Diversity in Humans CCR7 Expression and Memory T Cell

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CCR7 Expression and Memory T Cell Diversity in Humans James J. Campbell, Kristine E. Murphy, Eric J. Kunkel, Christopher E. Brightling, Dulce Soler, Zhimin Shen, Judie This information is current as Boisvert, Harry B. Greenberg, Mark A. Vierra, Stuart B. of September 29, 2021. Goodman, Mark C. Genovese, Andrew J. Wardlaw, Eugene C. Butcher and Lijun Wu J Immunol 2001; 166:877-884; ;

doi: 10.4049/jimmunol.166.2.877 Downloaded from http://www.jimmunol.org/content/166/2/877

References This article cites 33 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/166/2/877.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CCR7 Expression and Memory T Cell Diversity in Humans1

James J. Campbell,2*†‡ Kristine E. Murphy,§ Eric J. Kunkel,†‡ Christopher E. Brightling,¶ Dulce Soler,§ Zhimin Shen,§ Judie Boisvert,ʈ Harry B. Greenberg,‡ʈ Mark A. Vierra,# Stuart B. Goodman,** Mark C. Genovese,†† Andrew J. Wardlaw,¶ Eugene C. Butcher,†‡ and Lijun Wu3§

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose speciﬁcities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We ﬁnd that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules

␣ ␤ Downloaded from (cutaneous lymphocyte Ag 4 7 integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ.

Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio http://www.jimmunol.org/ of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue. The Journal of Immunology, 2001, 166: 877–884.

he mammalian cognate immune system uses a self/non- its cognate ligand, it will differentiate into a memory T cell and self recognition strategy that randomly generates ex- may begin to express homing receptors that both enable the cell to T tremely large numbers of receptors, with the assumption home to nonlymphoid tissues and prevent it from homing to other that novel Ags will be recognized by at least a few members of the types of tissues. repertoire. As only perhaps 1 in 109 T cells may recognize a po- Along with lymphocyte adhesion molecules, chemokines and tentially threatening immunological target, it is imperative that as their receptors appear to play vital roles in the trafficking cycle of by guest on September 29, 2021 many lymphocytes as possible be exposed to any foreign Ag. lymphocytes during inflammation and routine immunosurveillance Thus, routine immunosurveillance involves constant recirculation (Refs. 2 and 3; reviewed in Ref. 4). CCR7 is a very important of lymphocytes through tissues. Naive T cells follow a relatively player in the mechanism by which T lymphocytes enter secondary simple path of recirculation from the blood into secondary lym- lymphoid organs through high endothelial venule(s) (HEV).4 T phoid organs, then back into the blood via afferent lymph (re- cells unable to receive a signal through CCR7 do not adhere to viewed in Ref. 1). This cycle continues until the cell at some point HEV after the initial tethering and rolling process begins; hence, they (during its time within a secondary lymphoid organ) recognizes an are unable to enter lymph nodes or Peyer’s patches (Refs. 5 and 6; Ag for which its TCR is specific. Once a naive T cell recognizes reviewed in Ref. 4). Mice lacking the CCR7 gene form small, disor- ganized lymphoid tissues containing very few T or B cells (7). Ligands for CCR7 are expressed by the HEV of secondary lym- *Joint Program in Transfusion Medicine, Children’s Hospital, and Harvard Medical phoid organs (5, 6, 8), by parenchymal cells within T zones of School, Department of Pathology, Boston, MA 02115; †Laboratory of Immunology and Vascular Biology, Department of Pathology, and the Digestive Disease Center, lymph nodes (8), and by endothelial cells at the openings of lym- Department of Medicine, Stanford University Medical School, Stanford, CA 94305; phatic vessels within peripheral tissues (8). In addition, CCR7 ex- ‡Center for Molecular Biology and Medicine, Veterans Affairs, Palo Alto Health Care pression (or lack thereof) defines a subset of peripheral blood CD4 System, Palo Alto, CA 94304; §Millennium Pharmaceuticals, Inc., Cambridge, MA 02142; ¶Division of Respiratory Medicine, Institute for Lung Health, Leicester Uni- cells enriched in cells of unique “effector” phenotype (9). versity Medical School, Leicester, United Kingdom; and Departments of ʈMicrobi- Thus, due to the apparent biological significance of this mole- # ** †† ology and Immunology, Surgery, Functional Restoration, and Division of Im- cule, we found it of great importance to fully characterize the munology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305 expression of CCR7 on T lymphocytes during all identifiable Received for publication August 3, 2000. Accepted for publication October 20, 2000. phases of T lymphocyte trafficking. We have recently generated a The costs of publication of this article were defrayed in part by the payment of page series of anti-CCR7 mAbs, two of which recognize epitopes that charges. This article must therefore be hereby marked advertisement in accordance correlate directly with functional responsiveness to CCR7 ligands. with 18 U.S.C. Section 1734 solely to indicate this fact. We report our findings with these new Abs below. 1 This work was supported by National Institutes of Health Grants AI46784 (to J.J.C.); and GM37732, AI37832, and GM56527 (to E.C.B.). 2 Address correspondence and reprint requests to Dr. James J. Campbell, Assistant 4 Abbreviations used in this paper: HEV, high endothelial venule(s); CXCR, CXC Professor of Pathology, Harvard Medical School; Children’s Hospital, Division of chemokine receptor; SDF, stromal cell-derived factor; MIP, macrophage-inflamma- Transfusion Medicine, 300 Longwood Avenue, Room BD-401, Boston, MA 02115. tory protein; SLC, secondary lymphoid-tissue chemokine; BCA-1, Burkitt’s lym- E-mail address: [email protected] phoma receptor-1 ligand chemokine; CLA, cutaneous lymphocyte Ag; RA, rheuma- 3 Please address mAB requests to L.W. toid arthritis.

