successful and continu-ing migration (55) of BM-derived pro-T cell precursors acrossthymic endothelium into the thymus (56). The adhesion molec-ules which facilitate this migration sequence have not beenidentified although a number of candidates have been pro-posed, including the ctg integrins (57,58), CD44 (59, 60) andL-selectin (58). p2-Microglobulin was thought to act as achemoattractant (61) although colonization of the thymus wasunimpaired in Pj-microglobulin-knockout mice (62). Stem cellmigration across basement membrane in vitro was blockedby antibodies and the synthetic peptide RGDS which inhibitsfibronectin recognition (63). Interestingly, fibronectin providesan alternative ligand for a ^ (40,54,64). The present experi-ments using mAb against the 04 subunit and VCAM-1 showedthat entry of CD45RC" CD4 T cells to the thymus from theperiphery depended on 04 integrin-VCAM-1 interaction. Toour knowledge, this is the first report which identifies theadhesion molecules involved in the in vivo migration of T cellsacross the vascular endothelium into the thymus. It is a mootpoint whether the presence of mature T cells has any role toplay during the differentiation and development of T cells inthe thymus. It will be interesting, however, to determinewhether pro-T cells gain access to the thymus via c^pVVCAM-1 interaction.

Acknowledgements

We wish to thank Simon Oldroyd and Samantha Hayes for technicalassistance and Graham Preece for purifying and testing the HP2/1mAb. This work was supported by project grants to E. B. B. fromthe Medical Research Council and the Arthritis and RheumatismCouncil (B0174).

Migration pathways of CD4 T cell subsets 1869

Abbreviations

BMCLNHEVLNMLNPPRTESPTDL

bone marrowcervical lymph nodehigh endothelial venuleslymph nodesmesenteric lymph nodePeyer's patchesrecent thymic emigrantssingle positivethoracic duct lymphocytes

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