1993 81: 1-8   

 D Dunon and BA Imhof Mechanisms of thymus homing

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BLOOD Thejournal of The American Society of Hematology

VOL 81, NO 1 JANUARY 1, 1993

BRIEF REVIEW

Mechanisms of Thymus Homing

By Dominique Dunon and Beat A. lmhof

HYMUS COLONIZATION during embryogenesis starts T with the accumulation of basophilic cells in the jugular vein, in capillaries, and in the mesenchyme surrounding the thymus. In birds the extrinsic origin of these basophilic cells, which are considered to be hematopoietic precursors, was established by the construction of quail-chick chimeras.lS2 Using this technique, Jotereau and Le Douarin3 and Coltey et a14 showed that the thymus of birds is colonized mainly in three waves during embryogenesis and the first few days after hatching. In mice, the thymus is colonized by hema- topoietic precursors at days 10 and 13 of embryogenesis.' A similar process, albeit at much lower level, may occur throughout life? The major cellular events of thymus homing are illustrated in Fig 1 .

In birds, T-cell precursors first originate from para-aortic hematopoietic foci at the level of the ducts of Cuvier in 3- day-old quail embryos.',' During the second and the third waves of thymus colonization, most T-cell precursors come from the bone marrow (BM). In mammals, the yolk sac con- tain precursors to T lymphocytes at a time of development when no lymphoid precursors could be found in the embryo proper' (and Palacios R, Imhof BA: submitted). In older em- bryos, intra-embryonic T-cell precursors are found in the 1 i ~ e r . I ~ ' ~ After birth they are mainly produced in the BM.'49'5

The homing of these precursors to the thymus has been extensively studied. Hematopoietic cells are transported via blood circulation from their site ofemergence to the thymus.16 Early progenitors enter the nonvascularized thymic rudiment by the capsule, but pro-T cells could also enter the organ at the cortico-medullary j~nc t ion . '~ After extravasation the T- cell precursors find themselves in a perivascular space rich in mesenchymal cells embedded in extracellular matrix. Fi- nally, after invasive migration through the epithelial basal membrane, T-cell precursors interact with the thymic epi- thelium and other cells that constitute this complex microen- vironment and consequently undergo differentiation along the T-cell pathway. The entire process of thymus colonization seems to occur rapidly, because fluorescein-labeled BM cells can be found in the thymus within 3 hours after intravenous (IV) injection.''

In this review, we summarize current knowledge on the molecules involved in thymus colonization identified so far and propose a model for the molecular mechanisms partic- ipating in this process (Fig 1).

ADHESION OF PRO-T CELLS TO VASCULAR ENDOTHELIUM

The interaction of mature lymphocytes with endothelium during lymphocyte recirculation as well as at sites of inflam- mation has been a subject of intense study by several groups. Adhesion molecules on lymphocytes and/or their counter- parts on endothelial cells have been identified by monoclonal antibodies (MoAbs) that block lymphocyte binding to en- dothelial cells of specific lymphoid organs. These molecules include members of distinct adhesion receptor families in- cluding selectins, integrins of the P I , B2, and p7 classes, mem- bers of the Ig family, and CD44, a member of a family of hyaluronate receptors generated by alternative splicing. 19-23

Selectins bind carbohydrate receptors by their N-terminal domain and two of them have been identified as homing receptors: Lselectin expressed on lymphocytes as the pe- ripheral lymph node recept01-s~~ and E-selectin as a skin homing receptor for cutaneous lymphocyte antigen (CLA) positive memory T cells during inflammati~n.~' Integrins bind different extracellular matrix molecules and members of the Ig superfamily: ICAM-1 and VCAM are ligands for LFAl (a& and VLA-4 ((~4pI) integrins, respectively. In lymphocyte-endothelium interactions, integrins as well as CD44 could act as secondary adhesion effectors, playing a general role in adhesion ~ t r e n g t h e n i n g . ~ ~ , ~ ~ * ~ ~ Nevertheless, a4Pp (LPAM), a P7 integrin, is thought to function as a specific Peyer's patches receptor.27

For long time, the few T-cell progenitors present in BM and fetal liver and the lack of pro-T cell lines have precluded analysis of the interaction of T-cell progenitors with endo- thelium. Very recently the pro-T cell line FTF1, a nontrans- formed T-cell progenitor clone isolated from fetal thymus, was shown to bind to frozen sections of thymus and liver from newborn mice as well as to an endothelial cell lir~e.~'.~'

