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The Journal of Immunology, 2007, 178: 291-300.
Copyright © 2007 by The American Association of Immunologists, Inc.

CD25+CD4+ Regulatory T Cell Migration Requires L-Selectin Expression: L-Selectin Transcriptional Regulation Balances Constitutive Receptor Turnover1

Guglielmo M. Venturi*, Rochelle M. Conway{dagger}, Douglas A. Steeber{dagger} and Thomas F. Tedder2,*

* Department of Immunology, Duke University Medical Center, Durham, NC 27710; and {dagger} Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The molecular mechanisms controlling regulatory CD25+Foxp3+CD4+ T cell (Treg) migration are central to in vivo immune responses. Treg cell subsets differentially express L-selectin, an adhesion molecule mediating lymphocyte migration to peripheral LNs (PLNs) and leukocyte rolling during inflammation. In this study, L-selectin was essential for Treg cell migration and normal tissue distribution. Specifically, there was a 90% reduction in PLN Treg cells in L-selectin–/– mice with a compensatory increase in spleen Treg cell numbers. Unexpectedly, however, 40% of the CD4+ T cells remaining within PLNs of L-selectin–/– mice were Treg cells. The migratory properties of Treg cells were nonetheless markedly different from those of naive CD4+ T cells, with 3- to 9-fold lower migration of Treg cells into PLNs and ~2-fold lower migration into the spleen. Treg cells also turned over cell surface L-selectin at a faster rate than CD25–CD4+ T cells, but maintained physiologically appropriate L-selectin densities for optimal migration. Specifically, Treg cells expressed 30–40% more cell surface L-selectin when its endoproteolytic cleavage was blocked genetically, which resulted in a 2-fold increase in Treg cell migration into PLNs. However, increased L-selectin cleavage by Treg cells in wild-type mice was accompanied by 2-fold higher L-selectin mRNA levels, which resulted in equivalent cell surface L-selectin densities on Treg and naive T cells. Thus, Treg cells and CD25–CD4+ T cells share similar requirements for L-selectin expression during migration, although additional molecular mechanisms constrain Treg cell migration beyond what is required for naive CD4+ T cell migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cells (Treg)3 with a CD25+CD4+ phenotype that express the Foxp3 transcription factor are essential for a broad spectrum of immune processes ranging from immune tolerance, inflammatory responses, maintenance of graft tolerance, and T cell homeostasis (1, 2, 3, 4, 5, 6). Treg cells and self-reactive Treg cells in particular, appear to be generated as a unique T cell lineage within the thymus (7, 8, 9, 10). Suppressive Treg cells may also be generated in lymph nodes (LNs) and other lymphoid tissues during adaptive tolerogenic responses (11, 12, 13, 14, 15). Treg cells found within most lymphoid tissue environments modulate naive T cell activation and proliferation, and thereby influence immune responses (1, 6, 7, 16) through cell-cell contact (17) and the cytokines they secrete (18, 19). Treg cells also have the capacity to migrate in vivo (6, 15, 20, 21, 22), with the majority of Treg cells expressing L-selectin (1, 20). Both L-selectin+ and L-selectin– subsets have been phenotypically characterized, with the L-selectin+ subset proliferating and maintaining suppressive function far better than the L-selectin– subset in cases of autoimmunity (23, 24) and graft-vs-host disease following alloengraftment (25, 26). Naive- and effector/memory-like CD4+ Treg cell subsets with complex patterns of L-selectin and {alpha}Ebeta7 integrin expression ({alpha}E–CD25+, {alpha}E+CD25+, and {alpha}E+CD25– subsets) have also been described (21). Likewise, the sphingosine 1-phosphate receptor agonist FTY720 that affects naive T cell migration, also influences the recirculation and function of Treg cells (24, 27, 28). Because understanding the trafficking abilities of Treg cell subsets is central to understanding their in vivo function, identifying the molecular mechanisms that regulate their migration is necessary.

L-selectin is a critical adhesion molecule for lymphocyte migration that is expressed by the majority of naive T cells after their exit from the thymus (29, 30). L-selectin is required for circulating lymphocyte entry into peripheral LNs (PLN) (31, 32, 33), and it contributes significantly to mesenteric LN (MLN) migration (34) and the migration of activated and memory/effector lymphocyte subsets (35, 36). In addition, L-selectin cooperates with other selectins and integrins to support leukocyte rolling on inflamed vascular endothelium before their firm adhesion and transmigration into lymphoid and nonlymphoid tissues (37, 38, 39). Lymphocyte migration into PLN is virtually absent in L-selectin–/– mice, and leukocyte migration into sites of inflammation is severely attenuated (29, 34, 40, 41). A direct role for L-selectin in Treg cell migration has only been assessed in a limited number of in vivo studies (6, 15, 20, 21, 22).

One unique feature that distinguishes L-selectin from other known adhesion molecules is that it is rapidly cleaved from the cell surface after cellular activation by cis-acting cell surface protease(s) (42, 43, 44, 45, 46). L-selectin endoproteolytic cleavage reduces L-selectin cell surface density on newly activated leukocytes, concomitant with the up-regulation of multiple other adhesion/activation molecules (43, 44, 47). Regulation of L-selectin endoproteolytic cleavage is essential because subtle changes in L-selectin cell surface density can significantly alter lymphocyte recirculation and migration (48, 49). Long-term lymphocyte activation also results in decreased L-selectin expression by some subsets due to the extinction of L-selectin gene transcription (35, 48, 50, 51), although some memory/effector T cell subsets express L-selectin and continue to migrate in an L-selectin-dependent manner (52, 53). These processes are thought to redirect activated lymphocytes away from L-selectin-dependent lymphoid tissues and into L-selectin-independent lymphoid and extra-lymphoid tissues (54). With the key role played by L-selectin in T cell recirculation, its expression and constitutive or activation-induced cleavage will undoubtedly affect the ability of Treg cells to migrate through lymphoid and nonlymphoid tissues under diverse in vivo conditions. To address this issue, L-selectin expression and Treg cell tissue localization and migration were assessed in L-selectin–/– mice and in L(E)SAME mice where L-selectin is not subject to endoproteolytic release from the cell surface (48).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

