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Originally published online as doi:10.1189/jlb.0608389 on October 27, 2008

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(Journal of Leukocyte Biology. 2009;85:251-261.)
© 2009 Society for Leukocyte Biology

CD44 promotes progenitor homing into the thymus and T cell maturation

Mohini Rajasagi*, Mario Vitacolonna*, Bojan Benjak*, Rachid Marhaba*,1 and Margot Zöller*,{dagger},1,2

* Tumor Cell Biology, Department of Surgery, University of Heidelberg, and Department of Tumor Progression and Tumor Defense, German Cancer Research Center, Heidelberg, Germany; and
{dagger} Department of Applied Genetics, University of Karlsruhe, Karlsruhe, Germany

2 Correspondence: Tumor Cell Biology, Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 365, D-69120 Heidelberg, Germany. E-mail: m.zoeller{at}dkfz.de

ABSTRACT

Regain of immunocompetence after myeloablation and bone marrow cell (BMC) reconstitution essentially depends on T progenitor homing into the thymus and intrathymic T cell maturation. CD44 facilitates progenitor homing and settlement in the bone marrow and is known as a T progenitor marker. In search for improving regain of immunocompetence after BMC reconstitution, we explored whether the CD44 standard (CD44 s) and/or variant isoforms CD44v6 and CD44v7 contribute to thymus repopulation and thymocyte maturation. Antibody-blocking studies and cells/mice with a targeted deletion of CD44v6/7 or CD44v7 revealed that CD44s, but not CD44v6 and CD44v7, has a major impact on progenitor cell homing into the thymus. Instead, CD44v6 strengthens apoptosis resistance and expansion of early thymocytes. CD44v6-induced apoptosis resistance, most strong in double-negative (DN) thymocytes, is accompanied by Akt activation. CD44v6-induced proliferation of DN cells proceeds via activation of the MAPK pathway. At later stages of T cell maturation, CD44 acts as an accessory molecule, initiating and supporting TCR/CD3 complex-mediated signal transduction in double-positive and single-positive thymocytes. Thus, CD44 plays a major role in thymus homing. In addition, CD44v6 is important for survival and expansion of early thymocytes. These findings suggest that strengthening CD44v6 expression on lymphoid progenitors could well contribute to accelerated regain of immunocompetence.

Key Words: rodent • hematopoiesis • thymocytes • maturation • adhesion molecules

INTRODUCTION

The option of bone marrow cell (BMC) reconstitution relies on the self-renewing capacity of hematopoietic stem cells located in the bone marrow [1 ]. A subpopulation of lymphoid progenitors leaves the marrow and migrates toward the thymus, where T lineage progenitors mature and pass the positive and negative selection processes, essential for establishing self-tolerance [2 , 3 ]. Thymus involution, starting soon after birth, does not create health problems in adults as a result of maintenance of rudimentary function [4 ] and the higher proportion of memory T cells [5 , 6 ]. However, when adults require a therapeutic replacement of the hematopoietic system, the failure of appropriate T cell education poses a serious problem on this therapeutic setting [7 8 9 ]. Thus, repopulation of the thymus and intrathymic T cell maturation are important in BMC reconstitution.

The earliest lymphoid progenitors, c-kithigh, Sca-1high, Rag1+, and EBF+ [2 , 10 ], may differentiate into common lymphoid progenitors (CLP), which are lin–c-kitlowSca-1lowIL-7R{alpha}+. These CLP give rise to B, T, NK, and dendritic cells [11 ]. However, these cells are not found in the thymus. As the most primitive thymocytes are double-negative (DN; CD4–CD8–), lin–CD44+CD25–CD4lowc-kithigh [12 ], it has been speculated that CLP proceed further toward T cell differentiation before leaving the marrow. In fact, two CLP subpopulations were described in the marrow: CLP1 cells, which are c-kit+B220–, and CLP2, c-kit–B220+CD19– [12 13 14 ]; only the CLP2 subpopulation colonizes the thymus efficiently [12 ]. P-selectin and its ligand, {alpha}4β1/VCAM-1, LFA-1/ICAM-1, CD44 [11 , 12 , 15 ], and chemokines and their receptors [16 , 17 ], particularly CCL25/CCR9, are important for the attraction of CLP2 into the thymus [12 , 15 , 18 ].

Progenitor T cells enter the thymus at the corticomedullary junction, migrate to the mid and outer cortex and the subcapsular zone, and cross the cortex in the reverse direction toward the medulla. During this migration, thymocytes proceed through four DN stages: DN1 (CD44highCD25–) to DN2 (CD44highCD25+) to DN3 (CD44lowCD25+) and DN4 (CD44lowCD25–) to the double-positive (DP; CD4+CD8+) stage. The DP thymocytes express the TCR and the CD3 complex and become subject to the positive-selection process, which is accompanied by the loss of CD4 or CD8. Thus, positively selected thymocytes are single-positive (SP) CD4+ or CD8+ cells, which enter the medulla, pass the negative selection, and leave the thymus after an additional maturation period [19 ]. T cell maturation and migration depend on the organization of the thymic epithelium [15 , 20 21 22 ]. Thymocyte markers and cytokines/chemokines and their receptors, which orchestrate thymocyte migration, are well defined [15 , 20 ]. Signals induced by the thymocyte–thymic epithelial cross-talk are not yet elucidated fully [15 , 22 ].