From the Basel Institute for Immunology, Basel, Switzerland. Submitted June 30, 1992; accepted August 25, 1992. The Basel Institute for Immunology was founded and is supported

Address reprint requests to D. Dunon, PhD, Basel Institute for Im-

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2

Blood Vessel Extracellular Matrix Network

DUNON AND IMHOF

Thymus

Adhesion molecules Y

. Chemotactic molecule

Y 1 Basal membrane

Fig 1. Colonization of the thymus by pro-T cells. This scheme depicts only thymus colonization during ontogeny before vascularization of the thymus, but the model may also be valid after thymus vascularization, when the intimate extravascular space is filled by leukocytes and fibroblasts surrounded by a tight monolayer of thymic epithelial cells. Pro-T cells are transported to the vicinity of the thymus via blood circulation (1 1. Several adhesion molecules are responsible for the attachment of pro-T cells to thymic endothelium (2) and extravasation. Migration in the perivascular space (3) needs interaction of pro-T cells with extracellular matrix and chemotactic molecules. Once in the organ, pro-T cells recognize the basement membrane of thymic epithelia cells (4) before they enter the thymus.

This property of pro-T cells appeared restricted to vessels in hematopoietic tissues. The EA-1 MoAb raised against an embryonic mouse endothelial cell line was found to block the binding of pro-T cells to thymus derived-endothelium, but it did not affect the adhesion of mature T lymphocytes and myeloid cells. The EA-1 MoAb was subsequently found to recognize integrins (a& and a6&,30 and Ruiz P, Dunon D, Sonnenberg A, Imhof BA, submitted). Attempts to in- crease the binding of FTFl pro-T cells by treatment of the embryonic or thymus-derived endothelial cell lines with in- terleukin- 1 (IL- 1) and interferon-y (IFN-y) failed29; this sug- gests that homing of pro-T cells to the thymus depends on their interaction with a set of adhesion molecules different from that mediating cell adhesion to endothelia at inflam- mation sites, such as P selectin (GMP-140, PADGEM), E- selectin (ELAM-l), or ICAM-l .31

Nevertheless, the integrin LFA-1 and its ligand ICAM-1 may play a marginal role in pro-T cell homing. Although anti-LFA1 antibody does not inhibit the binding of pro-T cells to thymic endothelium, it slightly increased the inhibitory effect of MoAb EA- 1, a fact which suggests that LFA- 1 plays

at best an accessory role in the endothelium binding of pro T cells mediated by a6 in t eg r in~ .~~ As recently suggested for the interaction of platelets or neutrophils with endothelium at sites of i n f l a m m a t i 0 n , 2 ~ , ~ ~ , ~ ~ - ~ ~ the adhesion process may occur in at least two steps. The pro-T cell might first recognize the perithymic endothelium by means of a combination of specific molecules, after which adhesion strength might be increased by integrins.

a6 Integrins are expressed on vascular endothelium of many tissue^.^^.^^ The apparent discrepancy between the ubiquitous distribution of as integrin and its putative function as a thy- mus vascular addressin for pro-T cells can be explained in different ways (Fig 2). The tissue specificity of pro-T cell binding could be ensured by a particular combination of ubiquitous adhesion molecules (Fig 2A). The specific in- volvement of (Y6 integrin in pro-T cell binding to thymic en- dothelium could also depend on the binding affinity of these molecules or their level of expression (Fig 2, B and C). It has been described that phorbol esters or physiological agonists can activate the binding capacity of ,8,, p2, or p3 integrins without changing their level of expression. Likewise, acti-

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n THYMUS HOMING J

Fig 2. Model for a specific molecular interaction of pro-T cells with the thymic endothe- lium. Several adhesion mole- cules may be necessary for the attachment of pro-T cell to the thymic vascular system and subsequent extravasation. (A) Pro-T cells express different adhesion molecules, their li- gands might all be present on thymic endothelia (ie. a. inte- grins, a', c'), absence of one may resuk in the loss ofthis function. CD44 could correspond to a or c. (B) All ligands might be pres- ent on all endothelial cells but aa integrins would only be acti- vated on thymic vessels during colonization periods. lntegrin activation may proceed over the secretion of cytokines by the thymic epithelium. (C) Func- tional pro-T cell interaction would only proceed after expression of ae integrin on thymic endothelium is in- creased. This could be stimu- lated by thymus derived cyto- kines or by triggering of other adhesion molecules also in- volved in this step.