L-selectin–/– mice were backcrossed with C57BL/6 mice for 10 generations (29) and L(E)SAME mice were as described (48). All studies and procedures were approved by the Duke University Animal Care and Use Committee. Mice were housed under specific pathogen-free conditions and were used for studies between 2 and 3 mo of age.

Lymphocyte isolation and immunofluorescence analysis

Single-cell leukocyte preparations from spleen, PLN (brachial, axillary, inguinal, and cervical), and MLN (superior mesenteric) were isolated and stained with fluorochrome-conjugated CD103 (M290), CD44 (IM7), CD8 (53-6.7), CD4 (RM4-5), CD25 (PC61), and Foxp3 (FJK-16s) mAbs (from BD Pharmingen and eBioscience) and FITC-conjugated LAM1-116 mAb (55, 56) as described previously (34). Erythrocytes were lysed in Tris-buffered 100 mM ammonium chloride solution. Leukocyte numbers were quantified by hemocytometer following red cell lysis, with cell frequencies from all tissues determined by immunofluorescence staining with flow cytometry analysis. Leukocytes were stained at 4°C using predetermined optimal concentrations of Abs for 30 min. Ab binding was analyzed on a FACScan flow cytometer (BD Biosciences) by gating on cells with the forward and side light scatter properties of lymphocytes. Nonreactive, isotype-matched Abs (Caltag Laboratories/BD Pharmingen) were used as controls for background staining.

Lymphocyte migration assays

In vivo lymphocyte migration assays were performed as described previously (29, 48). Pooled PLN and MLN lymphocytes or splenocytes from wild-type (control) and L(E)SAME or L-selectin–/– mice (test populations) were labeled with 0.1 µM and 0.01 µM Vybrant carboxyfluorescein diacetate, succinimidyl ester (Molecular Probes), respectively, according to the manufacturer’s recommendations. After labeling, the cells were resuspended in PBS. Equal numbers of lymphocytes from wild-type and L(E)SAME or L-selectin–/– mice were mixed with ~16 x 106 lymphocytes injected i.v. into wild-type C57BL/6 recipients. Wild-type lymphocyte migration served as an internal standard population (control) for each mouse. Aliquots of the injected cell mixtures were also labeled using PE-conjugated CD25 mAb (BD Pharmingen) and Cy-Chrome-conjugated CD4 mAb (BD Pharmingen) and analyzed by flow cytometry to calculate the relevant ratios of labeled cells and/or frequency of each cell subset (Ri). Twenty-four hours following lymphocyte injections, single-cell suspensions from spleen, PLN (inguinal, axillary, and brachial), and MLN were isolated, and 5 x 106 lymphocytes were labeled using PE-conjugated CD25 mAb and Cy-Chrome-conjugated CD4 mAb. The recovered cells were analyzed by flow cytometry to calculate the relevant ratios of labeled cells and/or frequency of each cell subset (Ro), with a minimum of 1,000 viable fluorescence bright cells counted. Nonfluorescent cells and cells presenting light scattering properties of dead cells were excluded from the analysis. Results were expressed as described (29, 48) as ratios between the indicated cell populations before (Ri) and after (Ro) recovery from tissues as described in the figure legends.

Real-time reverse transcriptase-PCR analysis

L-selectin expressing CD25+CD4+ and CD25–CD4+ lymphocyte subsets from pooled PLNs and MLNs of wild-type and L(E)SAME mice were isolated by fluorescence-based cell sorting based on CD4, L-selectin, and either CD25+ or CD25– expression. Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies). cDNA synthesis was performed using SuperScript III RNase H– Reverse Transcriptase with random hexamer priming (Invitrogen Life Technologies). Forward (5'-CTG TGATG CAGGG TATTA CGGG-3') and reverse (5'-CTCTC TTCCC TCAGA ACAGT TG-3') primers were used for L-selectin cDNA amplification. beta-Actin primers were used as internal controls (forward, 5'-ATGTT TGAGA CCTTC AACAC-3' and reverse, 5'-GTGCA GTGTG AAGTA CTACC-3'). Real-time quantitative PCR amplification used the LightCycler FastStart DNA MasterPLUS SYBR Green I reaction mix and the LightCycler System (Roche Applied Science) as per the manufacturer’s recommendations. Relative expression software tool (REST; provided by M. W. Pfaffl (Institute of Physiology, Technical University of Munich, Munich, Germany) and G. W. Horgan (Biomathematics and Statistics Scotland, Rowett Research Institute, Aberdeen, U. K.)) was used for calculating relative transcript quantities and the relative fold increase in L-selectin transcript levels between Treg cells and CD25–CD4+ T cells.

In vitro activation assays

Single-cell lymphocyte preparations from wild-type and L(E)SAME PLNs were isolated and resuspended at a concentration of 1 x 106 lymphocytes/ml in RPMI 1640 culture medium (Sigma-Aldrich), supplemented with 10% FBS (Sigma-Aldrich), HEPES, 2-ME, and MEM nonessential amino acids (all obtained from Invitrogen Life Technologies). A total of 1.5 µg/ml CD3 mAb (clone 500A2; BD Pharmingen) was added to the lymphocyte preparations, and the suspensions were incubated at 37°C in a humidified environment for 4 h. Before and following activation, lymphocytes were labeled using Cy-Chrome-conjugated CD4 mAb, PE-conjugated CD25 mAb, and FITC-conjugated LAM1-116 mAb.