Materials and Methods Lymphocytes from the lamina propria of human jejunum were isolated mAbs to human CCR7 as described previously (16) with slight modification (i.e., the DTT step to degrade mucous was eliminated because we found that DTT destroyed the ␣ ␤ L1/2 cells were transfected with human CCR7 and used to immunize mice. epitopes recognized by the anti- 4 7 mAb ACT-1). Lymphocyte yield was mAbs to CCR7 were generated as described (10) (the mAbs were produced not reduced by eliminating this step (data not shown). Lymphocytes were and purified by the staff of Millennium Pharmaceuticals, Cambridge, MA). isolated from normal human skin by first using a razor blade to separate the mAbs that recognized CCR7 transfectants but not wild-type L1/2 cells epidermis and a portion of the dermis from the lower dermis and s.c. fat. were tested against the following panel of L1/2 cells transfected with var- The epidermal pieces were cut into strips and incubated in cold 5 mM ious other known (or suspected) chemokine receptors: CCR1, CCR2, EDTA/HBSS for 120 min with vigorous stirring. The supernatant from this CCR3, CCR4, CCR5, CCR6, CCR8, CCR9, CXC chemokine receptor step was spun down to obtain released lymphocytes, and the remaining (CXCR) 1, CXCR2, CXCR3, CXCR4, CXCR5, CX3CR1, CR1, Bonzo, strips were crushed through a 50-␮m pore size strainer to obtain additional BOB, APJ, RDC, AF014958, and LyGPR. No appreciable cross-reactivity lymphocytes. Lymphocytes were isolated from normal lung by finely minc- was observed for any of these receptor transfectants. Several clones spe- ing the tissue and sieving the resulting suspension through gauze. Lym- cific for CCR7 transfectants were derived, and a few of them, including phocytes were isolated from explant livers by first cutting the liver into 7H12 (murine IgG2b) and 3D9 (murine IgM), recognized a proportion of small 1-cm2 pieces, washing in RPMI 1640 supplemented with 10% FBS, PBLs nearly identical with the proportion capable of responding to known then homogenizing into a cell suspension with a Stomacher 400 (Seward, CCR7 ligands. These two mAbs gave essentially identical staining patterns London, U.K.). Lymphocytes were isolated from synovial tissue by minc- on all lymphocyte subsets tested, and only these clones were used for the ing and treating with EDTA as described above for epidermis. Synovial studies reported here. The staining patterns of these two novel mAbs were fluid and bronchoalveolar lavage fluid were diluted with FACS buffer (PBS very similar to that of the CCR7 mAb used in Ref. 9 on T cells. In further supplemented with 0.1% azide and 1% BSA), centrifuged at 250 ϫ g, and support of the specificity of these mAbs for CCR7, the ability to stain PBLs resuspended in FACS buffer. Cell suspensions from liver were layered over with the 7H12 and 3D9 mAbs was lost after treatment with the CCR7 Ficoll (Amersham Pharmacia Biotech, Piscataway, NJ), and the mononu- ligand macrophage-inflammatory protein (MIP)-3␤, but not after treatment clear cell layer was used for FACS analysis. We found that the treatments Downloaded from with the CXCR4 ligand stromal cell-derived factor (SDF)-1␣ (J.J.C., un- required to dissociate these tissues did not affect CCR7 expression, and published data). Thus, MIP-3␤ may either directly block binding of the other lymphocyte markers such as LFA-1 were positive on all isolated mABs to their cell surface epitope or cause internalization of their cell lymphocyte populations (data not shown). surface epitope (directly or indirectly). Indirect triggering of internalization seems unlikely, as in the reciprocal experiment pretreatment of PBL with Results the CXCR4 ligand SDF-1␣ abolished binding of two commercially avail- able anti-CXCR4 mAbs without affecting the binding of our CCR7 mAbs Functional expression of CCR7 on tissue-specific memory CD4 http://www.jimmunol.org/ (J.J.C., unpublished data). cells from peripheral blood Four-color flow cytometry We tested the hypothesis (9, 17) that CCR7 expression (essential for homing of naive T-cells to secondary lymphoid tissues; Refs. Four-color flow cytometry was performed as in Ref. 2. mAbs were either directly conjugated to FITC, PE, or APC, or visualized with biotinylated 5–7) is lost in memory T cells that express adhesion receptors horse anti-mouse IgG (Vector Laboratories, Burlingame, CA) followed by necessary for homing to nonlymphoid tissues. We generated two streptavidin-peridinin chlorophyll protein (Becton Dickinson, San Jose, novel mAbs to human CCR7 (7H12 and 3D9, see Materials and ␣ ␤ CA). Purified Igs from the following hybridoma clones were used: 4 7 Methods). integrin (ACT-1), CCR5 (2D7), CD4 (RPA-T4), CD8 (RPA-T8), CD11B/ The best studied peripheral blood memory CD4 T cells dedi- MAC-1 (BEAR-1), CD27 (M-T271), CD45RA (HI-100), CD62L/L-selectin (DREG-56), CD69 (FN50), and cutaneous lymphocyte Ag (CLA) cated to specific nonlymphoid tissues are the skin-homing cells by guest on September 29, 2021 ␣ ␤ (HECA-452), (CLA(pos)/ 4 7(neg)) and the gut-homing cells (CLA(neg)/ ␣ ␤ (pos)) (reviewed in Ref. 1). Four-color flow cytometry was Chemotaxis 4 7 used to examine CCR7 expression on these two populations, as All migration assays were performed in Costar 24-well plate tissue-culture well as on naive CD4 cells and CLA(neg)/␣ ␤ (neg) or double ␮ ϫ 5 4 7 inserts with 5- m-pore polycarbonate filters. Cells (5 10 ) were placed negative memory CD4 cells of unknown tissue-homing specificity in the upper chamber in 100 ␮l, and 600 ␮l of a given chemokine dilution was placed in the lower well. Migration was carried out in migration me- (Fig. 1A). Naive and double negative memory CD4 cells were dium (RPMI 1640/0.5% BSA fraction V; Sigma, St. Louis, MO). Migrated nearly 100% positive for CCR7 expression (Fig. 1B). Skin- and cells were counted as described previously (2, 11–14). Migrating cells were gut-homing memory CD4 cells were somewhat enriched in CCR7 kept at 37°C with 8% CO2. cells (Fig. 1B), but the vast majority of both populations were MIP-3␤ was synthesized by one of us (D.S.) or purchased in recombinant form from PeproTech (Rocky Hill, NJ). Both synthetic and recombi- CCR7(pos). A survey of peripheral blood CD4 subsets from 10 nant forms behaved identically in parallel experiments. Recombinant sec- healthy donors (Fig. 1C) shows that skin- and gut-homing popu- ondary lymphoid-tissue chemokine (SLC) was purchased from PeproTech. lations were ϳ80% CCR7(pos), whereas naive and double nega- Synthetic SDF-1␣ was purchased from Gryphon Sciences (South San Fran- tive memory cells were nearly 100% CCR7(pos). cisco, CA). Recombinant Burkit’s lymphoma receptor-1 ligand chemokine Migration of these four peripheral blood CD4 populations to (BCL-1) was purchased from R&D Systems (Minneapolis, MN). known CCR7 ligands (MIP-3␤ and SLC) was similar to their mi- Human peripheral blood and tissue lymphocytes gration to SDF-1␣, a nearly universal lymphocyte chemoattractant Human peripheral blood was collected in heparinized tubes from healthy (11, 18) (Fig. 1D). Thus, the vast majority of naive, memory, and donors. Granulocytes were removed as described (15). Monocytes were tissue-dedicated CD4 cells from peripheral blood express CCR7, removed by two 30-min rounds of adherence to a T-175 culture flask respond to CCR7 ligands, and can probably home to secondary (Nunc, Roskilde, Denmark) at 37°C and 8% CO2 in RPMI 1642 medium supplemented with 10% calf serum. Fresh tonsils were obtained after rou- lymphoid organs. tine adenoid and tonsillectomy procedures. Tonsil lymphocytes were dispersed through stainless steel mesh and incubated in the same way as PBL CCR7 vs other markers on peripheral blood CD4 cells above to remove adherent cells. As CCR7 loss correlated only poorly with expression of tissue- Normal human jejunum, lung, facial skin, inflamed liver, and inflamed synovial tissue were obtained from patients undergoing various surgical dedicated homing receptors, we set out to identify other features of procedures. Synovial fluid was obtained from patients undergoing diag- CCR7(neg) CD4 T subsets. Fig. 2 shows flow cytometry of CCR7 nostic arthroscopy. Bronchial alveolar lavage cells were obtained by fiber vs several other markers on CD4(pos) T cells from peripheral optic bronchoscopy. Subjects were premedicated with nebulized salbuta- blood. As expected from Fig. 1, the CCR7(neg) population con- mol before being lightly sedated with midazolam, and the upper airway ␣ ␤ high was anesthetized with 2% lignocaine. Normal saline (180 ml) was inserted tained subsets of CLA(pos) and of 4 7 cells (Fig. 2, top). All through the bronchoscope into the right lower lobe and aspirated using CCR7(neg) cells were contained within the CD45RA(neg) mem- gentle suction. Recovery was between 20 and 46%. ory population (Fig. 2, second row left). Most CCR7(neg) cells The Journal of Immunology 879