pc;~L~l ~ H Y M I C 1 ENDOTHELIUM

CYTOIK~NES 1

IC) >clJ t 8% 0

P I +

vation of PI integrins can be induced by antibodies against CD3 1, an adhesion molecule of the Ig family.37 A PI chain- specific antibody was found to directly modulate the binding affinity of (Y4&, or 0(6& integrins by causing conforma- tional changes in the p r ~ t e i n ? ~ ~ ~ ~ ~ ~ ~ Antibodies directed against the fibrinogen receptor (aIIbP3 integrin), the ICAM receptor LFA-1, or the a4 chain of VLA-4 ( ( ~ 4 6 1 ) integrin were found to also enhance bindingw3 The strength of adhesion of pro- T to endothelial cells could also depend on the density of (Y6

integrin on the cell surface of thymus endothelium. Cytokines could regulate synthesis of a 6 integrin. For instance, tumor necrosis factor a (TNFa) was found to induce a1 integrin sub- unit expression on foreskin microvascular endothelium but it inhibited the synthesis of the P3-chain in human umbilical vein endothelial cells (HUVEC)."Z~~

+

P I +

Discrepancies between tissue distribution of adhesion molecules and their putative function in specific homing have also been found for homing receptors of mature lymphocytes. For instance, L-selectin is expressed on lymphocytes as well as on neutrophils and monocytes46 and VLA-4 is also ex- pressed by neural crest cells.47

and possibly a 6 6 4 integnns are laminin re- ~ e p t o r s , " ~ , ~ ~ the EA-1 antibody does not inhibit binding of

cells to laminin fragments (Ruiz P, Dunon D, Sonnen- berg A, lmhof BA, submitted). One interpretation of this observation is that a novel ligand for (Y6 integrins on pro-T cells might exist. Such putative ligands for 016 integrins (and/ or for other thymic vascular addressins) could be the pro-T cell-specific markers Joro 75 and Joro 37-5, which are known to be present on pro-T cells colonizing the thymus.'3350 The

Although

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4 DUNON AND IMHOF

expression of Joro 75 and 37-5 seems to be restricted to T- cell progenitors as they are switched off as pro-T cells differ- entiate into mature thymocytes.

CD44 could be a receptor for a thymic vascular addressin yet to be found, because anti-CD44 antibodies inhibit homing of fluorescent-labeled BM cells to the thymus and CD44 is expressed on BM cells able to repopulate the thymus of ir- radiated Likewise, L-selectin might belong to this group of receptors because it is expressed on BM cells, pre- sumably on pro-T cells.54

Because endothelial cells cocultured with thymic epithelial cells doubled their capacity for pro-T cell binding, expression of, as yet unidentified, ligands for pro-T cells expressed on perithymic vascular endothelium could be regulated by the thymic epithelium.28 The HECA-452 antigen could be one of such putative ligands; it is present on a subpopulation of venules predominantly located near the corticomedullary junction in human fetal thymus.53 In the adult, HECA-452 antigen is expressed on HEV of lymphoid tissues and induced on HEV-like vessels of inflammatory tissues, suggesting that it plays a role in extravasation of lymphocyte^.^^ In addition, the HECA-452 MoAb recognizes CLA, which is present on a subset of memory T cells homing to skin and is a ligand for E se1ecti1-1.~~ The epitope recognized by HECA-452 MoAb contains neuraminidase sensitive carbohydrate and thereby this antibody could recognize such antigenic determinant on different molecules on human fetal venules and CLA+ mem- ory lymphocytes in adult.

INVOLVEMENT OF CHEMOTACTIC MOLECULES IN THE

ENDOTHELIUM TO THYMIC EPITHELIUM MIGRATION OF PRO-T CELLS FROM VASCULAR

The role of chemotactic factors in thymus homing seems to be restricted to the migration of T-cell progenitors from the vascular endothelium through the penthymic mesen- chyme to the thymic epithelium.’ Evidence for the chemo- taxis of quail hematopoietic precursors isolated from thymus or BM was initially obtained by observing the migration of

individual cells in a modified Zigmond ~hamber .~ ’ . ’~ ,~~ BM cells from 1 1.5-day-old quail embryos migrated up a gradient of soluble molecules secreted by a chicken embryonic thymus during the first period of thymic colonization. When BM cells were exposed to medium conditioned by a receptive thymus, two parameters of their locomotion were modified: their migration was oriented (chemotaxis) and their speed was increased (chemokinesis). The chemotactic migration was lost when the rudiment placed into the Zigmond chamber was taken from an embryo not being colonized in vivo. However, putative receptors for chemotactic factors must be expressed on T-cell progenitors throughout embryogenesis because colonization of a grafted thymus can occur during a refractory (for colonization) period of the thymus recipi- ent.”