Statistical analysis

All data are shown as means ± SEM. The unpaired Student’s t test was used to determine the significance of differences between population means, unless noted otherwise.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
L-selectin expression by tissue Treg cells

L-selectin expression by Treg cells in wild-type mice was quantified to determine whether this might influence Treg cell migration. Within PLNs, 67% of Treg cells expressed L-selectin at high levels, whereas significantly more CD25–CD4+ cells expressed high-level L-selectin (94%; p < 0.05; Fig. 1A). Within MLN and spleen, only 48 and 44% of Treg cells expressed L-selectin at high levels, respectively, whereas significantly higher fractions of CD25–CD4+ T cells expressed high-level L-selectin (81 and 70%, respectively; p < 0.05). For Treg and CD25–CD4+ T cell subsets that expressed L-selectin at high densities, the mean fluorescence intensity of L-selectin staining was comparable for both cell subpopulations.


Figure 1
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  		FIGURE 1. Representative L-selectin, CD25, and Foxp3 expression by CD4+ T cells in L-selectin–/– and L(E)SAME mice, compared with wild-type littermates. A, L-selectin expression by Treg cells. The relative frequency of Treg and CD25–CD4+ T cells expressing high-level L-selectin was quantified for PLN, MLN, and spleen by three-color immunofluorescence staining with flow cytometry analysis. Representative flow cytometry histograms demonstrate L-selectin expression (heavy lines) for CD4+ T cells from wild-type mice. Dashed lines indicate isotype-matched control mAb staining. The vertical dashed lines are provided for reference. Results are representative of ≥3 independent experiments. Mean values significantly different between CD25+ and CD25– cells are indicated; *, p < 0.05. B, The relative frequency of CD4+ cells coexpressing Foxp3 and CD25 was quantified for spleen, PLN, and MLN by three-color immunofluorescence analysis. Values shown for the indicated quadrants represent mean (±SEM) frequencies from ≥3 three independent experiments. Mean values significantly different between wild-type and L-selectin–/– mice are indicated; *, p < 0.05.

 
Because CD25 is expressed by activated CD4+ T cells in addition to Treg cells, the frequency of Foxp3-expressing cells among CD25+CD4+ T cells within tissues was determined using wild-type and L-selectin–/– littermates. In wild-type mice, the majority of PLN (79%), MLN (80%), and spleen (75%) CD25+CD4+ T cells expressed Foxp3 (Fig. 1B). The majority of PLN (86%), MLN (80%), and spleen (76%) CD25+CD4+ T cells in L-selectin–/– mice also expressed Foxp3. Thus, the majority of CD25+CD4+ T cells in tissues of L-selectin–/– and wild-type mice were Foxp3+ Treg cells, most of which expressed L-selectin at high densities.

Role for L-selectin in Treg cell tissue localization

The role of L-selectin in Treg cell migration was quantified using L-selectin–/– mice (34). The number of Treg cells within PLNs of L-selectin–/– mice was reduced by ~90% when compared with wild-type littermates, which was similar to the 96% decrease in CD4+ T cells and overall 97% decrease in total lymphocytes (Table I and data not shown). There was no significant change in Treg cell numbers within the MLNs of L-selectin–/– mice compared with wild-type littermates. However, Treg cell exclusion from PLNs by the loss of L-selectin expression resulted in an ~30% increase in numbers of spleen Treg cells in L-selectin–/– mice, compared with an 80% increase in CD4+ T cells and overall 20% increase in total splenocyte numbers. Thus, L-selectin expression was required for the normal tissue distribution of Treg cells, consistent with previous studies demonstrating a similar redistribution of CD4+ T cells in L-selectin–/– mice (49).


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  		Table I. Tissue distribution of Treg cells in wild-type and L-selectin–/– littermatesa

 
Despite the 90% reduction in numbers of Treg cells within PLN of L-selectin–/– mice, the relative frequency of CD25+Foxp3+ cells was increased almost 4-fold among CD4+ T cells when compared with wild-type littermates (Fig. 1B). As a result, 35–40% of PLN CD4+ T cells were Treg cells in L-selectin–/– mice (Table I). The relative frequency of Treg cells was also slightly increased in MLNs of L-selectin–/– mice; 25% compared with 14% in wild-type littermates. The overall number of Treg cells within the spleens of L-selectin–/– mice increased by 29% compared with wild-type littermates (Table I). However, due to the overall increase in lymphocytes in the spleens of L-selectin–/– mice, the relative frequency of Treg cells was reduced in the spleens of L- selectin–/– mice relative to wild-type littermates, particularly when CD25 was used as a marker for this CD4+ T cell subset (Table I and Fig. 1B). Thus, L-selectin expression regulated the tissue distribution of Treg cells, but also had a dramatic influence on the relative proportions of different T lymphocyte subsets within individual tissues.