We are particularly concerned about potential functions of CD44 in thymus reconstitution after allogeneic BMC reconstitution. Although a targeted deletion of CD44 can be compensated [23 , 24 ], it is well established that CD44 contributes to hematopoietic progenitor [25 ] and leukemic stem cell [26 27 28 ] settlement and survival in the marrow and progenitor homing into the thymus [29 , 30 ]. However, it is unknown which progenitor subpopulation(s), particularly whether the CLP2 subpopulation, make(s) use of CD44 as a homing receptor for the thymus. It is also unknown whether CD44 variant isoforms (CD44v) contribute to progenitor homing into the thymus. Furthermore, CD44 also acts as a scaffolding protein during T cell maturation and activation [31 ], where CD44 ligation can promote proliferation [32 ], apoptosis, or apoptosis resistance [31 , 33 ]. These processes, repeatedly associated with CD44v6 and CD44v7 expression [31 32 33 34 35 36 37 ], are quite important during thymus repopulation. We here provide evidence that the CD44 standard isoform (CD44 s) is involved in progenitor homing into the thymus. Importantly, within the thymus, CD44v6 protects thymocytes at early stages of their maturation from apoptosis and drives their expansion.

MATERIALS AND METHODS

Mice and tumor lines
BALB/c (H-2d), SVEV (H-2b), CD44v7–/–, and CD44v6/7–/– (back-crossed to SVEV) mice [38 ], kindly provided by Ursala Günthert (Department of Microbiology, University of Basel, Switzerland), were bred at the Central Animal Facilities of the German Cancer Research Center (Heidelberg, Germany). Eight- to 10-week-old mice were used for experiments.

Antibodies
Hybridoma supernatant of anti-CD3{epsilon}, -CD4, -CD8, -CD11a, -CD11b, -panCD44 (IM7), -CD45, -SCA-1 (European Association of Animal Cell Cultures, Salisbury, UK), and -CD49d [39 ] was purified by affinity chromatography. Unlabeled, biotinylated, or dye-labeled anti-Ter-119, -CD49b, -Ly6C/G, -CD11c, -CD18, -CD19, -CD29, -CD44v6, -CD44v7, -CD62 ligand (CD62L), -CD95, -CD95L, -CD103, -CD106, -CD117, -CD123, -CD127, -IL-3, -IL-7, -CCL19, -phosphorylated (p)-Akt, -Bcl-2, -p-lck, -p-ZAP70, -p-ERK1/2, -protein tyrosine kinase (PTK), and -actin; biotinylated, dye- or HRP-labeled secondary antibodies; and streptavidin were obtained commercially (BD PharMingen, Heidelberg, Germany; Dianova, Hamburg, Germany; Biotrend, Köln, Germany; Bender Medsystems, Vienna, Austria; and Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Hematopoietic cell preparation
BMC were obtained by flushing femur and tibia with PBS. Thymus and bone marrow were teased through fine gauze. BMC were T cell-depleted by panning on anti-CD4- and anti-CD8-coated Petri dishes (purity: ~98%). Lineage-negative cells were enriched by anti-Ter119, -DX5, -Ly6C/G, -CD3{epsilon}, -CD8, -CD11c, and -CD19 magnetic bead depletion (Miltenyi Biotec, Bergisch Gladbach, Germany). Lineage-negative cells served as the starting population to separate CLP1 (c-kit+B220–) and CLP2 (c-kit–B220+) by incubation with the respective magnetic bead-coated antibodies. DP (CD4+CD8+) and DN (CD4–CD8–) thymocytes were enriched by magnetic bead-coated anti-CD4, followed by magnetic bead-coated anti-CD8 (regain DP: ~60%; regain DN: ~5%; purity: 95–98%). DN1, DN2, DN3, and DN4 were enriched by incubation with anti-CD44/anti-rat IgG-coated magnetic beads. Adherent/nonadherent fractions were separated further by incubation with anti-CD25-PE/anti-PE-coated magnetic beads (regain: <1%; purity: 85–95%). Where indicated, cells were CFSE (Invitrogen, Karlsruhe, Germany)-labeled.

Flow cytometry
Cells (5x105) were stained according to routine procedures. For intracellular staining (cytokines, chemokines), cells were fixed and permeabilized in advance. Samples were processed in a FACSCalibur using the CellQuest program for analysis (Becton Dickinson, Heidelberg, Germany).

RT-PCR
Total RNA was extracted using TRIzol reagent. RT-PCR for detecting CCL25 and CCR9 mRNA was performed as described [40 ].