Medium conditioned by thymic epithelial cells taken from 1 1.5-day-old quail embryos (second wave of colonization) stimulated migration of hematopoietic precursors in Boyden chambers.60 This medium contained chemotactic peptides in the 1 to 4 Kd range (Table 1). An additional peak of che- motactic and chemokinetic activity of a molecular weight greater than 5 Kd was found as well. These results are in agreement with the size of factors detected in chicken em- bryonic thymus during the first wave of co l~niza t ion .~~

It has been suggested that chemotaxis also plays a role in thymic colonization in mammals.6’962 Chemotactic molecules secreted by thymic stromal cells have been described (Table 1). Thymic microenvironmental factors (TMFs), specifically attracting hematopoietic stem cells from fetal liver, were as- sayed by their migration through agar.63 Other thymic che- motactic factors (TCFs) have been characterized in mice and rats; these molecules fall into three ranges of molecular weight 100 to 150 Kd, 10 to 20 Kd, and <10 Kd.64965 In vitro, TCFs attract thymocytes, BM cells, fetal liver cells, and nonadherent lymphocytes from blood and spleen in Boyden chamber as- says. However, none of these molecules have been charac- terized so far.

Table 1 . Thymic Factors Involved in Chemotactic Migration of Hematopoietic Precursors

Chemotactic Molecular Factors Species Weight (Kd) Producers Migrating Cells References

Quail peptides Quail >5 3 < 5 43

Chicken peptides Chicken 250 < I 2

D2m Chicken 11.5 Rat 11 Mouse? 11

TCFs Rat, mouse 100 < <200 10 < <30 < I O

TMFs Mouse 140 50

Thymus during second colonization period

Embryonic thymus Thymus during first colonization period

Thymus during second colonization period IT45 thymic epithelial cell line Recombinant mouse p2m

Thymic stromal cells

Thymic stromal cells

Hematopoietic precursors from BM 60

Hematopoietic precursors from BM 58

Lymphoid precursors from BM 72-74 67-71

70

64-65 Thymocytes, BM cells, blood, and spleen lymphocytes

Lymphoid cells from fetal liver 63

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THYMUS HOMING 5

To date, the best characterized chemotactic molecule in- volved in thymus homing is the soluble &microglobulin (O2m), which is the common small subunit of major histo- compatibility complex (MHC) class I antigens.@ By following its chemotactic activity on BM cells from young rats, this molecule has been purified from medium conditioned by a rat thymic epithelial cell line.67-69 This chemotactic activity was also found with plasma rat &m as well as human &m and recombinant mouse &m, suggesting that no additional maturation of this protein is necessary for chemotactic ac- tivity?' Rat BM cells migrating toward 02m were resting cells and could acquire T-cell markers in coculture experiments with thymic stroma.71 Also, chicken B2m attracts BM cells of 13day-old chicken embryos (second wave of colonization) in Boyden chamber assay^.^*-^^ The responsive D2m cells are able to colonize a 13-day-old thymus in ovo. Moreover, dur- ing chicken embryogenesis, peaks of P2m RNA transcripts and of free Pzm protein synthesis were only detected in the thymus. The peak of free &m protein synthesis in the thymus and the increased number of BM cells responding to P2m occur concomitantly with the second wave of colonization. In addition, thymus colonization in ovo by injected BM cells could be partly inhibited by a simultaneous injection of &m or anti-&m specific antibody (Dunon D, Imhof BA, in prep aration). These results suggest that O2m participate in the process of thymus colonization in the chick. It is unclear whether the Pzm also plays a chemotactic role in adult birds and mammals; &m is expressed on most cells as subunit of MHC class I antigens and in body fluids at high concentration in these inspnces. The thymus colonization in adult P2m- deficient mice is normal.75 This suggests that other molecular entities with chemotactic properties must exist. The P2m- deficient mice may prove valuable in the identification of other chemotactic factors.