FIGURE 1. Peripheral CD4 homing phenotype vs CCR7 expression. A, Four-color ﬂow cytometry dividing peripheral blood CD4 cells into naive (population 1), double negative memory (population 2), skin-homing memory (population 3), and intestinal-homing memory (population 4). B, Flow cytometry of each population above with the anti-CCR7 MAb 7H12 (solid lines) overlaid on isotype-matched control (dotted lines). C, Bar graph showing proportion of cells within each population expressing CCR7 with 7H12. Mean and SD for 10 donors. D, Chemotaxis of each population to titrations of CCR7 ligands and SDF-1␣. Mean and range from two donors shown, representative of 15 donors tested at only the Downloaded from optimal concentrations.

lacked L-selectin, consistent with the notion that these cells are 2, bottom). Moreover, over 50% of the CD27(neg) CD4 cells also

incapable of homing to secondary lymphoid organs (Fig. 2, third lacked CCR7. http://www.jimmunol.org/ row left). Interestingly, CCR7(neg) cells were nearly 100% positive for CCR5, the receptor for MIP-1␤, RANTES, and several The CD27(neg) subset of peripheral blood memory CD4 cells other CC chemokines (the anti-CCR5 mAb 2D7 was used for these The enrichment of CD27(neg) cells in the CCR7(neg) CD4 T cell studies because it was shown to correlate best with genetic expres- compartment was of great interest to us because CD27 loss has sion of CCR5 when compared with other CCR5 mAbs; Ref. 19) been reported to mark both CD4 (20, 21) and CD8 (22) T cells of (Fig. 2, third row right). Interestingly, CCR7(neg) cells were also effector phenotype. As Sallusto et al. had reported that the ␤ enriched in CD11b (MAC-1), a 2 integrin commonly expressed in CCR7(neg) CD4 population is enriched in an effector-like pheno- the myeloid lineages and B1 B cells found in serosal cavities, but type, we set out to more fully define the relationship between rare on T lymphocytes (Fig. 2, second row right). CCR7(neg) cells CCR7 and CD27 expression on peripheral blood CD4 cells. Fig. by guest on September 29, 2021 often lacked CD27 (a member of the TNF-␣ receptor family; Fig. 3A shows that staining of CD4 cells with CD45RA and CD27 cleanly divides them into three distinct subsets: CD45RA(pos)/ CD27(pos) naive cells; CD45RA(neg)/CD27(pos) conventional memory cells; and CD27(neg) memory cells. Expression of CCR7, CCR5 (2D7), MAC-1, and L-selectin was examined individually on each of these populations (Fig. 3B). Naive cells all expressed both L-selectin and CCR7, consistent for their known tropism for secondary lymphoid tissues (Fig. 3A, top). Conventional memory cells contained a subpopulation of CCR7(neg) and L-selectin(neg) cells, but the vast majority was positive for both. Some conventional memory cells expressed CCR5, and a very small number expressed MAC-1 (Fig. 3A, middle). The CD27(neg) memory population was very different from the naive and conventional memory subsets (Fig. 3A, bottom). Most CD27(neg) memory cells expressed neither CCR7 nor L-selectin. Many of these cells expressed CCR5 and MAC-1, markers that were never seen on naive cells. A survey of 10 healthy donors showed that CCR7(neg) cells were quite rare (as a percentage) in the naive and conventional memory subpopulations, but common within the CD27(neg) memory population (Fig. 3C). Responsive- ness to known CCR7 ligands in chemotaxis assays correlated with CCR7 expression (Fig. 3D). Most naive and conventional memory cells responded well to CCR7 ligands. Less than half of the CD27(neg) cells responded to CCR7 ligands. All three populations respond equally well to SDF-1␣ (Fig. 3D, right). FIGURE 2. CCR7 vs other markers on peripheral blood CD4 cells. Flow cytometry of CD4-gated PBLs showing CCR7 staining (y-axis) vs Diversity of memory CD4 cells lacking CCR7, CD27, or L-selectin ␣ ␤ either CLA, 4 7 integrin, CD45RA, MAC-1, L-selectin, CCR5, or CD27 (x-axis). All plots are from a single donor. Representative of full profiles on The above analysis of CD27(neg) memory cells shows them to be two other donors and partial profiles on 12 donors. See Materials and a population greatly enriched in cells of unusual (presumptive lym- Methods for mAb clones used. phoid tissue-excluded) phenotype. However, this observation is 880 CCR7 AND T CELL DIVERSITY Downloaded from