Proliferation and apoptosis assay
Cells (2x105) were stimulated with anti-CD3{epsilon} (1 µg or 10 µg/ml) and/or anti-panCD44 or anti-CD44v6 (10 µg/ml). Where indicated, cells were treated with the MEK1/2 inhibitor SL327 (5 µM) or the PI-3K inhibitor LY294002 (5 µM; Calbiochem, Darmstadt, Germany). Proliferation was determined after 48 h by 3H-thymidine uptake. Apoptosis was measured by Annexin V-FITC/propidium iodine (PI) staining. The percentage of respiratory active cells was evaluated by MTT staining.

Thymocyte activation and Western blot (WB)
Thymocytes were seeded on antibody (anti-CD3 and/or anti-panCD44/-CD44v6)-coated 96-well plates. Cells were lysed after 15 min or overnight culture (Bcl-2) in Laemmli buffer, sonicated, and boiled (5 min). Lysates (30 µl) were resolved on 10% or 12% SDS-PAGE. Proteins were transferred to nitrocellulose membranes (30 V, 16 h, 4°C) and detected by WB with indicated antibodies using the ECL detection system.

Reconstitution
Lethally irradiated BALB/c (8Gy) or SVEV (9.5Gy) mice were i.v.-reconstituted with 5 x 106 BMC or subpopulations at the indicated dose, 24 h after irradiation. Mice received i.v. injections of 100 µg anti-CD44 (IM7) or rat IgG (control) twice per week starting at the day of reconstitution. For short-term reconstitution, mice received an i.v. injection of 1 x 107 CFSE-labeled BMC and/or 1–5 x 106 BMC or thymocyte subpopulations, as indicated in the individual experiments. Mice were killed at the indicated time-points. Thymocytes were isolated, and the recovery of fluorescent cells was evaluated by flow cytometry. Depending on the percentage of fluorescent cells, up to 500,000 events were collected. Animal experimentations were approved by the governmental authorities of Baden-Wuerttemberg (Germany).

Statistical analysis
Significance of differences was calculated according to the Wilcoxon rank sum test (in vivo assays) or the Student’s t-test (in vitro studies). Mean values ± SD of in vivo experiments are derived from three to five experiments with two to three mice/group in each experiment, corresponding to six to 15 mice/group. Mean ± SD of in vitro studies, repeated three times, is based on three to four replicates.

RESULTS

The importance of CD44 in hematopoietic stem-cell homing and settlement is well documented [25 26 27 28 ]. Besides evidence that CD44 supports pre-T cell homing [29 ], less is known about CD44 contributing to thymus repopulation [30 ]. Noting that anti-CD44 interferes with thymus repopulation in allogeneically reconstituted mice [9 ], it became important to define the impact of CD44 on T cell maturation during reconstitution.

CD44 expression in early T cell progenitors
Before pursuing the impact of CD44 on thymus reconstitution, we defined CD44 expression in lymphoid progenitor cells as well as on thymocytes at different stages of maturation.

Both subpopulations of common lymphoid progenitors, CLP1 and CLP2, express CD44 at a high level. Roughly 16% of CLP1 cells and 25% of CLP2 cells express CD44v6. Less than 3% of both subpopulations express CD44v7. Within the thymus, between 60% and 70% of the four major subpopulations, DN, DP, CD4 SP, and CD8 SP thymocytes, are stained by a panCD44-specific antibody. CD44v6 expression is high in DN, low in DP, and increases again in SP thymocytes. CD44v7 expression is very low in all thymocyte subpopulations (Fig. 1A ). DN thymocytes are subdivided into four subpopulations according to CD44 and CD25 expression. As described, the intensity of CD44 expression decreases during maturation from the DN1 to the DN4 stage but is increased again in DP and SP cells. CD44v6 expression, too, is weak in DN3, DN4, and DP cells but higher again in SP thymocytes (Fig. 1B) .


Figure 1
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  		Figure 1. Adhesion molecule, cytokine, chemokine, and cytokine/chemokine receptor expression in thymocyte subpopulations. (A) Subpopulations of BALB/c thymocytes were stained with anti-panCD44, anti-CD44v6, and anti-CD44v7. The mean percentage ± SD (five assays) of stained cells is shown. (B) Examples on the intensity of CD44/CD44v6 expression on thymocyte subpopulations. Single fluorescence overlays with the negative control are shown. (C) DN, DP, and CD4+ and CD8+ thymocytes were double-stained with anti-CD44v6 and antibodies against the indicated adhesion molecules. The percentage of SP and DP cells (mean values from three to five assays) is shown. The percentage of double-stained cells, taking the maximally possible percentage as 100%, is shown. (D) CCL25 and CCR9 mRNA in thymocyte subpopulations was evaluated by RT-PCR. Actin served as internal control. A representative example is shown. (E) DN, DP, and CD4+ and CD8+ thymocytes were stained with anti-IL-3, -IL-7, -IL-3R, or -IL-7R. The percentage of stained cells (mean values from three to five assays) is shown. (F) BALB/c, SVEV, SVEVv7–/–, and SVEVv6/7–/– thymocytes were stained with a panCD44-, a CD44v6-, and a CD44v7-specific antibody. The mean percentage ± SD of stained cells (mean values from five assays) is shown. *, Differences between BALB/c and SVEV mice.