Role for L-selectin in Treg cell migration into tissues

To quantify Treg cell migration relative to CD25–CD4+ T cells, lymphocytes from spleens of L-selectin–/– and wild-type littermates were differentially labeled with the fluorescent tracking dye CFSE, transferred into wild-type recipients, and recovered from PLN, MLN, and spleen 24 h later. The relative ratios of L-selectin–/– and wild-type Treg cells were then determined by flow cytometry (Fig. 2A). The relative migration of L-selectin–/– Treg cells to PLN and MLN was significantly reduced when compared with wild-type Treg cell migration, with both Treg cell subsets migrating to the spleen at similar frequencies. Similarly, the relative migration of L-selectin–/– CD25–CD4+ T cells to PLN and MLN was significantly reduced when compared with wild-type CFSE+CD4+ T cells, with L-selectin–/– CD25–CD4+ T cells preferentially migrating to the spleen. Thus, L-selectin–/– Treg cells did not migrate efficiently into PLN or MLN when compared with wild-type Treg cells.


Figure 2
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  		FIGURE 2. In vivo migration of L-selectin–/– and wild-type Treg cells in wild-type mice. A, Representative in vivo migration of L-selectin–/– spleen CD4+ cells (CFSElow) compared with wild-type CD4+ cells (CFSEhigh). Equal numbers of donor wild-type (R2 + R4 gates) and L-selectin–/– (R1 + R3 gates) spleen lymphocytes were differentially labeled with CFSE, mixed, and transferred into wild-type littermates. The injected cell mixture was also analyzed by flow cytometry, with the relative frequencies of L-selectin–/– and wild-type CD4+ T cells determined for each mixture. To compensate for small variations in CD4+ T cell numbers between wild-type and L-selectin–/– donor mice, a normalization factor was determined and used in subsequent calculations. After 24 h, PLN, MLN, and spleen lymphocytes were harvested from recipients, labeled for CD25 and CD4 expression, with the relative frequencies of CD4+ CFSE-labeled cells within the indicated gates (R1–R4) determined by flow cytometry analysis as shown. The bar graphs indicate the relative calculated (±SEM) differences in migration observed in three recipient wild-type mice. B, Representative in vivo migration of wild-type CD25+CD4+ (R1 or R2 gates) and CD25–CD4+ (R3 or R4 gates) T cells from spleen (R2 + R4 gates) and LNs (R1 + R3 gates). Equal numbers of donor tissue lymphocytes were differentially labeled with CFSE, mixed, and transferred into wild-type littermates. The injected cells were also analyzed by flow cytometry to determine the relative frequencies of cell subsets in the preinjected (Ri) cell mixture. After 24 h, PLN, MLN, and spleen lymphocytes were harvested from recipients, labeled for CD25 and CD4 expression, with the relative frequencies of CD4+ CFSE-labeled cells within the indicated gates (R1–R4) determined by flow cytometry as shown. The bar graphs indicate differences in migration observed in three independent experiments derived by comparing the ratio of indicated lymphocyte subsets recovered from tissues (Ro) after migration with the ratio of indicated lymphocyte subsets injected (Ri). These values were used to determine mean Ro:Ri ratios (±SEM). As a control, the relative migration of CFSE+ LN CD25–CD4+ T cells (R3) was compared with the migration of CFSE+ spleen CD25–CD4+ lymphocytes (R4) ({square}). *, Significant differences between mean Ro:Ri ratios for LN and spleen Treg cells are indicated; p < 0.05. {dagger}, Significant differences between mean Ro:Ri ratios for Treg cells compared with CD25–CD4+ T cells are indicated; p < 0.01.

 
Unexpectedly, wild-type spleen Treg cells did not appear to migrate as efficiently as wild-type CD25–CD4+ T cells when the relative ratios of these cells before adoptive transfer were compared with their relative ratios after recovery from tissues during in vivo migration assays (Fig. 2A and data not shown). Therefore, the relative migratory abilities of Treg and CD25–CD4+ T cells were compared directly. In addition, the relative migratory abilities of LN (pooled PLNs and MLNs) and spleen Treg cells were also assessed. To compare wild-type Treg cell migration, LN and spleen lymphocytes were differentially labeled with CFSE, transferred into wild-type recipients, and recovered from tissues 24 h later. The ratio of each T cell subset before injection (Ri) was determined by flow cytometry and compared with the ratio of each respective T cell subset recovered from tissues following migration (Ro). Ro:Ri ratios would be ~1.0 if the starting ratio of Treg cells:CD25–CD4+ T cells before injection was equivalent to the ratio of Treg cells:CD25–CD4+ T cells recovered after migration. By analyzing relative ratios in this way, population bias resulting from using donor cells with different frequencies of cells expressing L-selectin is eliminated, thereby allowing LN and spleen Treg cell migration to be compared directly. In each tissue, CD25–CD4+ T cells from LN and spleen migrated into PLN, MLN, and spleen similarly, generating Ro:Ri ratios approaching 1.0 (Fig. 2B). By contrast, the migration of both LN and spleen Treg cells into PLN, MLN, and spleen was significantly impaired when compared with CD25–CD4+ T cell migration. When combined, LN and spleen Treg cells had an ~3-fold reduction in migration into PLNs and MLNs and 2-fold lower migration into the spleen when compared with CD25–CD4+ T cells. In addition, spleen Treg cells were significantly less efficient at migrating into PLN, MLN, and spleen when compared with LN Treg cells. Thus, LN and spleen Treg cells were not predisposed to efficiently migrate to PLN, MLN, or spleen when compared with conventional CD4+ T cells.