FIGURE 4. Cells lacking CCR7, CD27, or L-selectin form an extremely heterogeneous network of mutually overlapping populations. PBLs were http://www.jimmunol.org/ stained simultaneously with anti-CD4, CCR7, CD27, and L-selectin mAbs. All plots are from the same donor on the same day, shown with various FIGURE 3. Comparison of CD45RA(neg)/CD27(neg) peripheral blood gating criteria. The figure is representative of the same experiment per- CD4 cells to conventional naive and memory CD4 cells. A, Four-color flow formed on two other donors. cytometry dividing peripheral blood CD4 cells into naive (population 1), conventional memory (population 2), and CD27(neg) memory (population 3). B, Flow cytometry of each population above with anti-CCR7, CCR5, CCR7 expression in tonsil T cell populations MAC-1, or L-selectin (solid lines); overlaid on isotype controls (dotted lines). C, Bar graph showing proportion of cells within each population As a representative secondary lymphoid organ for humans, tonsil- expressing CCR7 with 7H12. Mean and SD for 10 donors. D, Chemotaxis lar T cells were examined for CCR7 expression. As in peripheral by guest on September 29, 2021 of each population to titrations of CCR7 ligands and SDF-1␣. Mean and blood, tonsil T cells can be divided into naive and memory based range from two donors are shown, representative of titrations on four do- on CD45RA expression. However, unlike peripheral blood, most nors and nine donors tested at only the optimal concentrations. CD45RA(neg) T cells from the tonsil expressed the early activation marker CD69, suggesting that they were in an activated state only part of the story, and may be misleading. The relationship (Figs. 6A and 7A). Most CD45RA(pos) naive tonsil T cells did not between CCR7 and CD27 is only an enrichment, not a direct cor- express CD69 (Figs. 6A and 7A). relation. In fact, if we stain peripheral blood CD4 cells for CCR7, Tonsil CD4 cells. Chemokine receptor expression was examined CD27, and L-selectin together (Fig. 4), we see that by gating on separately on naive (CD45RA(pos)/CD69(neg)) and activated either CCR7(neg), CD27(neg), or L-selectin(neg) cells, we enrich (CD45RA(neg)/CD69(pos)) tonsil cells. Like naive CD4 cells in cells that are lacking either or both of the other two markers. from the circulation, essentially all naive tonsil CD4 cells ex- Thus, the message from these studies is that neither CCR7, CD27, pressed high levels of CCR7. This is consistent with the notion that nor L-selectin define a distinct population. There is extreme diver- CD4 cells require CCR7 for homing to secondary lymphoid tissues sity in the cells lacking any one of these markers, whereas there is through HEV. The tonsil contained almost no CD27(neg) cells relative uniformity among the majority of both memory and naive (Fig. 7), which may be unable to enter secondary lymphoid organs lymphocytes, which express all three of these markers together. because of their low CCR7 and L-selectin levels. The presence or absence of CCR7 has been implicated in de- CCR7 expression on peripheral blood CD8(pos) T cell subsets termining microenvironmental movements of activated T cells We next examined the CCR7 expression of peripheral blood CD8 within secondary lymphoid organs of mouse (23). Therefore, it T cell subsets. As mentioned above, CD45RA and CD27 can be was of great interest to compare CCR7 expression between naive used to identify naive, memory, and effector CD8 subsets (22) and activated cells in this organ. Unlike naive cells, activated cells (Fig. 5A). CCR7 expression and responsiveness to CCR7 ligands were markedly diverse in their CCR7 expression (Fig. 6B). The was tested on each of these peripheral blood CD8 populations. activated CD4 T cells express CCR7, on average, at lower levels Like naive CD4 cells, naive CD8 cells (CD45RA(pos)/CD27(pos)) than naive cells, and there was a large population of CCR7(neg) all expressed CCR7 and responded well to MIP-3␤ and SLC (Fig. cells. Responsiveness to CCR7 ligands again parallels CCR7 ex- 5, B and C). The memory CD8 population contained more pression between naive and activated cells (Fig. 6C). Another che- CCR7(neg) than conventional memory CD4 cells (ϳ50%), which mokine receptor, CXCR5 (BLR-1/EBI-2) has also been implicated correlated well with their lower responsiveness to CCR7 ligands. in microenvironmental T cell movements (24). Expression of Effector CD8 cells were nearly all negative for CCR7 expression CXCR5, originally cloned as a B cell chemokine receptor, may and did not respond to CCR7 ligands. All three peripheral blood allow activated T cells to migrate into B cell zones to provide T CD8 subsets responded equally well to SDF-1␣ (Fig. 5C, right). cell help (25). We found that expression of CXCR5 was nearly The Journal of Immunology 881