Additional adhesion molecules described to be involved in thymus homing or thymocyte maturation are CD49d ({alpha}4) and its ligand CD106 (VCAM-1), CD11a (LFA-1) and its ligand CD54 (ICAM-1), CD103 ({alpha}E), and CD62L (L-selectin). CD49d and CD54 are expressed at a high percentage of thymocytes throughout their maturation. CD62L expression decreases transiently in DP thymocytes. High CD103 expression is only seen in DN thymocytes. CD11a and CD106 expression is low in all thymocyte subpopulations. All of these markers are coexpressed with CD44 (data not shown). Coexpression with CD44v6 is particularly high for CD49d and CD11a, high for CD54 and CD62L, and somewhat lower for CD103. Except for reduced CD103/CD44v6 and a more pronounced CD54/CD44v6 coexpresssion in more mature DP and SP thymocytes, coexpression of these adhesion molecules with CD44v6 does not vary considerably during thymocyte maturation (Fig. 1C) .

CCL25/CCR9 are important for the attraction of CLP2 into the thymus [12 , 15 , 18 ], and IL-3 and IL-7 are major thymocyte growth factors. As revealed by flow cytometry and semiquantitative RT-PCR, CCR9 expression, indeed, is particularly strong in DN and DP but is strongly reduced in SP thymocytes. CCL25 expression is comparably strong in CD4 SP but is not detected in DN and CD8 SP thymocytes (Fig. 1D) . IL-3 and IL-7 are expressed throughout the thymocyte maturation process, and expression is strongest in CD4+ thymocytes. Notably, IL-3R and IL-7R are most strongly expressed in DN and hardly in DP thymocytes (Fig. 1E) .

Although not well documented for human, CD44 expression in hematopoietic cells varies widely between different mouse strains. Therefore, we considered it important to see whether a potential impact of CD44 on thymus reconstitution would account similarly for strains with CD44high and CD44low expression. BALB/c mice express CD44 at a high level. This also accounts for thymocytes. Significantly less SVEV than BALB/c thymocytes are stained by a panCD44-specific antibody. Instead, CD44v6 and CD44v7 expression does not differ between BALB/c and SVEV thymocytes. Notably, too, a targeted deletion of CD44v7 or CD44v6/7 has no influence on panCD44 expression on thymocytes. SVEVv7–/– and SVEVv6/7–/– thymocytes express unaltered levels of CD44. In SVEVv7–/– thymocytes, CD44v6 expression is also unaltered (Fig. 1F) .

Taken together, particularly BALB/c thymocytes express CD44 at relatively high frequency. CD44 and CD44v6 expression varies with the maturation stage with a transient decline in DP thymocytes. CD44v7 expression is low. CCR9, IL-3R, and IL-7R expression is highest in DN cells. Irrespective of the maturation stage, integrins, CD54 and CD62L are mostly coexpressed with CD44v6. CD103, and to some extent, CD62L (co-)expression with CD44v6 is strongest in early thymocytes.

Anti-CD44 treatment and a CD44v6 deficiency retard thymus repopulation
To obtain a first hint as to whether CD44v6 and/or CD44v7 are important for thymus repopulation, lethally irradiated SVEV mice, with/without a targeted deletion of CD44v7 or CD44v6/7, were reconstituted with BMC. Lethally irradiated and reconstituted BALB/c mice served as an additional control. Thymi of BALB/c and SVEV mice reconstituted with comparable efficacy, reaching normal numbers of thymocytes 6 weeks after reconstitution. However, a significant reduction in thymocytes was seen in reconstituted SVEVv6/7–/– but not in reconstituted SVEVv7–/– mice (Fig. 2A ). Furthermore, anti-panCD44 (100 µg i.v., twice/week) interfered with thymus repopulation. The effect was stronger in BALB/c than SVEV mice (Fig. 2B) .


Figure 2
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  		Figure 2. The impact of CD44 on thymus reconstitution. (A) Lethally irradiated BALB/c, SVEV, SVEVv7–/–, and SVEVv6/7–/– mice were reconstituted with 5 x 106 autologous BMC. (B) BALB/c and SVEV mice, reconstituted as described in A, received 100 µg control IgG or anti-CD44 i.v., twice/week. (C) Lethally irradiated SVEV mice were reconstituted with 5 x 106 SVEV, SVEVv7–/–, or SVEVv6/7–/– BMC; SVEVv7–/– and SVEVv6/7–/– mice were reconstituted with 5 x 106 SVEV or SVEVv7–/– or SVEVv6/7–/– BMC. (A–C) Three mice/group were killed after 1–6 weeks. The mean number ± SD of thymocytes is shown. *, Significant differences between SVEV and SVEVv7–/–/SVEVv6/7–/– mice (A and C) and between control IgG- and anti-CD44-treated mice (B).