PRO-T CELL MIGRATION INTO AND WITHIN THE PERIVASCULAR SPACE

Chemotactic factors and/or cytokines could also regulate the binding of pro-T cells to the endothelium, which might explain the increased binding of pro-T cells to endothelial cells when they were cocultured with thymic epithelial cells?8 Their effect could be complex like it was found for the neu- trophil chemoattractant IL-8. IL-8 downregulates L-selectin expression on neutrophils but stimulates transendothelial m i g r a t i ~ n . ~ ~ - ~ ~ Other neutrophil chemoattractants, such as fMLP or the C5a fragment of complement, activate Mac-1 and LFA-I but also cause a rapid loss of L-selectin that is involved in the initial binding to vascular endothelium under physiological flow ~onditions.~~,~'

Lymphocyte transendothelial migration also requires the rapid loss of several adhesion molecules. Proteases could play an important role during this transendothelial migration by breaching endothelial and subendothelial barriers. For in- stance, there is a rapid loss of L-selectin on lymphocytes caused by proteolysis induced by protein kinase C (PKC) activators.81.82 Moreover, a trypsin-like serine protease present on T cells releases high molecular weight products from the sulfated proteoglycans in the subendothelial extracellular

matrix.83 Another protease, the neutral endopeptidase (CD10/ NEP) that cleaves at the amino terminal side of hydrophobic residues in peptides such as enkephalins, substance P, neu- ropeptide and the neutrophil chemoattractant MLP, is ex- pressed on lymphoid leukemia cells and on very early lym- phoid precursor cells in fetal liver and fetal and adult BM and thymus.@ It suggests that proteases might also participate in the maintenance of a concentration gradient of chemotactic peptides in the vicinity of the thymus. Finally, immature CD4-CD8- thymocytes also express specific cell-surface-as- sociated exoaminopedidase activities: serine ectodipeptidyl peptidase IV or DPP IV, Leu-, and Ala-aminopeptida~es.85.~~

Unlike prokaryotes, eukaryotic cells need a solid support to migrate. Pro-T cell migration from the perivascular space toward the thymic epithelium requires extracellular matrix proteins as anchoring points. In the presence of thymic che- motactic factor, quail hematopoietic precursors were able to transverse a human amniotic basement membrane. l6 The inhibition of this process by fibronectin-specific antibodies or by synthetic peptides containing RGDS, a cell binding sequence of fibronectin, suggest that T-cell precursors interact with fibronectin during this migration. It has also been shown that laminin-specific antibodies inhibit this invasive process and that migrating precursors express 01 integrins including a low level ( ~ 6 p 1 integrin recognized by EA- 1 antibody. Other ligands for fibronectin and/or laminin, such as the DPP IV, might also be in~olved.'~ In addition, CD44, the receptor for the hyaluronic acid and LFA- 1, might also play a role in this

The last event before entering the thymus is the interaction with the basal membrane of thymic epithelial cells, as he- matopoietic precursors bind preferentially to extracellular matrix of thymic epithelial cells. T-cell differentiation depends on the subsequent interaction between the pro-T cell and the thymic epithelial cell itself.

CONCLUSION

The different steps of thymus homing described above use distinct molecular processes, some of which must be redun- dant. The (Yg integrins and CD44 are now known to partic- ipate in the adhesion of T-cell progenitors but other adhesion molecules that play a role in this step may exist and their identification is an important area of research in this field. Chemotactic factors guide the migration of pro-T cells from the blood vessels to the thymic epithelium where T-cell dif- ferentiation proceeds. The only characterized chemotactic factor so far is P2m, but the availability of P2m-deficient mice may facilitate the identification of other chemotactic factors in the near future. The molecular dissection of thymus hom- ing may also shed light on the metastasis process in cancer research. Indeed, thymus homing and metastasis seem to be similar processes as they both exhibit the same steps: blood transportation of cells, organ-specific recognition, extrava- sation, and an invasive process. The ability of some cells to metastasize could be caused by abnormal regulation of the expression of adhesion molecules that are now known to par- ticipate in thymus homing, as it is the case for CD4489390 and (Y& integrins3' (and our unpublished observations).

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6 DUNON AND IMHOF

ACKNOWLEDGMENT

The authors thank Hanspeter Stahlberger for artwork, Hans Spal- inger and Beatrice Pfeiffer for photography, Drs Patricia Ruiz and Jean-Paul Thiery for helpful discussions, and Drs Ron Palacios, Charley Steinberg, and Jod-Carlos Guttierez-Ramos for critical reading of the manuscript.

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