FIGURE 5. CCR7 expression on peripheral blood CD8 T cell populations. A, Four-color ﬂow cytometry dividing peripheral blood CD8 T cells into naive (population 1), memory (population 2), and effector (population 3). B, Flow cytometry of each population above with the anti-CCR7 mAb 7H12 (solid lines) overlaid on isotype-matched control (dotted lines). C, Chemotaxis of each population to titrations of CCR7 ligands and SDF-1␣. Mean and range from two donors is shown, representative Downloaded from of four other donors tested. http://www.jimmunol.org/

opposite to that of CCR7. Naive tonsil cells expressed no CXCR5, tissues. The ratio of CD27(neg) to CD27(pos) cells varied greatly whereas most activated cells expressed CXCR5. In agreement with from tissue to tissue (Fig. 8) but was similar among donors for a this, activated but not naive cells migrated to the CXCR5 ligand given tissue (data not shown). CD27(neg) cells from all tissues

BLC/BCA-1 (Fig. 6C). Both naive and activated CD4 cells in the were consistently CCR7(neg). The CD27(pos) populations con- by guest on September 29, 2021 tonsil responded equally well to SDF-1␣ (Fig. 5C, right). tained both CCR7(neg) and CCR7(pos) subsets. Tonsil CD8 cells. Like CD4 cells, tonsil CD8 cells could be CD4 lymphocytes isolated from small intestine (jejunum) lam- cleanly subdivided into CD45RA(pos)/CD69(neg) naive and ina propria (n ϭ 3), liver (n ϭ 3), dispersed lung tissue (n ϭ 4), CD45RA(neg)/CD69(pos) activated subsets (Fig. 7A). However, and the bronchial space of lung (n ϭ 4) were consistently naive CD8 cells were much more abundant than activated CD8 CD27(neg)/CCR7(neg) (Fig. 8). Those isolated from normal skin cells, unlike CD4 cells in which the naive and activated popula- (from face lifts, n ϭ 3) and from synovium or synovial fluid of tions were nearly equal (Fig. 4A). This is consistent with the find- autoimmune arthritis patients (n ϭ 1 and 4, respectively) contained ing (Fig. 5) that a large proportion of memory CD8 cells from many more CD27(neg)/CCR7(neg) than patient-matched blood peripheral blood do not express CCR7 (and CD8 effector cells (data not shown), but the majority of infiltrating cells from these from blood are nearly devoid of CCR7) and thus may be unable to tissues were CD27(pos)/CCR7(pos) (Fig. 8). home to secondary lymphoid organs. CCR7 and CXCR5 expression is similar to that of the homologous CD4 populations. Naive cells express uniformly high levels of CCR7 and no CXCR5. Ac- Discussion tivated CD8 cells are heterogeneous for CCR7 expression and ex- Peripheral blood T cells lacking CCR7 press CXCR5 (Fig. 7B). Responsiveness to chemokines again parallels receptor expression (Fig. 7C). CCR7 expression is not lost upon differentiation into skin- or gut-homing phenotypes. We have examined the expression and CCR7 expression in T lymphocytes infiltrating nonlymphoid function of CCR7 in lymphocytes from peripheral blood, second- tissues ary lymphoid tissues, and nonlymphoid tissues. An existing hy- To further understand the role of CCR7 in routine lymphocyte pothesis suggests that for a memory T cell to differentiate into a trafficking, immunosurveillance, and homing to inflamed sites, form capable of homing to nonlymphoid tissues, it must lose the CCR7 expression was examined on tissue-infiltrating lymphocytes ability (possessed by all naive T cells) to home to secondary lym- freshly isolated from various nonlymphoid tissues. CD8 cells were phoid tissue via HEV (9, 17). rare in most tissues examined, so our study focused on CD4 cells We have found that, like naive and most other memory T cells, only. Contamination of tissue-infiltrating lymphocytes with pe- the majority of tissue-specific memory T cells (i.e., skin- and gut- ripheral blood cells was not significant, as naive cells, which con- homing) still expressed CCR7, and are thus (because they also stitute nearly 50% of peripheral blood CD4 cells, were consistently express L-selectin and LFA-1; Ref. 26) likely still competent to absent from tissue populations (see Figs. 1 and 3A). enter secondary lymphoid tissues. Skin- and gut-homing popula- Unlike CD45RA(neg) T cells from secondary lymphoid tissue tions are moderately enriched in CCR7(neg) cells, indicating that (tonsil), CD27(neg) cells were quite common in nonlymphoid loss of CCR7 may occur after differentiation into tissue-specific 882 CCR7 AND T CELL DIVERSITY Downloaded from