To evaluate a particular impact of CD44v6 and CD44v7 on reconstitution, SVEVv7–/– and SVEVv6/7–/– BMC were transferred into CD44v-competent (CD44vcomp) SVEV mice and vice versa. Repopulation of the thymus was unimpaired when SVEV mice were transferred into SVEVv7–/– or SVEVv6/7–/– mice. When SVEVv7–/– BMC were transferred into CD44vcomp mice, thymus repopulation was improved marginally. Instead, when SVEVv6/7–/– BMC were transferred into CD44vcomp mice, recovery of SVEVv6/v7–/– thymocytes remained impaired (Fig. 2C) .

Thus, thymus homing is affected by a panCD44 blockade, and CD44v6/7–/– mice are burdened, in addition, by reduced thymocyte expansion, which is not compensated in the CD44v6/7comp host. The latter indicates a possible involvement of CD44v6 in thymocyte expansion and/or maturation. To consolidate these hypotheses, we evaluated the impact of CD44 on thymocyte homing and explored whether and by which signaling pathways CD44 might support thymocyte expansion.

CD44 influences thymus homing of early T cell progenitors
The importance of CD44 for lymphoid progenitor homing into the thymus was confirmed by the transfer of CFSE-labeled progenitor cells. Few CFSE-labeled BMC [11 ] and CLP1, but roughly 2% of input CLP2 and DN1 cells, homed into the thymus. DN2, DN3, and DN4 cells homed with reduced efficacy, but even DP thymocytes homed with higher efficacy than BMC. CD4 and CD8 SP thymocytes did not home into the thymus, and thymus homing of CLP2 and DN1 cells was strongly affected by anti-CD44. Surprisingly, thymus homing of DN3 and DN4 cells, which express CD44 at a reduced level, was still affected (Fig. 3A ).


Figure 3
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  		Figure 3. The impact of CD44 on thymocyte homing. (A) BALB/c-derived, CFSE-labeled BMC (1x107), thymocytes (TC; 1x107), and subpopulations (1x106–1x107) thereof were i.v.-injected into lethally irradiated BALB/c mice, concomitantly with 150 µg anti-panCD44. (B) SVEV- or SVEVv6/7–/–-derived, CFSE-labeled BMC (1x107), thymocytes (1x107), and subpopulations (1x106–1x107) thereof were i.v.-injected into lethally irradiated SVEV or SVEVv6/7–/– mice, which were killed after 48 h. The percent recovery of input CFSE-labeled cells in the thymus was evaluated by flow cytometry. Mean values ± SD of three mice/group are shown. *, Significant differences between SVEV and SVEVv6/7–/– or between control IgG- and anti-panCD44-treated mice are indicated.

With thymus repopulation impaired in SVEVv6/7–/– mice (Fig. 2) , the question arose as to whether homing or intrathymic expansion may be altered. Similar to BALB/c, unseparated SVEV BMC and thymocytes poorly homed into the thymus. CLP2 and DN1 cell homing was lower than in BALB/c mice but strongly increased as compared with thymus homing of unseparated BMC and thymocytes. However, a host CD44v6/7 deficiency exerted no major impact, and thymus homing of CD44v6/7–/– CLP2 and DN1 cells was not improved in the CD44v6/7comp host. Also, poor thymus homing of DN3 and DP cells was not affected significantly by a CD44v6/7 deficiency in the hematopoietic cells or the host thymus stroma (Fig. 3B) .

These findings confirm preferential CLP2 cell homing into the thymus [12 ]. Thymocyte subpopulations also home into the thymus, where the homing capacity inversely correlates with maturation. A blockade of panCD44 affects progenitor homing, but CD44v6/7 competence has a minor impact. As thymus repopulation is delayed in CD44v6/7–/– mice (Fig. 2) , we speculated that CD44v6 may contribute to early stages of T progenitor maturation.

CD44v6 promotes DN thymocyte expansion
When BALB/c, SVEV, SVEVv7–/–, and SVEVv6/7–/– thymocytes were cultured in the presence of anti-CD3 and/or anti-CD44, CD44 cross-linking provided a proliferative stimulus that exceeded the response to high-level CD3 cross-linking, only when cross-linked together with subthreshold levels of CD3. This was strain-independent but less pronounced in CD44v6/7–/– thymocytes (Fig. 4A ).


Figure 4
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  		Figure 4. CD44 promotes thymocyte proliferation. (A) Thymocytes or (B) thymocyte subpopulations were seeded in antibody-coated, 96-well plates. Proliferation was evaluated by 3H-Thymidine incorporation after 48 h. Mean values of triplicates ± SD are shown. *, Significant differences between (A) anti-CD3 (10 µg/ml) and anti-CD44 (10 µg/ml) plus anti-CD3 (1 µg/ml) or (B) between anti-CD3 (1 µg/ml) and anti-CD44 (10 µg/ml) or anti-CD44 (10 µg/ml) plus anti-CD3 (1 µg/ml).