Treg cell expression of {alpha}E integrins

Previous studies using pooled PLN and spleen T cells have suggested that {alpha}E integrin (CD103) expression identifies functionally distinct Treg subsets and preferentially directs their migration to extralymphoid tissues and to sites of inflammation (21). To assess whether the reduced migration of PLN and spleen Treg cells in adoptive transfer experiments resulted from differences in adhesion molecule expression, the relative densities of L-selectin and {alpha}E integrin on Treg cells was assessed for PLN, MLN, and spleen. First, the patterns of L-selectin and {alpha}E integrin expression by Treg cell subsets revealed a substantial degree of tissue specificity. Within PLNs and spleen, the majority (>80%) of Treg cells did not express {alpha}E integrins, whereas ~40% of Treg cells found within MLNs expressed {alpha}E integrins (Fig. 3). Second, the majority of PLN {alpha}Ehigh Treg cells expressed high-density L-selectin as did {alpha}EhighCD25lowCD4+ and {alpha}Elow Treg cells (Table II). By contrast, the majority of MLN and spleen {alpha}Ehigh Treg cells and {alpha}EhighCD25lowCD4+ T cells did not express L-selectin, whereas most {alpha}Elow Treg cells expressed high-density L-selectin. These findings suggest that L-selectin plays a major role in directing the migration of most Treg cells independent of {alpha}E integrin expression. Moreover, the low frequency of {alpha}E integrin expression by PLN and spleen Tregs indicates that the modest migratory behavior of Treg cells to PLN, MLN, and spleen does not result from {alpha}E integrin expression inducing their preferential migration to extralymphoid tissues or to sites of inflammation.


Figure 3
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  		FIGURE 3. L-selectin, CD25, and {alpha}E integrin (CD103) expression by CD4+ T cell subsets from PLN, MLN, and spleens of wild-type mice as determined by four-color immunofluorescence staining with flow cytometry analysis. L-selectin expression (right histograms) was assessed for each CD4+ T cell subset (regions R1, R2, and R3), with the frequency of L-selectinhigh cells determined (demarked by the gates shown). The vertical dashed lines are provided for reference. These results represent those obtained in ≥3 experiments.

 

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  		Table II. Percentage of cells expressing high L-selectin levelsa

 
L-selectin endoproteolytic cleavage in Treg cells

The extent that L-selectin expression by Treg cells is controlled by down-regulation of L-selectin gene transcription or increased receptor endoproteolytic release due to cellular activation was assessed using L(E)SAME mice. Importantly, L-selectin endoproteolytic release from the cell surface does not occur in L(E) or L(E)SAME mice, as described previously (48). However, L(E)SAME mice were genetically engineered and selected to express cell surface L-selectin at wild-type densities, with normal homeostatic lymphocyte migration. As a result of equivalent L-selectin expression, the frequency of Tregs within spleen, PLNs, and MLNs of L(E)SAME mice was comparable to that in wild-type mice and the majority expressed Foxp3 (Fig. 1B). However, within PLNs of L(E)SAME mice, 85% of Treg cells expressed high L-selectin levels, whereas only 67% of Treg cells in wild-type littermates expressed high-density L-selectin (p < 0.05; Fig. 4A). Also, 98% of PLN CD25–CD4+ T cells in L(E)SAME mice expressed high-density L-selectin, which was higher than the 94% observed in wild-type littermates. Most MLN Treg cells in L(E)SAME mice also expressed high-density L-selectin (65%) compared with 48% of Treg cells in wild-type littermates (p < 0.05). These results indicate that ~18% of PLN and MLN Treg cells have been recently activated to induce significant L-selectin cleavage from the cell surface. In spleen, comparable frequencies of Treg cells from L(E)SAME and wild-type littermates expressed high-density L-selectin. The significantly decreased fraction of L-selectinlow Treg cells observed in the PLN and MLN of L(E)SAME mice compared with wild-type littermates demonstrates that ~55 to 35%, respectively, of L-selectinlow Treg cells have removed L-selectin by endoproteolytic cleavage, with the remaining L-selectinlow Treg cells in PLNs, MLNs, and spleen representing lymphocytes that have lost L-selectin expression due to extinguished L-selectin transcription.


Figure 4
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  		FIGURE 4. L-selectin expression by Treg cells from L(E)SAME and wild-type littermates. A, The relative frequency of Treg and CD25–CD4+ T cells expressing high-level L-selectin was quantified for PLN, MLN, and spleen by three-color immunofluorescence staining with flow cytometry analysis. Representative flow cytometry histograms demonstrate L-selectin expression (heavy lines) for PLN CD4+ T cells from wild-type and L(E)SAME littermates. Dashed histograms indicate isotype-matched control mAb staining. The vertical dashed lines are provided for reference. Bar graphs represent mean (±SEM) frequencies of CD4+ T cells falling within the indicated L-selectinhigh gates as shown in the flow cytometry histograms and represent results from ≥3 independent experiments. B, L-selectin cell surface densities expressed by CD4+ T cells from PLN, MLN, and spleens of L(E)SAME and wild-type littermates assessed by three-color immunofluorescence staining. Bar graph values represent average (±SEM) mean fluorescence intensities of L-selectin on T cells expressing high-level L-selectin from three experiments. A and B, Mean values significantly different between L(E)SAME and wild-type mice are shown; *, p < 0.05. C, Real-time PCR analysis of L-selectin mRNA expression levels. Treg and CD25–CD4+ T cells were isolated from pooled PLN and MLN of wild-type (one mouse) and L(E)SAME (two mice) littermates by FACS, lysed, and their RNA subjected to quantitative real-time PCR analysis. Values represent light cycler-generated units of measure relative to actin RNA levels as controls, with relative units adjusted so that CD25–CD4+ T cell values equaled 1.0. Values significantly different between CD25–CD4+ and Treg cells are indicated; *, p < 0.05. D, L-selectin and CD44 expression by PLN CD4+ T cells from L(E)SAME and wild-type littermates. E, Representative L-selectin and CD44 expression on PLN CD4+ and CD8+ T cells isolated from wild-type and L(E)SAME littermates assessed by three-color immunofluorescence staining with flow cytometry analysis. The vertical dashed lines are provided for reference. D and E, Results are representative of three independent experiments.