FIGURE 7. CCR7 and CXCR5 expression are reciprocally controlled http://www.jimmunol.org/ FIGURE 6. CCR7 and CXCR5 expression are reciprocally controlled on the activation/memory phenotype of tonsil CD8 cells. A, Flow cytom- on the activation/memory phenotype of tonsil CD4 cells. A, Flow cytometry dividing human tonsil CD8 T cells into naive (population 1) and ac- etry dividing human tonsil CD4 T cells into naive (population 1) and activated (population 2). B, Flow cytometry of each population above with tivated (population 2). B, Flow cytometry of each population above with the anti-CCR7 mAb 7H12 (solid lines) overlaid on isotype-matched control the anti-CCR7 mAb 7H12 (solid lines) overlaid on isotype-matched control (dotted lines). C, Chemotaxis of each population to titrations of CCR7 (dotted lines). C, Chemotaxis of each population to titrations of CCR7 ligands, BCA-1, and SDF-1␣. Representative of titrations on two other ␣ ligands, BCA-1, and SDF-1 . Representative of titrations on two other donors. donors. by guest on September 29, 2021 phenotypes or that these two differentiation processes may be en- tirely independent. population may simply consist of cells that have recently been CCR7-negative peripheral blood CD4 cells. CCR7 was missing activated and have not yet up-regulated the receptors necessary for from only a minor subset of peripheral blood memory CD4 T cells. homing back to secondary lymphoid organs. This may be a defense All naive T cells, and Ͼ90% of memory CD4 T cells (defined by mechanism, preventing active cells (which are poised to secrete CD45RA phenotype) expressed high levels of CCR7. The activating cytokines) from entering lymphoid organs where they CCR7(pos) memory cells were relatively uniform in expression of could potentially cause unregulated activation of a large number of L-selectin and CD27, like the naive population (although, of cells. The expression patterns of CD27 and CCR7 on cells from course, they aren’t uniform in homing receptor expression). En- nonlymphoid tissues (discussed below) as well as the enrichment richment of CCR7(neg) cells in the CD27(neg) population was of CCR7(neg) cells in the tissue-specific phenotypes (Fig. 1) are intriguing, and was examined in detail. consistent with this notion. Additional experiments will be re- Memory T cells lacking CD27 have been previously associated quired to resolve these issues. with effector properties (20–22). We found that besides being en- Peripheral blood CD8 T cells and CCR7 expression. We found high riched in CCR7(neg) cells, the CD27(neg) population of CD4 cells that all naive CD8 cells (CD45RA /CD27(pos)) expressed was also enriched in cells lacking L-selectin and in cells express- CCR7, whereas almost no effector-phenotype CD8 cells ing MAC-1 and CCR5. At first, we proposed that because (CD45RA(pos)/CD27(neg)) expressed CCR7. The resting CD8 CCR7(neg) memory CD4 cells have been previously reported to memory population (CD45RA(neg)/CD27(pos)) contained an in- contain a subset with a functional effector phenotype (27), CD27 termediate level of CCR7-expressing cells. However, again, CD8 negativity may define a uniform effector immunophenotype as sug- cells of effector phenotype could also consist of recently activated gested in Refs. 20 and 21. However, the CD27-negative population cells that have not yet regained the ability to traffic through lym- was not uniform with respect to CCR7 (or L-selectin expression). phoid organs. And, in fact, CD27, CCR7, or L-selectin could be used to identify small, diverse populations of cells, each enriched in cells negative CCR7 in lymphocyte homeostasis and immunosurveillance for the other two markers, but each extremely heterogeneous. It There have been three (nonexclusive) hypotheses for the roles of remains to be seen whether CCR7, CD27, or L-selectin negativity CCR7 in lymphocyte homing to date: 1) recognition of SLC pre- will correlate best with the effector phenotype defined in Ref. (9) sented by HEV of secondary lymphoid tissues, triggering arrest of or whether (more likely) heterogeneity is in fact the essence of circulating T cells on endothelium as a prerequisite to migration effector-memory cells. into the tissue. In this role, CCR7 would be an HEV homing re- Because these markers do not define a distinct phenotype as ceptor, along with L-selectin and LFA-1. There is now ample sup- proposed in Ref. 9, we propose an alternative hypothesis. This port for this hypothesis in animal models (5, 6; reviewed in Ref. 4); The Journal of Immunology 883

FIGURE 8. Expression of CCR7 on CD27(neg) and CD27(pos) tissue-infiltrating CD4 T cells. CD27 vs CCR7 staining of CD4(pos)/CD45RA(neg)-gated tissue-infiltrating lymphocytes. Lymphocytes were isolated from tissues using gentle mechanical agitation and chelation, without enzymatic digestion (see Materials and Methods). Representative dot-plots for each organ type shown. Three jejunal lamina propria preparations (from gastric bypass operations) were tested, four bronchoalveolar lavages (from nonasthmatic donors), three dispersed lung-tissue, three dispersed lung-tissue speci- mens, three cirrhotic livers (two alcoholic and one hep- atitis C), three normal skin preparations (from face- lifts), four synovial fluid samples (two from RA and two from psoriatic arthritis), one RA synovium, and five tonsils (from routine adenoid and tonsillectomies). Downloaded from

2) migration through gradients of MIP-3␤ expressed within sec- ory T cells can be found at low levels in every tissue of the body ondary lymphoid organs, which coordinates proper microenviron- (34). We have examined the CCR7 expression of CD4 T cells mental localization within the organ. This hypothesis also has sup- isolated from several types of nonlymphoid tissues. One important port in animal models, as manipulation of CCR7 expression can ﬁnding from this work is that many tissue-inﬁltrating lymphocytes