To define at which stage of thymocyte maturation CD44/CD44v6 cross-linking supports proliferation, the experiment was repeated with DN, DP, and SP cells. CD4+ and CD8+ thymocytes responded weakly to CD44 or CD44v6 cross-linking alone. Instead, CD44 together with subthreshold CD3 cross-linking provided a strong stimulus for SP and DP thymocytes and a weak stimulus for DN3 and DN4 thymocytes. SP, DN3, and DN4 thymocytes also responded to CD44v6 together with subthreshold CD3 cross-linking. On the contrary and importantly, DN1 and DN2 cells responded to CD44 and CD44v6 cross-linking alone, and this response was not increased significantly by concomitant CD3 cross-linking (Fig. 4B) .

In view of the strong proliferation of DP and SP thymocytes in response to CD3/CD44 cross-linking, it is obvious that the response of the small population of DN1 and DN2 thymocytes to CD44 cross-linking became hidden when evaluating unseparated thymocytes. Nonetheless and importantly, these experiments provided first evidence that CD44 by itself induces proliferation of early (DN1 and DN2) T progenitors. This could well explain retarded thymus repopulation by CD44v6/7–/– progenitors.

CD44/CD44v6 promotes activation of the MAPK pathway in DN1 and DN2 thymocytes
To gain insight into molecular mechanisms accounting for CD44/CD44v6-dependent, early thymocyte expansion, we started to explore which signal transduction pathways may be involved. When stimulating unseparated thymocytes by CD3 and/or CD44 or CD44v6 cross-linking, tyrosine phosphorylation became increased strongly by CD3 plus CD44 but also by CD44v6 cross-linking. Accordingly, CD44/CD44v6 cross-linking promoted lck and ZAP70 phosphorylation, which was not strengthened significantly by concomitant subthreshold CD3 cross-linking in unseparated and DP thymocytes. Low-level ERK1/2 phosphorylation, induced by CD3 cross-linking, became more pronounced by concomitant CD44 cross-linking in unseparated DP and SP (shown for CD4+ thymocytes) thymocytes. However, in these thymocyte subpopulations, CD44/CD44v6 cross-linking alone did not initiate ERK activation (Fig. 5 A and B ).


Figure 5
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  		Figure 5. CD44-initiated signal transduction in thymocytes. (A and B) Unseparated DP and CD4+ thymocytes were stimulated for 30 min by seeding on antibody-coated plates. Cells were lysed, and lysates were separated by SDS-PAGE. After protein transfer, membranes were incubated with (A) antiphosphotyrosine or (B) anti-lck/anti-p-lck, anti-ZAP/anti-p-ZAP, and anti-ERK1/2/anti-p-ERK1/2. (B) The ratios of p-lck:lck, p-ZAP70:ZAP70, and p-ERK1/2:ERK1/2 are included. (C and D) Thymocyte subpopulations were cultured for 48 h on antibody-coated plates in the presence of 5 µM MEK1/2 inhibitor. 3H-Thymidine incorporation (mean values of triplicates±SD) is presented as percent of the DMSO control.

These results imply that CD44 cross-linking can induce the early activation events of lck and ZAP70 phosphorylation but does not proceed beyond this step (MAPK pathway activation) in the absence of CD3 signals in DP and SP thymocytes. On the contrary, the effect of CD44/CD44v6 cross-linking on DN1 and DN2 thymocytes (Fig. 4B) argues for CD3-independent, CD44/CD44v6-induced signal transduction at early maturation stages. To control for an involvement of the ERK pathway directly downstream of CD44/CD44v6, proliferation of thymocyte subpopulations was evaluated in the presence of a MEK1/2 inhibitor, which interfered most strongly with the proliferation of DN1 and DN2 cells in response to CD44/CD44v6 cross-linking (Fig. 5C) . The MEK1/2 inhibitor also suppressed proliferation of DP and SP thymocytes in response to low-level CD3 cross-linking but more efficiently upon concomitant CD44/CD44v6 cross-linking (Fig. 5D) .

Thus, CD44 ligation promotes lck and ZAP70 activation, partly independent of TCR/CD3 complex ligation. At later stages of thymocyte maturation, CD44 cooperates with the CD3 complex to proceed toward ERK1/2 phosphorylation, similar to its activity in mature T cells [31 , 35 ]. On the contrary, in early DN1 and DN2 thymocytes, CD44v6 cross-linking suffices for activation of the MAPK cascade.

CD44 contributes to apoptosis protection during thymocyte maturation
The low recovery of thymocytes after reconstitution with CD44v6/7–/– BMC could also have been a result of higher apoptosis susceptibility of CD44v6/7-deficient thymocytes. To control this hypothesis, we evaluated whether CD44, particularly CD44v6, is involved in apoptosis protection.

CD95 and CD95L expression did not differ significantly between SVEV and SVEVv6/7–/– thymocytes (data not shown). However, CD44/CD44v6 cross-linking protected SVEV but not SVEVv6/7–/– thymocytes to a significant degree from apoptosis. CD3 cross-linking did not induce apoptosis protection (Fig. 6A ). The increased apoptosis protection of SVEV thymocytes by CD44/CD44v6 cross-linking was accompanied by Akt phosphorylation and Bcl-2 up-regulation, which was not observed in SVEVv6/7–/– thymocytes (Fig. 6B) .