 
Enhanced L-selectin transcription by Treg cells

L-selectin density on Treg cells from L(E)SAME mouse PLNs, MLNs, and spleens was significantly higher than on CD25–CD4+ T cells (38 ± 2%; n = 3–11 mice; p < 0.01; Fig. 4, A and B). This was not observed for Treg cells from wild-type littermates. Increased L-selectin protein expression on L(E)SAME Treg cells also correlated with increased L-selectin transcription by Treg cells from both wild-type and L(E)SAME littermates. Specifically, Treg and CD25–CD4+ T cells were isolated from single-cell suspensions of PLN and MLN by FACS, lysed, and their RNA subjected to quantitative real-time PCR analysis. On average, Treg cells from both wild-type and L(E)SAME littermates expressed 2.2 ± 0.4-fold increases (Fig. 4C; p < 0.02) in L-selectin transcription when compared with CD25–CD4+ T cells. Thereby, increased L-selectin expression by Treg cells from L(E)SAME mice resulted from both higher L-selectin mRNA production and the absence of L-selectin cleavage from the cell surface. Thus, L-selectin cleavage and L-selectin transcription were both elevated in Treg cells of wild-type mice, but were precisely balanced to maintain an appropriate cell surface density of L-selectin.

Treg cells constitutively express elevated levels of CD44 (Fig. 4D) and other phenotypic markers that are characteristic of cell activation (21, 57). To determine whether the CD44high phenotype on Treg cells alone contributed to an increase in L-selectin expression, L-selectin levels on CD44highCD25– and CD44highCD25+ T cells were assessed in both wild-type and L(E)SAME mice. L-selectin expression was equivalent on both CD44highCD25– and CD44highCD25+ T cells in wild-type mice. By contrast, L-selectin expression on CD44highCD25+ T cells was selectively increased in L(E)SAME mice (Fig. 4D). L-selectin expression on CD4+ and CD8+ T cell subsets with a CD44high and a CD44low phenotype was also assessed. CD4+ T cells from PLN, MLN, and spleen of wild-type and L(E)SAME mice expressed L-selectin at equivalent densities regardless of CD44 expression (Fig. 4E and data not shown). By contrast, CD44highCD8+ T cells from PLN, MLN, and spleen of both wild-type and L(E)SAME mice characteristically expressed L-selectin at ~30% higher densities than CD44lowCD8+ T cells (Fig. 4E and data not shown). Thus, the CD44high phenotype of Treg cells alone does not account for their increased L-selectin expression in L(E)SAME mice. Instead, Treg cells differ from both CD25–CD4+ T cells and CD8+ T cells in how they regulate L-selectin cell surface densities.

L-selectin surface density on Treg cells before and following activation

Treg cells may be particularly prone to cleaving L-selectin from the cell surface following stimulation because wild-type Treg cells cycled cell surface L-selectin at a higher rate than CD25–CD4+ T cells (Fig. 4), the frequency of L-selectinlow expressing Tregs was higher than that of CD25–CD4+ T cells (Fig. 1), and mean L-selectin cell surface densities for the total Treg cell population in PLN of wild-type mice was 62% (n = 3; p < 0.01) lower than for CD25–CD4+ T cells (data not shown). To assess this, L-selectin expression on Treg and CD25–CD4+ T cells from wild-type and L(E)SAME PLNs was quantified following in vitro stimulation with CD3 mAb (Fig. 5). Following stimulation, the overall decrease in mean L-selectin densities was equivalent for Treg and CD25–CD4+ T cells, with ~30% of Treg and CD25–CD4+ T cells from wild-type mice having reduced L-selectin expression after 4 h of activation. However, this resulted in ~65% of wild-type Treg cells having down-regulated L-selectin expression, whereas only ~35% of CD25–CD4+ T cells had decreased L-selectin expression following activation. Reduced L-selectin expression in both cases resulted from endoproteolytic cleavage because L-selectin densities were not altered on stimulated L(E)SAME T cells. Thus, the L-selectin-dependent migration by the Treg and CD25–CD4+ T cell populations is likely to differ significantly following cellular activation because 50% of CD25+CD4+ T cells that originally had the potential to migrate to LNs using L-selectin have lost this capability as opposed to 30% of CD25–CD4+ T cells that have lost L-selectin expression. Moreover, reductions in L-selectin expression may affect Treg cells to a higher degree because their migration to PLNs was already dramatically lower than for CD25–CD4+ T cells (Fig. 2).


Figure 5
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  		FIGURE 5. L-selectin expression by Treg cells from wild-type and L(E)SAME littermates after activation in vitro, as assessed by three-color immunofluorescence staining with flow cytometry analysis. PLN lymphocytes from L(E)SAME and wild-type littermates were assessed for L-selectin expression (heavy lines) before (0 h) and after 4 h of culture with 1.5 µg/ml CD3 mAb. Dashed lines indicate isotype-matched control mAb staining. The mean percentage (±SEM; n ≥ 3 experiments) of cells expressing high levels of L-selectin is shown as delineated by the histogram gates. The vertical dashed lines are provided for reference.

 
L-selectin density regulates Treg cell migration into tissues

Total Treg cell numbers within PLN of L(E)SAME mice were 21% higher (p < 0.05) than in wild-type littermates (Table III). Although the relative percentage of Treg cells within PLN of L(E)SAME mice was lower than in their wild-type littermates, this reduction occurred because the total number of CD4+ T cells was higher in PLN of L(E)SAME mice. By contrast, total numbers of Treg cells localized within the MLN and spleens of L(E)SAME mice were significantly decreased by 28 and 19%, respectively, compared with wild-type littermates. No differences in {alpha}E+ Treg cell frequencies were observed between spleens, PLNs, and MLNs of wild-type (Fig. 3) and L(E)SAME littermates (data not shown). These results suggest that blocking L-selectin endoproteolytic cleavage affects Treg cell migration.