alter the microanatomic location of T cells within the spleen (23); express CCR7. This further argues against the hypothesis that CCR7- http://www.jimmunol.org/ 3) migration of tissue-infiltrating lymphocytes back into the lym- expressing cells do not infiltrate nonlymphoid tissues (9, 17). phatic system. This has been proposed because endothelial cells We have found a correlation between CCR7 and CD27 expres- lining the lymph vessels have been found to express mRNA for sion in tissue-infiltrating lymphocytes, which parallels our findings CCR7 ligands (8). This hypothesis has not yet been tested for in peripheral blood. CD27 is a member of the TNF-␣ receptor lymphocytes in experimental models, but is supported by studies family, and its modulation may mark previously unknown activa- of DC migration (27–31). We have examined CCR7 expression on tion or developmental states in tissue-infiltrating lymphocytes. T cells derived from each of the locations where it is thought to However, loss of CD27 expression does not correlate well with have influence (peripheral blood (discussed above), secondary expression of the activation marker CD69, which is was generally lymphoid tissue, and nonlymphoid tissue) to shed light on these expressed on all tissue-infiltrating lymphocytes studied (data not by guest on September 29, 2021 hypotheses. shown). Tonsil T cells. Secondary lymphoid tissues are very high-traffic Nearly all CD27(neg) tissue-infiltrating lymphocytes lacked zones for lymphocytes. T and B cells are constantly entering from CCR7 expression (Fig. 8). Lymphocytes from normal intestinal the blood through HEV. Most cells entering lymphoid organs do tissue, normal lung, and cirrhotic liver are nearly all CD27(neg)/ not encounter their cognate Ag, and pass through the organ to CCR7(neg). Lymphocytes from normal skin and from synovium or return to the circulation via afferent lymph. However, those that do synovial fluid of rheumatoid arthritis-afflicted joints are more het- encounter Ag and become activated must be directed to microen- erogeneous and contain many CD27(pos)/CCR7(pos) cells. vironmental locations where they can perform their helper or ef- Normal skin (with mostly CD27(pos)/CCR7(pos) memory cell fector functions. It has been proposed that modulation of chemo- infiltrates) and gut (with mostly CD27(neg)/CCR7(neg) memory kine responses may mediate proper microenvironmental infiltrates) are sites with very different levels of immunological localization (25). We have found that CCR7 is expressed at high activity. The gut tissue of normal individuals is thought to be con- levels on naive T cells (both CD4 and CD8) within the tonsil, stantly involved in immune reactions to food Ags and intestinal comparable to that of circulating naive cells. In contrast, many flora. This has led to the suggestion that intestinal tissue be con- cells activated in the lymphoid organ may lose CCR7, which may sidered a tertiary lymphoid organ (reviewed in Ref. 34). In con- allow them to enter microenvironments not normally entered by trast, skin presents a barrier to the outside world and would only unactivated cells. Consistent with this notion, activated T cells engage in inflammatory reactions when the barrier is broken. Thus, gain the expression of CXCR5 (and perhaps other chemokine re- normal skin would not be expected to contain much immune ceptors not expressed by resting or naive cells), thus gaining the activity. ability to respond to new chemotactic gradients not perceived by The patterns of CCR7 expression within these two (very differ- other cells. It is interesting that both CD4 and CD8 T cells undergo ent) tissues fits well with the notion that CCR7 ligands may direct very similar changes in CCR7 and CXCR5 expression after acti- tissue-infiltrating T cells back to the lymph (8). Based on the vation; this strongly implies the participation of other chemokines known levels of immune activity in these organs, CD27(neg) cells in fine-tuning the microenvironmental localization of functionally may represent a population that is actively engaged in Ag recog- distinct lymphocytes, as modeled in Refs. 32 and 33). Our findings nition. Therefore, this population is rare in skin but common in are consistent with the hypothesis that chemokine responsiveness intestine. It would not be desirable for such actively engaged cells patterns are actively altered in different subsets of lymphocytes, a to return to the lymph; thus, these cells do not express CCR7. mechanism that may orchestrate the complex interplay among However, in the skin, where Ag recognition is not common, most APCs, T cells, and B cells within lymphoid organs. lymphocytes are passing through the tissue in routine immunosur- Nonlymphoid tissues. Although naive T lymphocytes can be veillance, are not actively engaged in Ag recognition, and continue found almost exclusively in the blood and lymphoid organs, mem- to express CD27. Such cells, not encountering Ag, continue to 884 CCR7 AND T CELL DIVERSITY express CCR7 to guide them back to the lymph, completing their 10. Wu, L., W. A. Paxton, N. Kassam, N. Ruffing, J. B. Rottman, N. Sullivan, circuit through the tissue. H. Choe, J. Sodroski, W. Newman, R. A. Koup, and C. R. Mackay. 1997. CCR5 levels and expression pattern correlate with infectability by macrophage-tropic The arguments used above for gut-infiltrating lymphocytes HIV-1, in vitro. J. Exp. Med. 185:1681. would also apply to lung and cirrhotic liver, whose infiltrating 11. Campbell, J. J., E. P. Bowman, K. Murphy, K. R. Youngman, M. A. Siani, lymphocytes are phenotypically similar to gut (Fig. 8). The high D. A. Thompson, L. Wu, A. Zlotnik, and E. C. Butcher. 1998. 6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is CCR7 expression in rheumatoid synovium and synovial fluid re- an agonist for the MIP-3␤ receptor CCR7. J. Cell Biol. 141:1053. quires another explanation: perhaps an autoimmune chronically 12. Campbell, J. J., E. F. Foxman, and E. C. Butcher. 1997. Chemoattractant receptor inflamed situation may greatly enhance the amount of lymphocyte cross talk as a regulatory mechanism in leukocyte adhesion and migration. Eur. J. Immunol. 27:2571. trafficking through the inflamed tissue (with respect to its unin- 13. Campbell, J. J., J. Pan, and E. C. Butcher. 1999. Cutting edge: developmental flamed counterpart). Only a small proportion of cells passing switches in chemokine responses during T cell maturation. J. Immunol. 163:2353. through such a high-traffic area would be specific for the autoan- 14. Campbell, J. J., S. Qin, K. B. Bacon, C. R. Mackay, and E. C. Butcher. 1996. Biology of chemokine and classical chemoattractant receptors: differential re- tigen. Thus, just like normal skin, most cells infiltrating rheuma- quirements for adhesion-triggering versus chemotactic responses in lymphoid toid arthritis synovium may not see Ag, and continue to express cells. J. Cell Biol. 134:255. CD27 and CCR7. Alternatively, the number of active cells in sy- 15. Nielson, C. P., R. E. Vestal, R. J. Sturm, and R. Heaslip. 1990. Effects of selective phosphodiesterase inhibitors on the polymorphonuclear leukocyte respiratory novium and synovial fluid samples could be lower due to anti- burst. J. Allergy Clin. Immunol. 