Figure 6
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  		Figure 6. The involvement of CD44v6 in apoptosis resistance. (A) SVEV and SVEVv6/7–/– thymocytes were seeded in antibody-coated, 96-well plates. Annexin-FITC/PI staining was evaluated after 48 h. Mean values ± SD of triplicates are shown. *, Significant differences to BSA-coated plates. (B) Unseparated thymocytes were cultured for 24 h in antibody-coated plates, lysed, and after separation by SDS-PAGE, blotted with anti-Akt/anti-p-Akt, anti-Bcl2, and anti-actin. (C and D) Thymocyte subpopulations were seeded in antibody-coated, 96-well plates. Annexin-FITC/PI staining was evaluated after 48 h. Mean values ± SD of triplicates are shown. *, Significant differences to BSA-coated plates. (E) Thymocyte subpopulations were cultured in antibody-coated, 96-well plates in the presence of 5 µM LY294002 (PI-3K inhibitor). After 48 h, respiratory active cells were evaluated by MTT staining. Mean values of triplicates ± SD are presented as percent of the DMSO control. *, Significant differences.

Apoptosis protection of SVEV thymocytes by CD44/CD44v6 cross-linking was most pronounced in DN1 thymocytes; DP thymocytes were partly protected; and CD4+ and CD8+ SP thymocytes were driven into apoptosis (Fig. 6C) . CD3 cross-linking or less-pronounced, low-level CD3 plus CD44 cross-linking promoted apoptosis of DP and SP thymocytes (Fig. 6D) . Apoptosis resistance correlated inversely with susceptibility to the PI-3K inhibitor LY294002; i.e., DN1 thymocytes, which were protected from apoptosis by CD44 cross-linking, remained respiratory active in the presence of a medium dose of LY294002. On the contrary, DP and SP thymocytes, which were driven into apoptosis by CD3 cross-linking, were highly susceptible toward the PI-3K inhibitor (Fig. 6E) .

Taken together, CD44v6 promotes thymus reconstitution by MAPK activation, which supports DN thymocytes expansion, and by PI-3K/Akt activation, which renders DN thymocytes less apoptosis-susceptible.

DISCUSSION

Thymus reconsitution and central tolerance induction are most important in allogeneic BMC reconstitution to avoid persisting graft-versus-host disease and to allow for supportive vaccination. However, T cell maturation is impeded in adults by thymus involution. As we noted that anti-CD44 interferes with bone marrow homing as well as thymus repopulation in the allogeneically reconstituted host [9 ], we strived for a more profound understanding of the CD44 contribution to thymus reconstitution and T cell maturation as a first step toward therapeutic exploitation.

CD44 promotes thymus homing of progenitor T cells
Extremely few CFSE-labeled BMC home into the thymus. Thus, the majority of BMC obviously does not enter the thymus directly. Furthermore, only long-term anti-CD44 treatment influences thymus repopulation. In fact, as described [12 , 13 ], only CLP2 cells home with good efficacy into the thymus. In addition, DN1, the most early stage of thymocytes, home as efficiently as CLP2 cells. The homing efficacy of thymocytes declines with their maturation stage, and SP thymocytes do not home. Anti-panCD44 treatment nearly abolishes CLP2 and DN1 cell homing into the thymus. Notably, the anti-CD44-mediated blockade of thymocyte homing does not correlate with the intensity of CD44 expression. A possible explanation could be that IM7, which does not block the hyaluronan-binding site of CD44 [41 ], interferes with a CD44-integrin/ICAM/selectin interaction that may be particularly important for thymus homing; e.g., it has been described that BMC adhere to CD44 on stromal cells via CD62E [42 ]. We currently explore the patterns of CD44-associated adhesion molecules on thymocyte subpopulations. CD49d and/or CD62L could be possible candidates.

Taken together, thymus-homing of CLP2, DN1, and DN2 cells is strongly supported by CD44. There has been no evidence that CD44v6/v7 or CD44v7 on T progenitors or the thymus stroma contributes to settlement in the thymus; i.e., homing of CD44v7–/– or CD44v6/7–/– progenitors into the thymus was indistinguishable from that of CD44v6/7comp progenitors. Thus, the delayed thymus reconstitution with CD44v6/7–/– T progenitors could not be explained by impaired homing and points toward CD44v6/7 contributing to intrathymic maturation.

CD44 promotes thymocyte expansion
Considering the impact of CD44 on thymocyte expansion, we have to differentiate between early and late stages of thymocyte maturation. A contribution of CD44 to the latter was supported by the finding that CD44 together with subthreshold CD3 cross-linking provide a stronger proliferative stimulus than CD3 cross-linking by itself for DP and CD4+ and CD8+ SP thymocytes. As described for peripheral T cells [31 , 43 ] and similar to CD28 [44 ], CD44 acts as an accessory molecule on these more mature thymocytes such that it cooperates with the CD3 complex. Accordingly, in DP and SP thymocytes, CD44 together with CD3 cross-linking is accompanied by lck and ZAP70 phosphorylation, and the proliferative response is inhibited by a MEK1/2 inhibitor. In addition, in DP and SP thymocytes, CD44/CD44v6 cross-linking alone is accompanied by lck and ZAP70 but not ERK1/2 phosphorylation. This implies that in DP and SP thymocytes, early signaling events downstream of CD44 cannot proceed toward activation of the Ras pathway in the absence of CD3 engagement.