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  		Table III. Tissue distribution of Treg cells in L(E)SAME and wild-type littermatesa

 
To assess whether differences in Treg cell numbers relative to CD25–CD4+ T cells in L(E)SAME mice resulted from differences in their migration, pooled PLN and MLN lymphocytes from wild-type and L(E)SAME littermates were differentially labeled with CFSE and transferred into wild-type recipients. The ratio of Treg cells:CD25–CD4+ T cells before injection (Ri) was determined and compared with the ratio of Treg cells:CD25–CD4+ T cells recovered from tissues following migration (Ro; Fig. 6A). In each tissue, CD25–CD4+ T cells from wild-type mice (CFSEhigh) and L(E)SAME mice (CFSElow) migrated into tissues similarly, generating Ro:Ri ratios of ~1.0 (Fig. 6). By contrast, the overall migration of wild-type Treg cells to PLN and MLN was ~9-fold lower and to spleen was 2-fold lower when compared with CD25–CD4+ T cell migration (Fig. 6B), as noted earlier (Fig. 2B). However, the 30–40% increase in L-selectin density on L(E)SAME Treg cells (Fig. 4B) resulted in a 2-fold increase in PLN migration compared with wild-type Treg cells (Fig. 6B). Increased L-selectin density on L(E)SAME Treg cells also had a small effect on MLN migration, but not spleen migration. Therefore, subtle differences in L-selectin cell surface density had a significant effect on Treg cell migration to PLN.


Figure 6
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  		FIGURE 6. In vivo migration of Treg cells from wild-type and L(E)SAME littermates. A, Representative in vivo migration of Treg cells. Equal numbers of L(E)SAME (R1 and R3 gates) and wild-type (R2 and R4 gates) PLN lymphocytes were differentially labeled with CFSE, mixed, and transferred into wild-type littermates. After 24 h, the relative frequencies of CFSE-labeled cells within the indicated gates (R1–R4) were determined by flow cytometry analysis. Results represent ≥3 independent experiments. B, The relative migration of Treg (R1 or R2) to CD25–CD4+ (R3 or R4, respectively) T cells was assessed by comparing the ratio of Treg:CD25–CD4+ lymphocytes in the preinjected (Ri) cell mixture with the ratio of cells recovered from tissues (Ro) after migration. These values were used to determine mean Ro:Ri ratios (±SEM) for three independent experiments. As a control, the relative migration of L(E)SAME CD25–CD4+ naive lymphocytes (R3) was compared with the migration of wild-type CD25–CD4+ naive lymphocytes (R4) ({square}). *, Significant differences between mean Ro:Ri ratios for L(E)SAME and wild-type Treg cells are indicated; p < 0.05. {dagger}, Significant differences between mean Ro:Ri ratios for Treg cells compared with CD25–CD4+ T cells are indicated; p < 0.01.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study demonstrates directly that Treg cells require L-selectin expression for normal tissue localization, and that appropriate L-selectin function is maintained through a unique balance between enhanced L-selectin transcription and its accelerated endoproteolytic release by Treg cells. Specifically, there was a 90% reduction of PLN Treg cells in L-selectin–/– mice with a compensatory increase in spleen Treg cell numbers (Table I). L-selectin deficiency had an even more significant effect on total CD4+ T cell migration, as described previously (29). This observation is likely to explain why 40% of the remaining CD4+ T cells within PLNs of L- selectin–/– mice were Treg cells (Fig. 1B and Table I). Small changes in the percentages of Treg cells are sufficient to alter the proliferation of activated lymphocytes in vitro (58). Thereby, the increased frequency of Treg cells within PLNs of L-selectin–/– mice is likely to significantly alter T cell regulation within these tissues, in addition to the functional effects resulting from decreased lymphocyte entry and numbers. Regardless, even without exogenous Ag-stimulation, LN Treg cells differed markedly in their migratory properties when compared with CD25–CD4+ T cells, with ~3- to 9-fold lower migration into PLNs and ~2-fold lower migration into the spleen in adoptive transfer experiments (Figs. 2B and 6). The inability of Treg cells to migrate effectively in vivo was only partially attributable to the increased frequency (37%) of L-selectin low or negative cells or accelerated L-selectin endoproteolytic release because L(E)SAME Treg cells were also significantly impaired in their ability to migrate into lymphoid tissues (Fig. 6). Thus, Treg cells and CD25–CD4+ T cells share similar requirements for L-selectin expression during migration, although the molecular mechanisms that regulate Treg cell migration extend far beyond the simple requirement for L-selectin expression.