86:801. inflammatory treatments the donors may have received. 16. Fiocchi, C., and K. Youngman. 1997. Isolation of human intestinal mucosal mononuclear cells. In Current Protocols in Immunology. J. E. Collogan, Conclusions. In this study we 1) analyzed in detail the population A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds. Wiley, of CCR7(neg) cells, which have been the focus of great interest New York, p. 7.30.1. recently; and 2) investigated the expression of CCR7 on cells at 17. Mackay, C. R. 1999. Dual personality of memory T cells. Nature 401:659. Downloaded from various points in the cycle of routine immunosurveillance in an 18. Bleul, C. C., R. C. Fuhlbrigge, J. M. Casasnovas, A. Aiuti, and T. A. Springer. 1996. A highly efficacious lymphocyte chemoattractant, stromal cell-derived fac- attempt to shed light on the role of CCR7 and its ligands in these tor 1 (SDF-1). J. Exp. Med. 184:1101. processes. We found that CCR7, CD27, and L-selectin, together 19. Hill, C. M., D. Kwon, M. Jones, C. B. Davis, S. Marmon, B. L. Daugherty, with MAC-1 and CCR5, mark an intensely heterogeneous series of J. A. DeMartino, M. S. Springer, D. Unutmaz, and D. R. Littman. 1998. The amino terminus of human CCR5 is required for its function as a receptor for overlapping populations. No single one of these markers can be diverse human and simian immunodeficiency virus envelope glycoproteins. Vi- used to identify a uniform population with respect to the others. It rology 248:357. http://www.jimmunol.org/ 20. De Jong, R., M. Brouwer, B. Hooibrink, T. Van der Pouw-Kraan, F. Miedema, is likely that a combination of these markers is required to identify Ϫ ϩ and R. A. Van Lier. 1992. The CD27 subset of peripheral blood memory CD4 all true effector memory cells. lymphocytes contains functionally differentiated T lymphocytes that develop by We also found that the CCR7 expression patterns on T cells are persistent antigenic stimulation in vivo. Eur. J. Immunol. 22:993. consistent with known or hypothesized roles for each of the three 21. Hintzen, R. Q., R. de Jong, S. M. Lens, M. Brouwer, P. Baars, and R. A. van Lier. 1993. Regulation of CD27 expression on subsets of mature T-lymphocytes. J. Im- major nexus points of immunosurveillance: trafficking into secondary munol. 151:2426. lymphoid organs, movement and organization within lymphoid or- 22. Hamann, D., P. A. Baars, M. H. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. R. Klein, and R. A. van Lier. 1997. Phenotypic and functional separation of gans, and trafficking out of nonlymphoid organs and into the lymph. ϩ memory and effector human CD8 T cells. J. Exp. Med. 186:1407. 23. Randolph, D. A., G. Huang, C. J. Carruthers, L. E. Bromley, and D. D. Chaplin. Acknowledgments 1999. The role of CCR7 in TH1 and TH2 cell localization and delivery of B cell by guest on September 29, 2021 We thank L. Pucher-Petersen and Dr. D. White (Surgecenter, Palo Alto, help in vivo. Science 286:2159. CA) for providing face-lift skin samples for our experiments. 24. Ansel, K. M., L. J. McHeyzer-Williams, V. N. Ngo, M. G. McHeyzer-Williams, and J. G. Cyster. 1999. In vivo-activated CD4 T cells upregulate CXC chemokine receptor 5 and reprogram their response to lymphoid chemokines. J. Exp. Med. 190:1123. References 25. Cyster, J. G. 1999. Chemokines and cell migration in secondary lymphoid organs. 1. Butcher, E. C., M. Williams, K. Youngman, L. Rott, and M. Briskin. 1999. Science 286:2098. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72:209. 26. Warnock, R. A., S. Askari, E. C. Butcher, and U. H. von Andrian. 1998. Mo- 2. Campbell, J. J., G. Haraldsen, J. Pan, J. Rottman, S. Qin, P. Ponath, lecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. D. P. Andrew, R. Warnke, N. Ruffing, N. Kassam, et al. 1999. The chemokine Med. 187:205. receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T 27. Sallusto, F., B. Palermo, D. Lenig, M. Miettinen, S. Matikainen, I. Julkunen, cells. Nature 400:776. R. Forster, R. Burgstahler, M. Lipp, and A. Lanzavecchia. 1999. Distinct patterns 3. Zabel, B. A., W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, and kinetics of chemokine production regulate dendritic cell function. Eur. J. Im- E. C. Ebert, N. Kassam, S. Qin, M. Zovko, et al. 1999. Human G protein-coupled munol. 29:1617. receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal 28. Barratt-Boyes, S. M., M. I. Zimmer, L. A. Harshyne, E. M. Meyer, S. C. Watkins, homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required S. Capuano, 3rd, M. Murphey-Corb, L. D. Falo, Jr., and A. D. Donnenberg. 2000. for thymus-expressed chemokine-mediated chemotaxis. J. Exp. Med. 190:1241. Maturation and trafficking of monocyte-derived dendritic cells in monkeys: im- 4. Campbell, J. J., and E. C. Butcher. 2000. Chemokines in tissue-specific and plications for dendritic cell-based vaccines. J. Immunol. 164:2487. microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12:336. 29. Saeki, H., A. M. Moore, M. J. Brown, and S. T. Hwang. 1999. Cutting edge: 5. Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, and U. H. von Andrian. 2000. The (CCR7) participate in the emigration pathway of mature dendritic cells from the CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lym- skin to regional lymph nodes. J. Immunol. 162:2472. phoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associ- 30. Sozzani, S., P. Allavena, G. D’Amico, W. Luini, G. Bianchi, M. Kataura, T. Imai, ated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node O. Yoshie, R. Bonecchi, and A. Mantovani. 1998. Differential regulation of che- high endothelial venules. J. Exp. Med. 191:61. mokine receptors during dendritic cell maturation: a model for their trafficking 6. Warnock, R. A., J. J. Campbell, M. E. Dorf, A. Matsuzawa, L. M. McEvoy, and properties. J. Immunol. 161:1083. E. C. Butcher. 2000. The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer’s patch high endothelial venules. J. Exp. Med. 191:77. 31. Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, 7. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, and F. Briere, A. Zlotnik, S. Lebecque, and C. Caux. 1998. Selective recruitment of M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing immature and mature dendritic cells by distinct chemokines expressed in different functional microenvironments in secondary lymphoid organs. Cell 99:23. anatomic sites. J. Exp. Med. 188:373. 8. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, and 32. Foxman, E. F., J. J. Campbell, and E. C. Butcher. 1997. Multistep navigation and L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139:1349. venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. 33. Foxman, E. F., E. J. Kunkel, and E. C. Butcher. 1999. Integrating conflicting Natl. Acad. Sci. USA 95:258. chemotactic signals: the role of memory in leukocyte navigation. J. Cell Biol. 9. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two 147:577. subsets of memory T lymphocytes with distinct homing potentials and effector 34. Butcher, E. C., and L. J. Picker. 1996. Lymphocyte homing and homeostasis. functions. Nature 401:708. Science 272:60.