On the contrary, the response of DN1 and DN2 thymocytes to CD44 cross-linking by itself is affected rather exclusively by a MAPK pathway inhibitor. As the effect is not seen in CD44v6/7–/– thymocytes, we interpret the data in the sense that CD44v6 cross-linking provides a proliferative stimulus for early thymocytes that proceeds via the MAPK pathway in the absence of TCR expression. This hypothesis is in line with the tightly regulated CD44 expression at the DN stage and the proliferative activity of DN1 and DN2 thymocytes, which could be a result of CD44 interacting with an unknown ligand in the thymus. The molecular mechanisms linking CD44 directly to the Ras pathway only at the DN stage are still missing. However, the data are in line with the study of Graham et al. [30 ], demonstrating a role for CD44 in T cell development by a direct competition between CD44+ and CD44– cells and with the finding that transgenic mice expressing CD44v4-v7 on T cells (Thy1 promoter) show accelerated thymus repopulation [34 , 45 ]. In a CD44v6-overexpressing thymoma line, CD44v6 is also linked to the ERK1/2 pathway and promotes proliferation via activation of a yet-undefined phosphatase [35 ]. This finding might extend to DN thymocytes. Work in progress attempts to identify the CD44v6-associated phosphatase and signal transduction pathway. Uncoupling Ras activation from early activation events such as lck and ZAP70 phosphorylation is described for anergic CD4 T cells, and there are several candidate molecules that could mediate this uncoupling [46 ]. Irrespective of whether DN thymocytes, indeed, use CD44v6 coupled to the Ras pathway to proliferate in advance of TCR expression, our findings clearly demonstrate the importance of CD44v6 in early thymocyte expansion.

CD44 promotes apoptosis protection of early thymocyte
CD44 together with low-level CD3 cross-linking can promote thymocyte apoptosis [31 ]. On the contrary, in the absence of CD3 cross-linking, CD44 or CD44v6 cross-linking provides apoptosis protection. CD44-induced apoptosis protection is not seen in SVEVv6/7–/– mice, reduced apoptosis resistance possibly contributing to impaired thymus repopulation. In line with the different effects of CD44 cross-linking on SVEV and SVEVv6/7–/– thymocytes, strong up-regulation of p-Akt and increased Bcl-2 protein expression by CD44 engagement are only seen in SVEV thymocytes. Apoptosis protection by CD44/CD44v6 cross-linking differs in thymocyte subpopulations, a strong, protective effect restricted to the DN stage. On the contrary, CD44 without concomitant CD3 cross-linking promotes apoptosis of SP thymocytes. Apoptosis resistance/susceptibility correlates mostly with susceptibility to the PI-3K inhibitor LY294002. PI-3K, an upstream activator of Akt [47 ], plays an important role in cell growth and survival [48 ] including the critical checkpoint of T cell development at the transition from DN3 to DP thymocytes [49 , 50 ]; i.e., DN3 thymocytes of Akt1–/–Akt2–/– mice are more apoptosis-susceptible, and maturation may be blocked at the DN stage [49 ].

We propose that delayed thymus reconstitution with CD44v6/7–/– T progenitors is a consequence of early thymocyte requirement of CD44v6 for activation of the MAPK pathway, which promotes proliferation, and for activation of the PI-3K/Akt pathway with up-regulation of Bcl-2, which prevents apoptosis. At more mature stages, the impact of CD44v6 vanishes, and CD44 acts similar to peripheral T cells in concert with CD3. The delayed repopulation of the thymus in SVEVv6/7–/– mice and the accelerated T cell maturation in mice expressing CD44v4-v7 as a transgene on T lineage cells [34 ] provide strong evidence for the physiological relevance of these CD44v6-mediated activities. After BMC transplantation in the adult, where inefficient T cell maturation and expansion as a result of thymus involution pose a major drawback, CD44v6-induced, early thymocyte expansion and apoptosis protection become particularly important.

Taken together, CD44 supports homing of CLP2 and DN cells into the thymus. CD44v6 has a strong impact on DN thymocyte expansion and apoptosis protection. These activities of CD44 s and CD44v6 should be therapeutically exploited to accelerate re-establishment of a competent immune system after the transfer of hematopoietic stem/progenitor cells.

ACKNOWLEDGEMENTS

This investigation was supported by the Carreras Foundation (M. Z.) and the Tumorzentrum Heidelberg/Mannheim (M. Z.). We cordially thank Ms. J. McAlear for editing.

FOOTNOTES

1 These authors contributed equally to this work. Back

Received June 30, 2008; revised September 15, 2008; accepted October 2, 2008.

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