LN Treg cells or their precursors required L-selectin expression for their initial localization within PLNs (Table I). However, isolated LN Treg cells were impaired in their ability to migrate back into PLNs despite their retention of high-level L-selectin expression (Figs. 1 and 4). This suggests that some LN Treg cells may not be intrinsically capable of migrating effectively when harvested from tissues and transferred ectopically into recipient hosts i.v. and may become trapped within postcapillary venules and vascular networks. It has also been suggested that some Treg subsets preferentially migrate to nonlymphoid tissues after adoptive transfer as a result of {alpha}E integrin expression (21). However, most Treg cells isolated from PLN and spleen did not express {alpha}E integrins, and MLN Treg cells varied markedly in their expression patterns of both L-selectin and {alpha}E integrins (Fig. 3 and Table II). In either case, Treg cell subsets express increased densities of adhesion molecules important for migration (CD44, CD54, and CD11a/CD18, {alpha}4beta7, {alpha}Ebeta7, and beta1 integrins, and E/P-selectin ligands), as well as diverse chemokine (CCR2, CXCR3, CCR4, CCR5, CCR6, and CCR8) receptors (1, 9, 21, 59, 60), with increased responsiveness toward CXCL9 and CCL17 chemokines (61). This complexity was further demonstrated by the finding that Treg cells isolated from PLN, MLN, and spleen varied markedly in their expression patterns of both L-selectin and {alpha}E integrins (Fig. 3 and Table II). Moreover, CCR7 is predominantly expressed on the L-selectinhigh subset of Treg cells, whereas levels of CCR2, CCR4, and CXCR3 are higher on L-selectinlow Treg cells (23). Thus, due to their complex patterns of adhesion molecule and chemokine receptor expression, Treg cell subsets present within different lymphoid tissues are likely to have very complex recirculation patterns during adoptive transfer experiments, as well as during homeostatic migration and the regulation of inflammatory immune responses.

Treg cells used a unique approach for maintaining physiologically appropriate L-selectin densities for optimal migration to LNs and sites of inflammation. Unexpectedly, Treg cells expressed 2-fold higher levels of L-selectin mRNA than CD25–CD4+ T cells (Fig. 4C). In combination, the endogenous rate of L-selectin endoproteolytic cleavage was significantly higher in Treg cells when compared with CD25–CD4+ T cells (Fig. 4, A and B). In fact, Treg cells from L(E)SAME mice expressed cell surface L-selectin at 30–40% higher densities than their CD25–CD4+ counterparts. Higher cell surface L-selectin densities on L(E)SAME Treg cells resulted in a 2-fold increase in Treg cell migration to PLN when compared with wild-type Treg cells (Fig. 6) and resulted in a consistent 20% reduction in the number of Treg cells in the spleens of L(E)SAME mice (Table III). Enhanced constitutive L-selectin endoproteolytic release was unique to Treg cells (Fig. 4D). Thereby, equivalent L-selectin cell surface protein densities on Treg and CD25–CD4+ T cells in wild-type mice is explained by increased L-selectin cleavage by Treg cells that is balanced by increased L-selectin mRNA levels. Maintaining appropriate levels of L-selectin on the surface of Treg cells is crucial due to their poor migratory potential to PLNs. If increased L-selectin cleavage was not counterbalanced by increased L-selectin transcription, L-selectin levels would likely drop to levels that would altogether exclude circulating Treg cells from entering PLNs. Finally, Treg and CD25–CD4+ T cells may differ significantly in their migratory potential and activities following Ag encounter in vivo, because the majority of PLN Treg cells became L-selectinlow or negative following in vitro activation. Relative differences in L-selectin cell surface densities are functionally important because a ~50% reduction in L-selectin surface expression on T lymphocytes results in a ~70% reduction in their migration to PLNs (49). Furthermore, the rapid loss of cell surface L-selectin following in vivo stimulation of naive Vbeta8+ T cells with staphylococcal enterotoxin B impairs their ability to migrate to PLNs (48). Thus, even relatively small changes in L-selectin cell surface density and endoproteolytic release would result in functionally significant differences in Treg cell migration.

The appropriate migration of Treg cells to lymphoid tissues is fundamentally important. The current findings document a major role for L-selectin in directing Treg cell migration independent of constitutively increased CD44, CD54, CD11a/CD18, {alpha}4beta7, {alpha}Ebeta7, and beta1 integrin expression (1, 9, 21, 59). Although Treg cell and naive CD4+ T cell migration were distinct, many of the same molecular mechanisms are likely to be used. Understanding the complex migratory options available to control Treg migration will be crucial in light of the roles that Treg cells play in tumor immunity and autoimmunity (2). Maintaining physiologically appropriate densities of cell surface L-selectin expression by the mechanisms described in this study may relate to the fact that thymus-derived Treg cells may need to migrate to PLNs to expand and proliferate in peripheral tissues in response to antigenic stimulation, or to be generated from naive CD4+ T cells by Ag stimulation (62, 63, 64). In addition, antitumor effector T cells and Treg cells capable of abrogating the antitumor reactivity of effector T cells are primed in the same LNs during tumor progression, with Treg cells generated in tumor-draining LNs retaining a L-selectinhigh phenotype (62). Likewise, tolerance-inducing Treg cells require expansion within the draining LNs of mice tolerant to transplanted hearts (15). Therefore, a better understanding of Treg cell migration and the regulation of L-selectin expression on these cells following Ag encounter will help further explain how Treg cells influence immune responses.


   Acknowledgment
 
We thank Dr. Thusitha Dissanayake for flow cytometry help.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
T. Tedder is a consultant for Medimmune, Incorporated and Angelica Therapeutics, Incorporated. He also holds equity in Angelica Therapeutics, Inc.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants CA96547, CA105001, CA098492, and AI56363, and a Basic Immunology Training Grant T32 A1052077 (to G.M.V.). Back

2 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Box 3010, Department of Immunology, Room 353, Jones Building, Research Drive, Duke University Medical Center, Durham, NC 27710. E-mail address: thomas.tedder{at}duke.edu Back

3 Abbreviations used in this paper: Treg, CD25+CD4+ regulatory T cell; LN, lymph node; PLN, peripheral LN; MLN, mesenteric LN. Back

Received for publication June 16, 2006. Accepted for publication October 20, 2006.


   References
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 Abstract
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 Results
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